Imunologie medicala

S E C O N D E D I T I O N

REALLY ESSENTIAL

MEDICALIMMUNOLOGYArthur Rabson, Ivan M.Roitt, Peter J. Delves

SECOND EDITION

Really EssentialMedical Immunology

Arthur RabsonMB, BCh, FRCPath

Department of PathologyTufts University School of Medicine

BostonUSA

Ivan M. RoittDSc, HonFRCP, FRCPath, FRS

Department of Immunology & Molecular PathologyRoyal Free & University College Medical School

LondonUK

Peter J. DelvesPhD

Department of Immunology & Molecular PathologyRoyal Free & University College Medical School

LondonUK

© 2005 A. Rabson, I.M. Roitt, P.J. DelvesPublished by Blackwell Publishing LtdBlackwell Publishing, Inc., 350 Main Street, Malden, Massachusetts 02148-5020, USABlackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UKBlackwell Publishing Asia Pty Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia

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First published 2000Reprinted 2001Second edition 2005

Library of Congress Cataloging-in-Publication DataRabson, Arthur.

Really essential medical immunology / Arthur Rabson, Ivan M. Roitt, Peter J. Delves.— 2nd ed.

p. ; cm.Rev. ed. of: Really essential medical immunology / Ivan Roitt and

Arthur Rabson. 2000.Includes bibliographical references and index.ISBN 1-4051-2115-7

1. Clinical immunology. [DNLM: 1. Immunity. QW 540 R116r 2005] I. Roitt, Ivan M. (Ivan Maurice) II. Delves, Peter J. III. Roitt, Ivan M. (Ivan Maurice). Really essential medical immunology. IV. Title.

RC582.R65 2005616.07’9—dc22

200400393

ISBN 1-4051-2115-7

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iii

Contents

Part 1 The Basis of Immunology

1 Innate Immunity, 1

2 Specific Acquired Immunity, 17

Part 2 The Recognition of Antigen

3 Antibodies, 27

4 Membrane Receptors for Antigen, 41

5 The Primary Interaction with Antigen, 50

Part 3 The Acquired Immune Response

6 The Anatomy of the Immune Response, 62

7 Lymphocyte Activation, 75

8 The Production of Effectors, 82

9 Control Mechanisms, 97

10 Ontogeny, 105

Part 4 Immunity to Infection

11 Adversarial Strategies During Infection, 114

12 Prophylaxis, 127

Part 5 Clinical Immunology

13 Immunodeficiency, 135

14 Hypersensitivity, 148

15 Transplantation, 164

16 Tumor Immunology, 177

17 Autoimmune diseases, 188

Glossary, 211

Index, 213

1

EXTERNAL BARRIERS AGAINST INFECTION

The simplest way to avoid infection is to prevent themicroorganisms from gaining access to the body. Themajor line of defense is of course the skin which, when

intact, is impermeable to most infectious agents. Whenthere is skin loss, as for example in burns, infection becomes a major problem. Additionally, most bacteriafail to survive for long on the skin because of the directinhibitory effects of lactic acid and fatty acids in sweatand sebaceous secretions and the low pH which theygenerate. An exception is Staphylococcus aureus, whichoften infects the relatively vulnerable hair follicles andglands.

Mucus secreted by the membranes lining the innersurfaces of the body acts as a protective barrier to blockthe adherence of bacteria to epithelial cells. Microbialand other foreign particles trapped within the ad-hesive mucus are removed by mechanical stratagemssuch as ciliary movement, coughing and sneezing.Among other mechanical factors that help protect theepithelial surfaces, one should also include the wash-ing action of tears, saliva and urine. Many of the secret-ed body fluids contain bactericidal components, suchas acid in gastric juice, spermine and zinc in semen, lactoperoxidase in milk, and lysozyme in tears, nasalsecretions and saliva.

Atotally different mechanism is that of microbial an-tagonism where the normal bacterial flora of the bodysuppresses the growth of many potentially pathogenicbacteria and fungi. This is due to competition for essential nutrients or by the production of microbici-dal substances. For example, pathogen invasion of thevagina is limited by lactic acid produced by commen-sal organisms which metabolize glycogen secreted by

CHAPTER 1

Innate immunity

External barriers against infection, 1Phagocytic cells kil l microorganisms, 2

The polymorphonuclear neutrophil, 2The macrophage, 2Pattern recognition receptors (PRRs) on phagocytic cells

recognize and are activated by pathogen-associatedmolecular patterns (PAMPs), 4

Toll-like receptors (TLRs) recognize PAMPs and cause cytokinerelease, 5

Microbes are engulfed by phagocytosis, 5Killing by reactive oxygen intermediates (ROIs), 6Other killing mechanisms, 7

Complement facil i tates phagocytosis, 7Complement and its activation, 7

Complement has a range of defensive biological functions, 9Complement can mediate an acute inflammatory

reaction, 10Macrophages can also do it, 10

Humoral mechanisms provide a second defensivestrategy, 11Acute phase proteins increase in response to infection, 12

Extracellular kil l ing, 13Natural killer (NK) cells are part of the innate immune

system, 13Natural killer cells also produce cytokines which regulate

inflammation and acquired immune function, 14Eosinophils, 14

We live in a potentially hostile world filled with abewildering array of infectious agents againstwhich we have developed a series of defensemechanisms at least their equal in effectivenessand ingenuity. It is these defense mechanisms thatcan establish a state of immunity against infection(Latin immunitas, freedom from) and whoseoperation provides the basis for the delightfulsubject called “Immunology.”

A number of nonspecific antimicrobial systems(e.g. phagocytosis) have been recognized whichare innate in the sense that they are not intrinsicallyaffected by prior contact with the infectious agentand are usually present before the onset of theinfectious agent. The innate response is notenhanced by previous exposure to the foreignorganism and the response time is very rapidusually occurring in minutes or hours. We shalldiscuss these systems and examine how, in thestate of specific acquired immunity, theireffectiveness can be greatly increased.

the vaginal epithelium. When protective commensalsare disturbed by antibiotics, susceptibility to oppor-tunistic infections such as Candida albicans and Clostrid-ium difficile is increased.

If microorganisms do penetrate the body, two fur-ther innate defensive operations come into play, thedestructive effect of soluble chemical factors such asbactericidal enzymes and the mechanism of phagocy-tosis — literally “eating” by the cell (Milestone 1.1).

PHAGOCYTIC CELLS KILL MICROORGANISMS

The polymorphonuclear neutrophil

This cell shares a common hematopoietic stem cell pre-cursor with the other formed elements of the blood andis the dominant white cell in the bloodstream. It is a nondividing, short-lived cell with a multilobed nu-cleus (figures 1.1 & 1.2a,b) and an array of granuleswhich are of two main types (figure 1.1): (i) the primaryazurophil granule, which develops early and containsmyeloperoxidase together with most of the nonoxida-tive antimicrobial effectors, including defensins, bactericidal/permeability-increasing (BPI) proteinand cathepsin G, and (ii) the peroxidase-negative sec-ondary specific granules, containing lactoferrin and

much of the lysozyme, alkaline phosphatase (figure1.2c) and membrane-bound cytochrome b558.

The macrophage

These cells derive from bone marrow promonocyteswhich, after differentiation to blood monocytes (figure 1.2a), finally settle in the tissues as maturemacrophages where they constitute the mononuclearphagocyte system (figure 1.2d). They are presentthroughout the connective tissue and around the basement membrane of small blood vessels and areparticularly concentrated in the lung (figure 1.2f, alveolar macrophages), liver (Kupffer cells), and liningof spleen sinusoids and lymph node medullary si-nuses, where they are strategically placed to filter offforeign material. Other examples are mesangial cells in the kidney glomerulus, brain microglia and osteo-clasts in bone. Unlike the polymorphonuclear neu-trophils, they are long-lived cells with significantrough-surface endoplasmic reticulum and mitochon-dria. Whereas the neutrophils provide the major de-fense against pyogenic (pus-forming) bacteria, as arough generalization it may be said that macrophagesare at their best in combating those bacteria (figure1.2e), viruses and protozoa that are capable of livingwithin the cells of the host.

PART 1—The basis of immunology2

Figure 1.1 Ultrastructure of neutrophil.The multilobed nucleus and two maintypes of cytoplasmic granules are well dis-played. (Courtesy of Dr D. McLaren.)

CHAPTER 1—Innate immunity 3

Milestone 1.1—Phagocytosis

The perceptive Russian zoologist, Elie Metchnikoff (1845–1916), recognized that certain specialized cells mediate de-fense against microbial infections, so fathering the wholeconcept of cellular immunity. He was intrigued by themotile cells of transparent starfish larvae and made thecritical observation that a few hours after the introductionof a rose thorn into these larvae these motile cells sur-rounded it. A year later, in 1883, he observed that fungalspores can be attacked by the blood cells of Daphnia, a tinymetozoan which, also being transparent, can be studieddirectly under the microscope. He went on to extend his investigations to mammalian leukocytes, showing theirability to engulf microorganisms, a process which hetermed phagocytosis.

Because he found this process to be even more effectivein animals recovering from infection, he came to a some-what polarized view that phagocytosis provided the main,if not the only, defense against infection. He went on to de-fine the existence of two types of circulating phagocytes:the polymorphonuclear leukocyte, which he termed a“microphage,” and the larger “macrophage.”

Figure M1.1.1 Reproductions of some of the illustrations inMetchnikoff’s book, Comparative Pathology of Inflammation(1893). (a) Four leukocytes from the frog, enclosing anthrax bacilli.Some are alive and unstained; others, which have been killed,have taken up the vesuvine dye and have been colored. (b) Draw-ing of an anthrax bacillus, stained by vesuvine, in a leukocyte ofthe frog. The two figures represent two phases of movement of thesame frog leukocyte which contains stained anthrax bacilli withinits phagocytic vacuole. (c and d) A foreign body (colored) in astarfish larva surrounded by phagocytes which have fused toform a multinucleate plasmodium, shown at higher power in (d).(e) This gives a feel for the dynamic attraction of the mobile mes-enchymal phagocytes to a foreign intruder within a starfish larva.

Figure M1.1.2 Caricature of Professor Metchnikoff (fromChanteclair, 1908, 4, p. 7). (Reproduction kindly provided by TheWellcome Institute Library, London.)

a

c

b

d

e

Pattern recognition receptors (PRRs) onphagocytic cells recognize and are activated by pathogen-associated molecular patterns(PAMPs)

Phagocytes must have mechanisms to enable them todistinguish friendly self-components from unfriendlyand potentially dangerous microbial agents . Phago-cytic cells have therefore evolved a system of receptorscalled pattern recognition receptors (PRRs) capable of

recognizing PAMPs expressed on the surface of infec-tious agents. These PAMPs are essentially polysaccha-rides and polynucleotides that differ minimally fromone pathogen to another but are not found in the host.By and large the PRRs are lectin-like and bind multiva-lently with considerable specificity to exposed micro-bial surface sugars. Engagement of the PRR generatesa signal through a NFkB (nuclear factor-kappa B) tran-scription factor pathway which alerts the cell to dangerand initiates the phagocytic process.


Figure 1.2 Cells involved in innate immunity. (a) Monocyte, show-ing “horseshoe-shaped” nucleus and moderately abundant pale cytoplasm. Note the three multilobed polymorphonuclear neu-trophils and the small lymphocyte (bottom left) (Romanowsky). (b)Four polymorphonuclear neutrophils and one eosinophil. The mul-tilobed nuclei and the cytoplasmic granules are clearly shown, thoseof the eosinophil being heavily stained. (c) Polymorphonuclear neutrophil showing cytoplasmic granules stained for alkaline phos-phatase. (d) Inflammatory cells from the site of a brain hemorrhageshowing the large active macrophage in the center with phagocy-tosed red cells and prominent vacuoles. To the right is a monocytewith horseshoe-shaped nucleus and cytoplasmic bilirubin crystals(hematoidin). Several multilobed neutrophils are clearly delineated

(Giemsa). (e) Macrophages in monolayer cultures after phagocytosisof mycobacteria (stained red) (Carbol-Fuchsin counterstained withMalachite Green.) (f) Numerous plump alveolar macrophages within air spaces in the lung. (g) Basophil with heavily staining granules compared with a neutrophil (below). (h) Mast cell frombone marrow. Round central nucleus surrounded by large darklystaining granules. Two small red cell precursors are shown at the bot-tom (Romanowsky). (i) Tissue mast cells in skin stained with Tolui-dine Blue. The intracellular granules are metachromatic and stainreddish purple. (The slides for (a), (c), (d), (g) and (h) were very kind-ly provided by Mr M. Watts. (b) was kindly supplied by Professor J.J. Owen; (e) by Professors P. Lydyard and G. Rook; (f) by Dr MerylGriffiths and (i) by Professor N. Woolf.)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Toll-like receptors (TLRs) recognize PAMPs andcause cytokine release

Toll-like receptors are a family of at least 10 trans-membrane proteins that recognize various microbialproducts. For example TLR2 recognizes Gram-positive bacterial peptidoglycan, TLR4 is specializedfor the recognition of Gram-negative bacteriallipopolysaccharide (LPS) (endotoxin) and TLR3 andTLR5 are important in the recognition of virus deriveddouble-stranded RNA. When the TLRs are activatedthey trigger a biochemical cascade with activation of NFkB and ultimately synthesis of proinflammatorycytokines and other antimicrobial peptides that lead to the development of adaptive immunity (figure 1.3).

Microbes are engulfed by phagocytosis

Before phagocytosis can occur, the microbe must first adhere to the surface of the polymorph ormacrophage through recognition of a PAMP. The resulting signal initiates the ingestion phase by activating an actin–myosin contractile system whichresults in pseudopods being extended around the particle (figures 1.4 & 1.5a). As adjacent receptors sequentially attach to the surface of the microbe, theplasma membrane is pulled around the particle justlike a “zipper” until it is completely enclosed in a vacuole called a phagosome (figures 1.4 & 1.5b). Within 1 min the cytoplasmic granules fuse with thephagosome and discharge their contents around theimprisoned microorganism (figure 1.5c), which is now


Toll-likereceptor

Serine/threoninekinase

TNF receptor associatedfactor

NFkB inducingkinase

Kinase

P

Translocation tonucleus

Ubiquitindegradation

Expression ofproinflammatory

genes

Transcriptionfactor

CD14

LBP

PHAG

OCY

TIC

CELL

INGESTION

Adaptor

LPS

LBP LPS

IRAK

TRAF

NIK

IKK

TLR

MyD88

IkBNFkB

IkBNFkB

Figure 1.3 Activation of a phagocytic cell by a Gram-negativelipopolysaccharide (LPS) (endotoxin) danger signal. CirculatingLPS is complexed by LPS-binding protein (LBP) and captured by theCD14 surface scavenging receptor. This signals internalization of thecomplex and activates the Toll-like receptor (TLR), which then initi-ates a phosphorylation cascade mediated by different kinase en-zymes. As a result the transcription factor nuclear factor-kappa B(NFkB) is released from its inhibitor IkB and translocates to the

nucleus, where it upregulates genes encoding defensive factors suchas tumor necrosis factor (TNF), antibiotic peptides and the nicoti-namide adenine dinucleotide phosphate (NADPH) oxidase whichgenerates reactive oxygen intermediates (ROIs). The TLR appears tocontrol the type of defensive response to different microbes. ThusTLR4 engineers the response to Gram-negative bacteria and LPSwhile TLR2 plays a key role in yeast and Gram-positive infections.

subject to a formidable battery of microbicidal mechanisms.

Killing by reactive oxygen intermediates (ROIs)

Trouble starts for the invader from the moment phagocytosis is initiated. There is a dramatic increasein activity of the hexose monophosphate shunt gener-

ating reduced nicotinamide adenine dinucleotidephosphate (NADPH). Electrons pass from theNADPH to a unique plasma membrane cytochrome(cyt b558), which reduces molecular oxygen directly tosuperoxide anion (figure 1.6). Thus, the key reactioncatalysed by this NADPH oxidase, which initiates the formation of ROIs, is:

NADPH O NADP O superoxide anion)oxidase2+ æ Æææ + ◊+ -

2 (


1 2 3 4

5 6 7 8

BACTERIUM

PHAGOCYTE

Chemotaxis Adherence throughPAMP recognition

Membrane activationthrough ‘danger’ signal

Initiation ofphagocytosis

Phagosomeformation Fusion Killing and

digestionRelease of degradation

products

GRANULES

Figure 1.4 Phagocytosis and killing of abacterium.

Figure 1.5 Adherence and phagocytosis. (a) Phagocytosis of Candi-da albicans by a polymorphonuclear neutrophil. Adherence to thesurface initiates enclosure of the fungal particle within arms of cyto-plasm (¥15000). (b) Phagolysosome formation by a neutrophil30 min after ingestion of C. albicans. The cytoplasm is already partly

degranulated and two lysosomal granules (arrowed) are fusing withthe phagocytic vacuole. Two lobes of the nucleus are evident (¥5000).(c) Higher magnification of (b) showing fusing granules dischargingtheir contents into the phagocytic vacuole (arrowed) (¥33000).(Courtesy of Dr H. Valdimarsson.)

(a) (b) (c)

The superoxide anion undergoes conversion to hydrogen peroxide under the influence of superoxidedismutase, and subsequently to hydroxyl radicals·OH. Each of these products has remarkable chemicalreactivity with a wide range of molecular targets making them formidable microbicidal agents; ·OH inparticular is one of the most reactive free radicalsknown. Furthermore, the combination of peroxide,myeloperoxidase (MPO) and halide ions constitutes apotent halogenating system capable of killing bothbacteria and viruses (figure 1.6).

Other killing mechanisms

Nitric oxide (NO) can be formed by an inducible NOsynthase (iNOS) in many cells of the body. Inmacrophages and human neutrophils it generates apowerful antimicrobial system. Whereas NADPH oxi-dase is dedicated to the killing of extracellular organ-isms taken into phagosomes by phagocytosis, the NOmechanism can operate against microbes that invadethe cytosol. It is not surprising therefore that iNOS capability is present in many nonphagocytic cells that may be infected by viruses and other parasites.

If microorganisms are not destroyed by these systems, they will be subjected to a family of peptides called defensins, which reach very high levelswithin the phagosome and act as disinfectants against

a wide variety of bacteria, fungi and enveloped viruses. Further damage is inflicted on the bacterialmembranes by neutral proteinase (cathepsin G) actionand by the bactericidal or bacteriostatic factors,lysozyme and lactoferrin. Finally, the killed organismsare digested by hydrolytic enzymes and the degrada-tion products released to the exterior (figure 1.4).

COMPLEMENT FACILITATESPHAGOCYTOSIS

Complement and its activation

Complement is the name given to a complex series ofover 30 proteins found in plasma and on cell surfaceswhich, along with blood clotting, fibrinolysis andkinin formation, forms one of the triggered enzymesystems found in plasma. These systems characteristi-cally produce a rapid, highly amplified response to atrigger stimulus mediated by a cascade phenomenonwhere the product of one reaction is the enzymic cata-lyst of the next. The activated or the split products ofthe cascade have a variety of defensive functions andthe complement proteins can therefore be regarded asa crucial part of the innate immune system.

Some of the complement components are desig-nated by the letter “C” followed by a number which isrelated more to the chronology of its discovery than to its position in the reaction sequence. The most abundant and the most pivotal component is C3.

C3 continuously undergoes slow spontaneous cleavage

Under normal circumstances, small amounts of C3 arecontinuously broken down into the split product C3b,or a functionally similar molecule designated C3bi. Inthe presence of Mg2+ this can complex with anothercomplement component, factor B, which then under-goes cleavage by a normal plasma enzyme (factor D) togenerate C3bBb—— . Note that conventionally a bar over acomplex denotes enzymic activity, and that on cleav-age of a complement component the larger product isgenerally given the suffix “b” and the smaller the suffix“a.”

C3bBb—— has an important new enzymic activity: it is aC3 convertase which can now split large amounts of C3to give C3a and C3b. We will shortly discuss the impor-tant biological consequences of C3 cleavage in relationto microbial defenses, but under normal conditionsthere must be some mechanism to restrain this processto a “tick-over” level since it can also give rise to evenmore C3bBb—— . That is, we are dealing with a potentially


.e

Cytosol

Granule

Membrane

Trigger

Phagocytic process

Fe2+02

MPOCl-

REACTIVE OXYGEN INTERMEDIATES

NADPH

NADP +

Flavo-cytochrome

.02 H202

.0H

HOCIchloramines

b558

Figure 1.6 Microbicidal mechanisms of phagocytic cells. Produc-tion of reactive oxygen intermediates (ROIs). Electrons from nicoti-namide adenine dinucleotide phosphate (NADPH) are transferredby the flavocytochrome oxidase enzyme to molecular oxygen toform the microbicidal molecular species shown in the boxes.

runaway positive-feedback or amplification loop(figure 1.7). As with all potentially explosive triggeredcascades, there are powerful regulatory proteins in the form of factor H and factor I which control thisfeedback loop.

During infection C3 convertase is stabilized and thealternative complement pathway is activated

A number of microorganisms can activate the C3bBb——

convertase to generate large amounts of C3 cleavageproducts by stabilizing the enzyme on their (carbo-hydrate) surfaces. This protects the C3b from factor Hand allows large quantities of C3bBb—— to build up andcleave C3. Another protein, properdin, acts sub-sequently on the bound convertase to stabilize it even

further. This series of reactions provoked directly bymicrobes leads to the clustering of large numbers ofC3b molecules on the microorganism and has beencalled the alternative pathway of complement activa-tion (figure 1.7).

Complement can be activated when carbohydrateson bacterial surfaces combine with a serum proteincalled mannose-binding lectin (MBL)

Mannose-binding lectin is found at low levels in normal serum and binds to mannose and other carbohydrates on bacterial surfaces. This initiates a series of reactions which culminate in complement activation. Mannose-binding lectin activates com-plement by interacting with two serine proteases


PROTECTED MICROBIAL SURFACE

UNPROTECTED HOST CELL SURFACE OR FLUID PHASE

MICROBIALPOLYSACCHARIDE

C3 PROPERDIN

C3bBb

FACTOR D

C3bB

C3b FACTOR B

FACTOR H

C3b Regulation

C3dgC3c

PROTEASES

FACTOR I

iC3b

LOOP

Stabilization

C3a

C3 convertase

Figure 1.7 Microbial activation of the alter-native complement pathway loop by stabi-lization of the C3 convertase (C3bBb

——), and

its control by factors H and I. When boundto the surface of a host cell or in the fluidphase, the C3b in the convertase is said to be“unprotected” in that its affinity for factor His much greater than for factor B and is there-fore susceptible to breakdown by factors Hand I. On a microbial surface, C3b binds factor B more strongly than factor H and istherefore “protected” from or “stabilized”against cleavage — even more so when sub-sequently bound by properdin. Although inphylogenetic terms this is the oldest comple-ment pathway, it was discovered after a sep-arate pathway to be discussed in the nextchapter, and so has the confusing designa-tion “alternative.” represents an acti-vation process. The horizontal bar above acomponent designates its activation.


called MASP1 and MASP2. It is known that MASP2cleaves and activates C4 and C2, generating a C3 convertase called C4b2a—— which we shall discuss inChapter 2. Activation of C3 initiates the alternativepathway loop and the formation of the membrane-at-tack complex.

The post-C3 pathway generates a membrane attackcomplex (MAC)

Recruitment of a further C3b molecule into the C3bBb—— enzymic complex generates a C5 convertase.This activates C5 by proteolytic cleavage releasing asmall polypeptide, C5a, and leaving the large C5b frag-ment loosely bound to C3b. Sequential attachment ofC6 and C7 to C5b forms a complex with a transientmembrane binding site and an affinity for C8. The C8sits in the membrane and directs the conformationalchanges in C9 which transform it into an amphipathicmolecule capable of insertion into the lipid bilayer andpolymerization to an annular MAC (figures 1.8 & 2.3).This forms a transmembrane channel fully permeableto electrolytes and water. Due to the high internal col-loid osmotic pressure of cells, there is a net influx ofNa+ and water, leading to cell lysis.

Complement has a range of defensive biological functions

1 C3b adheres to complement receptors

Phagocytic cells have receptors for C3b (CR1) and C3bi

(CR3) which facilitate the adherence of C3b-coated microorganisms to the cell surface and their subse-quent phagocytosis. This process is called opsoniza-tion and is perhaps the most important functionresulting from complement activation.

2 Biologically active fragments are released

C3a and C5a, the small peptides split from the parentmolecules during complement activation, have sever-al important actions. Both are anaphylatoxins in thatthey are capable of triggering the release of host de-fence mediators such as histamine, leukotriene B4 andtumor necrosis factor (TNF) from mast cells (figures1.2i, 1.9 & 1.10) and their circulating counterparts thebasophils. C5a acts directly on neutrophils, and bothC3a and C5a on eosinophils (described later in thischapter), to stimulate the respiratory burst associatedwith production of ROIs and to enhance the expressionof surface receptors for C3b. Importantly, C5a is also apotent neutrophil chemotactic agent. Both C3a andC5a have a striking ability to act directly on the capil-lary endothelium to produce vasodilatation and increased permeability, an effect that seems to be pro-longed by leukotriene B4 released from activated mastcells, neutrophils and macrophages.

3 The terminal complex can induce membrane lesions

As described above, the insertion of the MAC into amembrane may bring about cell lysis.

C3b

C3b

Bb

C5

C5 CONVERTASE

C6

C7

C5b

CELL SURFACE

C5b

C9

SOLUTES

SOLUTES

C8

ab g

C5aMAC

MAC

Figure 1.8 Post-C3 pathway generating C5a and the C5b–9 mem-brane attack complex (MAC). (a) Cartoon of molecular assembly. (b)Electron micrograph of a membrane C5b–9 complex incorporatedinto liposomal membranes clearly showing the annular structure.The cylindrical complex is seen from the side inserted into the mem-brane of the liposome on the left, and end-on in that on the right.(Courtesy of Professor J. Tranum-Jensen and Dr S. Bhakdi.)

(a)

(b)


Figure 1.9 The mast cell. (a) A resting cell with many membrane-bound granules containing preformed mediators. (b) A triggeredmast cell. Note that the granules have released their contents and aremorphologically altered, being larger and less electron dense. Al-though most of the altered granules remain within the circumfer-ence of the cell, they are open to the extracellular space (electronmicrographs ¥5400). (Reproduced from D. Lawson, C. Fewtrell, B.Gomperts and M.C. Raff (1975) Journal of Experimental Medicine 142,391–402. Copyright permission of The Rockefeller University Press.)

4 Complement plays a role in the induction ofantibody responses

As shall be described in detail later, B-cells proliferateand produce antibody when antigen binds to its sur-face receptors. This activation is modulated by co-receptors, including one for C3b. Therefore, when a B-cell is activated in the presence of C3b, the threshold foractivation is lowered, and much less antigen is required to activate the B-cell.

COMPLEMENT CAN MEDIATE AN ACUTEINFLAMMATORY REACTION

We can now put together an effectively orchestrateddefensive scenario initiated by activation of the alter-native complement pathway (figure 1.10).

In the first act, C3bBb—— is stabilized on the surface ofthe microbe and cleaves large amounts of C3. The C3afragment is released but C3b molecules bind copiouslyto the microbe. C3bBb—— activates the next step in the sequence to generate C5a and the MAC.

The next act sees the proinflammatory peptides, C3aand C5a (anaphylatoxins), together with the media-tors they trigger from the mast cell, recruiting polymorphonuclear neutrophils and further plasmacomplement components to the site of microbial inva-sion. Complement activation also causes the expres-sion of the adhesion molecules P-selectin and ICAM-1(intercellular adhesion molecule-1) on endothelialcells. Under the influence of the chemotaxins, neu-trophils slow down and the surface adhesion mole-cules they are stimulated to express cause them tomarginate to the walls of the capillaries. Here they firstadhere to the endothelial cells, then pass through gapsbetween these cells (diapedesis), and then move up theconcentration gradient of chemotactic factors untilthey come face to face with the C3b-coated microbe.C5a, which is at a relatively high concentration in thechemotactic gradient, activates the respiratory burst inthe neutrophils, with subsequent generation of toxicoxygen radicals and other phagocytic bactericidalmechanisms.

The processes of capillary dilatation (redness), exu-dation of plasma proteins and also of fluid (edema)due to hydrostatic and osmotic pressure changes, andaccumulation of neutrophils are collectively termedthe acute inflammatory response.

Macrophages can also do it

Tissue macrophages also play a crucial role in acute inflammatory reactions. They may be activated by the

direct action of C5a or certain bacterial toxins such asthe LPSs acting on the TLRs, or by the phagocytosis ofC3b-opsonized microbes. Following activation, themacrophages will secrete a variety of soluble media-tors which amplify the acute inflammatory response(figure 1.11). These include cytokines such as inter-leukin-1 (IL-1) and TNF, which upregulate the expres-sion of adhesion molecules for neutrophils on thesurface of endothelial cells, increase capillary perme-ability and promote the chemotaxis and activation ofthe polymorphonuclear neutrophils themselves.Thus, under the stimulus of complement activation,the macrophage provides a pattern of cellular eventswhich reinforces acute inflammation.

HUMORAL MECHANISMS PROVIDE ASECOND DEFENSIVE STRATEGY

Turning now to those defense systems which are medi-ated entirely by soluble factors, we recollect that manymicrobes activate the complement system and may be

lysed by the insertion of the MAC. The spread of infec-tion may be limited by enzymes released through tissue injury which activate the clotting system. Of the soluble bactericidal substances elaborated by thebody, perhaps the most abundant and widespread isthe enzyme lysozyme, a muramidase which splits the exposed peptidoglycan wall of susceptible bacteria.Interferons are a family of broad-spectrum antiviralagents induced by viruses and act to limit proliferationand spread of the infection. Interferon a (IFNa) is produced particularly by leukocytes, and inter-feron b (IFNb) especially by fibroblasts, although all nucleated cells can probably synthesize these mole-cules. Lastly, we may mention the two lung surfactant proteins SP-A and SP-D which, in conjunction withvarious lipids, lower the surface tension of the epithe-lial lining cells of the lung to keep the airways patent.They belong to a totally different structural group of molecules termed collectins, which contribute to innate immunity through binding of their lectin-likedomains to carbohydrates on microbes and their


C3

C3bBb

C5a C3a

BACTERIUM

C3bC5

C3b

VASCULARPERMEABILITY

MEDIATORS

C3bRECEPTOR

MC

Initiation

POLYMORPH

Exudation

KILL

1

3

7

65

4

2

ACTIVATION

CHEMOTACTICFACTORS

CAPILLARY

Figure 1.10 The defensive strategy of theacute inflammatory reaction initiated bybacterial activation of the alternative Cpathway. Directions: ➀ start with the acti-vation of the C3bBb—— C3 convertase by thebacterium, ➁ notice the generation of C3b(➂ which binds to the bacterium), C3a andC5a, ➃ which recruit mast cell mediators; ➄follow their effect on capillary dilatationand exudation of plasma proteins and ➅their chemotactic attraction of neutrophilsto the C3b-coated bacterium and triumphin ➆ the adherence and final activation ofneutrophils for the kill.

collagenous stem to cognate receptors on phagocyticcells , thereby facilitating the ingestion and killing ofthe infectious agents.

Acute phase proteins increase in response to infection

During an infection, microbial products such as endo-toxins (LPS) activate macrophages and other cells torelease various cytokines including IL-1, which is anendogenous pyrogen (incidentally capable of improv-ing our general defenses by raising the body tempera-ture), TNF and IL-6. These in turn act on the liver toincrease the synthesis and secretion of a number ofplasma proteins collectively termed acute phase pro-teins. These include C-reactive protein (CRP, the pla-sma concentration of which may increase 1000-fold),serum amyloid P component and MBL (Table 1.1). Wehave previously described the role that MBL plays inactivating the complement system. Other acute phaseproteins showing a more modest rise in concentrationinclude a1-antitrypsin, fibrinogen, ceruloplasmin, C9and factor B. Overall it seems likely that the acutephase response achieves a beneficial effect through en-hancing host resistance, minimizing tissue injury andpromoting the resolution and repair of the inflamma-tory lesion. For example, CRP can bind to numerous


Mf

BACTERIAL TOXIN C5a(?C3a) MICROBE

C3b C3b

MEDIATORS

ENDOTHELIAL CELL

BLOOD

NEUTROPHIL

BASEMENT MEMBRANEAttract + activate

neutrophilsIncrease vascular

permeability

Induceadhesionmolecules

Figure 1.11 Stimulation by complementcomponents and bacterial toxins such as lipopolysaccharide (LPS) inducesmacrophage secretion of mediators of anacute inflammatory response. Blood neu-trophils stick to the adhesion molecules onthe endothelial cell and use this to providetraction as they force their way between thecells, through the basement membrane(with the help of secreted elastase) and upthe chemotactic gradient.

Bind hemoglobin

scavengerO2• -

Acute phase reactant Role

C-reactive protein

Mannose binding lectin

�1-acid glycoprotein

Serum amyloid P component

Fixes complement, opsonizes

Fixes complement, opsonizes

Transport protein

Amyloid component precursor

�1-proteinase inhibitors

�1-antichymotrypsin

C3, C9, factor B

Ceruloplasmin

Inhibit bacterial proteases

Inhibit bacterial proteases

Increase complement function

Fibrinogen

Angiotensin

Haptoglobin

Fibronectin

Coagulation

Blood pressure

Cell attachment

Dramatic increasesin concentration:

Moderate increasesin concentration:

Table 1.1 Acute phase proteins.

microorganisms forming a complex that may activatethe complement pathway (by the classical pathway,not the alternative pathway with which we are at present familiar). This results in the deposition of C3bon the surface of the microbe which thus becomes opsonized (i.e., “made ready for the table”) for adher-ence to phagocytes. Measurement of CRP is a usefullaboratory test to assess the activity of inflammatorydisease.

EXTRACELLULAR KILLING

Natural killer (NK) cells are part of the innateimmune system

Viruses lack the apparatus for self-renewal so it is es-sential for them to penetrate the cells of the infectedhost in order to take over its replicative machinery. It is clearly in the interest of the host to find a way to kill such infected cells before the virus has had a chanceto reproduce. Natural killer cells appear to do just that.

Natural killer cells are large granular lymphocytes(figure 2.4a) with a characteristic morphology. Theypossess activating receptors which recognize struc-tures on glycoproteins on the surface of virally infectedcells or on tumor cells, and which bring killer and tar-get into close apposition (figure 1.12). Many of the ligands for the activating receptors can also be presenton noninfected normal cells, and therefore the NK cellsalso possess inhibitory receptors to prevent killing of normal cells. These inhibitory receptors, whichoverride the signals from the activating receptors,recognise ubiquitous molecules such as the major his-tocompatibility complex (MHC) class I glycoproteinnormally found on the surface of all nucleated cells.However, virally infected or tumor cells often lose ex-pression of MHC class I. Thus, only in the absence ofMHC class I is the killing of the target cell allowed toproceed (see figure 4.4). Activation of the NK cell en-sues and leads to polarization of granules between nu-cleus and target within minutes and extracellularrelease of their contents into the space between the twocells. This is followed by target cell death.

The most important of these granule contents is aperforin or cytolysin bearing some structural homolo-gy to C9. Like that protein, but without any help otherthan from Ca2+, it can insert itself into the membrane ofthe target forming a transmembrane pore with an an-nular structure, comparable to the complement MAC(figure 1.8). In addition to perforin, the granules contain lymphotoxin a and a family of serine proteasestermed granzymes, one of which, granzyme B, can

function as an NK cytotoxic factor by inducing apoptosis (programmed cell death) in the target cell.Very rapid nuclear fragmentation effected by a Ca-dependent endonuclease that acts on the vulnerableDNAbetween nucleosomes can be detected.

An alternative recognition system for NK-cell-mediated killing can involve engagement of upregu-lated Fas receptor molecules on the target cell surfaceby the FasL (Fas-ligand) on the effector NK cell, aprocess which also induces an apoptotic signal in theunlucky target. Tumor necrosis family members, inter-acting with TNF receptors on target cells can also me-diate cytotoxicity. One member of the family that is


Granzyme

TRIGGER

FasL

Fas

NK receptor

VIRALLYINFECTEDTARGETCELL

Virally induced structure

TNF

Per forin

NK CELL

DEATH SIGNAL

Figure 1.12 Extracellular killing of virally infected cell by naturalkiller (NK) cell. Binding of the NK receptors to the surface of the vi-rally infected cell triggers the extracellular release of perforin mole-cules from the granules. These polymerize to form transmembranechannels which may facilitate lysis of the target by permitting entryof granzymes, tumor necrosis factor (TNF) and other potentially cy-totoxic factors derived from the granules. (Model resembling thatproposed by D. Hudig, G.R. Ewoldt and S.L. Woodward in (1993)Current Opinion in Immunology 5, 90–6.) Engagement of the NK re-ceptor also activates a parallel killing mechanism mediated throughthe binding of the FasL (Fas-ligand) on the effector to the target cellFas receptor thereby delivering a signal for apoptosis.

expressed by activated NK cells is TRAIL (tumornecrosis factor-related apoptosis-inducing ligand).

Natural killer cells also produce cytokines whichregulate inflammation and acquired immune function

Not only do NK cells have the ability to lyse virally in-fected and tumor cells, but they also produce a widerange of cytokines once they are activated. These in-clude cytokines such as IL-1 and TNF, which play animportant role in inflammation, and granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon g (IFNg) and transforming growth factor-b(TGFb), which modulate the acquired immune re-sponse (see Chapter 2). Natural killer cells also expresscostimulatory molecules such as CD40L (CD40-ligand) and have been shown to regulate B-cell func-tion when they are activated.

Eosinophils

Large parasites such as helminths (worms) cannotphysically be phagocytosed and extracellular killing

by eosinophils would seem to have evolved to helpcope with this situation. Eosinophils, when releasedfrom the bone marrow, circulate in the peripheralblood and then traffic to peripheral tissue especially tothe lung and the gut. Their prominent location in thesesites suggests that they play an important role in hostdefence surveillance of mucosal surfaces. Eosinophilshave distinctive granules which stain avidly with aciddyes (figure 1.2b). They have surface receptors for cy-tokines, chemokines, adhesion molecules and comple-ment components, and on activation produce animpressive respiratory burst with concomitant gener-ation of active oxygen metabolites and proinflamma-tory cytokines.

Most helminths can activate the alternative complement pathway, but although resistant to C9 at-tack, their coating with C3b allows adherence ofeosinophils through the eosinophil C3b receptors. Ifthis contact should lead to activation, the eosinophilwill launch its extracellular attack, which includes therelease of major basic protein (MBP) present in theeosinophil granules which damages the parasite membrane.


REVISION

A wide range of innate immune mechanisms operatewhich do not improve with repeated exposure to infection.

Barriers against infection• Microorganisms are kept out of the body by the skin, the secretion of mucus, ciliary action, the lavagingand antibacterial action of fluids and microbial antagonism.• If penetration occurs, bacteria are destroyed by solublefactors such as lysozyme and by phagocytosis which is fol-lowed by intracellular digestion.• Phagocytic cells kill microorganisms.• The main phagocytic cells are polymorphonuclear neu-trophils and mononuclear macrophages. Organisms ad-here via their pathogen-associated molecular patterns(PAMPs) to pattern recognition receptors (PRRs) on thephagocytic cell surface.• Toll-like receptors (TLRs) are transmembrane proteins that recognize bacterial products. When activated they trigger the release of proinflammatory cytokines.

• Binding to PRRs activates the engulfment process andthe microorganism is taken inside the cell where it fuseswith cytoplasmic granules.• A formidable array of microbicidal mechanisms thencome into play including the conversion of oxygen to reac-tive oxygen intermediates (ROIs), the synthesis of nitricoxide and the release of multiple oxygen-independent fac-tors from the granules.• The complement system, a multicomponent triggeredenzyme cascade, is used to attract phagocytic cells to themicrobes and engulf them.• The most abundant component, C3, is split by a conver-tase enzyme to form C3b, which binds the adjacent microorganisms.• Mannose-binding lectin (MBL) binds to mannose on thesurface of microorganisms and initiates complement activation by binding the proteases MASP1 and MASP2.• Once C3 is split the next component, C5, is activatedyielding a small peptide, C5a. The residual C5b binds tothe surface of the organism and assembles the terminalcomponents C6–9 into a membrane attack complex


(MAC), which is freely permeable to solutes and can leadto osmotic lysis of the offending pathogen.

Complement has a range of defensive functions• C3b coated organisms bind to C3b receptor (CR1) onphagocytic cells and are more readily phagocytosed.• C5a is highly chemotactic for, and can activate, neu-trophils. Both C3a and C5a are potent chemotactic and activating agents for eosinophils and they both greatly increase capillary permeability.• C3a and C5a act on mast cells causing the release of fur-ther mediators such as histamine, leukotriene B4 andtumor necrosis factor (TNF) with effects on capillary per-meability and adhesiveness, and neutrophil chemotaxis;they also activate neutrophils.• Insertion of the MAC into an organism brings about celllysis.• C3b plays a role in facilitating antibody production byB-cells.

The complement-mediated acute inflammatory reaction• Following the activation of complement with the ensu-ing attraction and stimulation of neutrophils, the activatedphagocytes bind to the C3b-coated microbes by their sur-face C3b receptors and may then ingest them. The influx ofpolymorphs and the increase in vascular permeabilityconstitute the potent antimicrobial acute inflammatory response.• Complement activation induces endothelial cells to ex-press adhesion molecules which attach to leukocytes andcause them to move between endothelial cells into the areaof the microbes.• Phagocytic cells are activated by C5a to ingest and killinvading microbes.• Inflammation can also be initiated by tissue macro-phages, which can be activated by C5a or by bacterialproducts such as endotoxin acting on the TLRs. These cellssecrete cytokines including interleukin-1 (IL-1) and TNF which increase the adhesiveness of endothelial

cells thereby bringing more cells to the site of inflammation.

Humoral mechanisms provide a second defensive strategy• In addition to lysozyme, defensins and the complementsystem, other humoral defenses involve the acute phaseproteins such as C-reactive protein and mannose-binding ligand whose synthesis is greatly augmented by infection.• Recovery from viral infections can be effected by the interferons, which block viral replication.• Collectins bind to carbohydrates on organisms and alsoto receptors on phagocytic cells thereby facilitating phago-cytosis.

Acute phase proteins increase during infection• Cytokines such as IL-1 and TNF, released during acuteinflammation, act on the liver that synthesises plasma pro-teins called acute phase proteins.• These have a beneficial effect on host defence.• Measurement of C-reactive protein (CRP) is useful to assess the activity of inflammatory processes.

Extracellular killing• Natural killer (NK) cells possess killer activating recep-tors recognizing glycoproteins on the surface of the virally infected cell or tumor cell, and dominant in-hibitory receptors recognizing major histocompatibilitycomplex (MHC) class I on normal cells.• Virally infected cells can be destroyed by NK cells usingprogrammed cell death (apoptosis) through a perforin/granzyme pathway, or by FasL (Fas-ligand) on the NK cell engaging Fas on the target cell. • Extracellular killing by C3b-bound eosinophils may beresponsible for the failure of many large parasites to estab-lish a foothold in potential hosts.

See the accompanying website (www.roitt.com) for multiple choice questions

FURTHER READING

Beutler, B. & Hoffmann, J. (eds) (2004) Section on innate immunity.Current Opinion in Immunology, 16, 1–62.

Gregory, S.H. & Wing, E.J. (1998) Neutrophil–Kupffer cell interac-tion in host defenses to systemic infections. Immunology Today, 19,507–10.

Mollinedo, F., Borregaard, N. & Boxer, L.A. (1999) Novel trends inneutrophil structure, function and development. ImmunologyToday, 20, 535–7.

Nature Encyclopedia of Life Sciences. http://www.els.net

(constantly updated web-based resource, includes numerous immunology review articles at both introductory and advancedlevels).

Prussin, C. & Metcalfe, D. (2003) IgE, mast cells, basophils, andeosinophils. Journal of Allergy and Clinical Immunology, 111,S486–94.

Sabroe, I., Read, R.C., Whyte, M.K.B. et al. (2003) Toll-like receptors in health and disease: Complex questions remain. Journal of Immunology, 171, 1630–5.

Walport, M.J. (2001) Advances in immunology: Complement (sec-ond of two parts). New England Journal of Medicine, 344, 1140–4.


17

THE NEED FOR SPECIFIC IMMUNEMECHANISMS

Our microbial adversaries have tremendous opportu-nities through mutation to evolve strategies whichevade our innate immune defenses, and many organ-isms may shape their exteriors so as to avoid comple-ment activation completely. The body obviouslyneeded to “devise” defense mechanisms which couldbe dovetailed individually to each of these organismsno matter how many there were. In other words a verylarge number of specific immune defenses needed tobe at the body’s disposal. Quite a tall order!

ANTIBODY —THE SPECIFIC ADAPTER

Evolutionary processes came up with what can only bedescribed as a brilliant solution. This was to fashion anadapter molecule which was intrinsically capable notonly of activating the complement system and of attaching to and stimulating phagocytic cells, but alsoof sticking to the offending microbe. The adapter thushad three main regions; two concerned with commu-nicating with complement and the phagocytes (the biological functions) and one devoted to binding to anindividual microorganism (the external recognitionfunction). This latter portion would be complemen-tary in shape to some microorganism to which it couldthen bind reasonably firmly. Although the part of theadapter with biological function would be constant, aspecial recognition portion would be needed for each

of the hundreds and thousands of different organisms.The adapter is of course the molecule we know affec-tionately as antibody (figure 2.1).

Antigen–antibody complexes initiate a differentcomplement pathway (“classical”)

One of the important functions of antibody whenbound to a microbe is to activate the classical com-plement sequence. This occurs when the antigen–antibody complex binds a protein called C1q, which isthe first molecule in the classical complement sequence. C1q consists of a central collagen-like stembranching into six peptide chains each tipped by an antibody-binding subunit (resembling the blooms on abouquet of flowers). Changes in C1q consequent uponbinding the antigen–antibody complex bring aboutthe sequential activation of proteolytic activity in twoother molecules, C1r and then C1s. This forms a Ca2+-stabilized trimolecular C1 complex which dutifullyplays its role in an amplifying cascade by acting oncomponents C4 and C2 to generate many molecules ofC4b2a

—— , a new C3-splitting enzyme (figure 2.2).The next component in the chain, C4 (unfortunately

components were numbered before the sequence wasestablished), now binds to C1 and is cleaved enzymi-cally by C1s—. As expected in a multienzyme cascade,several molecules of C4 undergo cleavage into twofragments, C4a and C4b. Note that C4a, like C5a andC3a, has anaphylatoxin activity, although feeble, andC4b resembles C3b in its opsonic activity. In the pres-

CHAPTER 2

Specific acquired immunity

The need for specific immune mechanisms, 17Antibody —the specific adapter, 17

Antigen–antibody complexes initiate a different complementpathway (“classical”), 17

Cellular basis of antibody production, 19Antigen selects the lymphocytes that make antibody, 19The need for clonal expansion means humoral immunity must

be acquired, 19Acquired memory, 19

Secondary antibody responses are better, 20Acquired immunity has antigen specificity, 21

Discrimination between different antigens, 21Discrimination between self and nonself, 21

Vaccination depends on acquired memory, 22Cell-mediated immunity protects against

intracellular organisms, 22Cytokine-producing T-cells help macrophages to kill intracellular

parasites, 22Virally infected cells can be killed by antibody-dependent cellular

cytotoxicity (ADCC) and by cytotoxic T-cells, 23Immunopathology, 24

ence of Mg2+, C2 can complex with the C4b— to become anew substrate for the C1s—; the resulting product, C4b2a——,now has the vital C3 convertase activity required tocleave C3.

This classical pathway C3 convertase has the samespecificity as the C3bBb—— generated by the alternativepathway, likewise producing the same C3a and C3bfragments. Activation of a single C1 complex can bringabout the proteolysis of literally thousands of C3 mole-cules. From then on things march along exactly in par-allel to the post-C3 pathway, with one molecule of C3badded to the C4b2a——; to make it into a C5-splitting enzyme with eventual production of the membraneattack complex (figures 1.8 & 2.3). Just as the alterna-tive pathway C3 convertase is controlled by Factors Hand I, so the breakdown of C4b2a—— is brought about byeither a C4-binding protein (C4bp) or a cell surface C3breceptor called CR1.

The similarities between the two pathways are set out in figure 2.2 and show how antibody can supplement and even improve on the ability of theinnate immune system to initiate acute inflammatoryreactions.


PHAGOCYTE

ANTIBODY RECEPTOR

MICROBE

REC

BIOL

PHAGOCYTOSIS

Activation

RECOGNITION SITE

Activatecomplement

CHEMOTAXIS

INCREASEDVASCULAR

PERMEABILITY

Figure 2.1 The antibody adapter molecule. The constant part withbiological function (BIOL) activates complement and the phagocyte.The portion with the recognition unit for the foreign microbe (REC)varies from one antibody to another.

ALTERNATIVECLASSICAL

C1qrs

C4 C4b C2 C3b Factor B

PROPERDIN

STABILIZATION

C4b2 C3bB

C3

MICROBIALPOLYSACCHARIDE

ACTIVATION

ANTIBODY -MICROBECOMPLEX C4b2a C3bBb

Factor DC1qrs

Figure 2.2 Comparison of the alternative and classical comple-ment pathways. The classical pathway is antibody dependent, thealternative pathway is not. The molecular units with protease activ-

ity are highlighted. The mannose-binding lectin (MBL) pathway forcomplement activation (see p. 8) is not shown in this figure.

CELLULAR BASIS OF ANTIBODYPRODUCTION

Antigen selects the lymphocytes that make antibody

The majority of resting lymphocytes are small cellswith a darkly staining nucleus due to condensed chro-matin and relatively little cytoplasm containing theodd mitochondrion required for basic energy provi-sion (figure 2.4a). Each lymphocyte of a subset calledthe B-lymphocytes — because they differentiate in thebone marrow — is programmed to make one, and onlyone, specificity of antibody and it places these antibod-ies on its outer surface to act as receptors for the relevant antigen. These receptors can be detected byusing fluorescent probes. In figure 2.4c one can see themolecules of antibody on the surface of a human B-lymphocyte stained with a fluorescent rabbit anti-serum raised against a preparation of human

antibodies. Each lymphocyte has of the order of 105

identical antibody molecules on its surface.The molecules in the microorganisms that evoke

and react with antibodies are called antigens (gener-ates antibodies). When an antigen enters the body, it isconfronted by a dazzling array of lymphocytes allbearing different antibodies each with its own individ-ual recognition site. The antigen will only bind to thosereceptors with which it makes a good fit. Lymphocyteswhose receptors have bound antigen receive a trigger-ing signal causing them to enlarge, proliferate (figure2.4b) and develop into antibody-forming plasma cells(figures 2.4d & 2.5). Since the lymphocytes are pro-grammed to make only one antibody specificity, thatsecreted by the plasma cell will be identical with thatoriginally acting as the lymphocyte receptor, i.e. it willbind well to the antigen. In this way, antigen selects forthe antibodies that recognize it most effectively (figure2.6).

The need for clonal expansion means humoralimmunity must be acquired

Because we can make hundreds of thousands, maybeeven millions, of different antibody molecules, it is notfeasible for us to have too many lymphocytes producing each type of antibody; there just would notbe enough room in the body to accommodate them. Tocompensate for this, lymphocytes that are triggered bycontact with antigen undergo successive waves of pro-liferation (figure 2.4b) to build up a large clone of plas-ma cells which will be making antibody of the kind forwhich the parent lymphocyte was programmed. Bythis system of clonal selection, large enough concen-trations of antibody can be produced to combat infec-tion effectively (figure 2.6).

Because it takes time for the proliferating clone tobuild up its numbers sufficiently, it is usually severaldays before antibodies are detectable in the serum following primary contact with antigen. The newlyformed antibodies are a consequence of antigen expo-sure and it is for this reason that we speak of the acquired immune response.

ACQUIRED MEMORY

When we make an antibody response to a given infec-tious agent, by definition that microorganism mustexist in our environment and we are likely to meet itagain. It would make sense then for the immune mech-anisms alerted by the first contact with antigen to leave

CHAPTER 2—Specific acquired immunity 19

Figure 2.3 Multiple lesions in cell wall of Escherichia coli bacte-rium caused by interaction with immunoglobulin M (IgM) anti-body and complement. (Human antibodies are divided into fivemain classes: IgM, IgG, IgA, IgE and IgD, which differ in the speciali-zation of their “rear ends” for different biological functions such ascomplement activation or mast cell sensitization.) Each lesion iscaused by a single IgM molecule and shows as a “dark pit” due topenetration by the “negative stain.” This is somewhat of an illusionsince in reality these “pits” are like volcano craters standing proud ofthe surface, and are each single “membrane attack” complexes (cf.figure 1.8) (¥400000). (Kindly supplied by Drs R. Dourmashkin andJ.H. Humphrey.)

behind some memory system which would enable theresponse to any subsequent exposure to be faster andgreater in magnitude.

Our experience of many common infections tells usthat this must be so. We rarely suffer twice from suchdiseases as measles, mumps, chickenpox, whoopingcough and so forth. The first contact clearly imprintssome information, and imparts some memory so thatthe body is effectively prepared to repel any later inva-sion by that organism and a state of immunity is established.

Secondary antibody responses are better

By following the production of antibody on the firstand second contacts with antigen we can see the basisfor the development of immunity. For example, whenwe immunize a child with a bacterial product such astetanus toxoid, several days elapse before antibodiescan be detected in the blood; these reach a peak andthen fall (figure 2.7). If at a later stage we give a secondinjection of toxoid, the course of events is dramaticallyaltered. Within 2–3 days the antibody level in the bloodrises steeply to reach much higher values than were observed in the primary response. This secondary


B-CELL MEMBRANE

FLUORESCENT ANTI-Ig

SURFACEIg

(a) (b) (c) (d)

Figure 2.4 Cells involved in the acquired immune response. (a)Small lymphocytes. Condensed chromatin gives rise to heavy stain-ing of the nucleus. The bottom cell is a typical resting agranular T-cellwith a thin rim of cytoplasm. The upper nucleated cell is a large granular lymphocyte (LGL); it has more cytoplasm and azurophilicgranules are evident. B-lymphocytes range from small to intermedi-ate in size and lack granules (Giemsa). (b) Transformed lymphocytes(lymphoblasts) following stimulation of lymphocytes in culturewith a polyclonal activator. The large lymphoblasts with their rela-tively high ratio of cytoplasm to nucleus may be compared in sizewith the isolated small lymphocyte. One cell is in mitosis(May–Grünwald–Giemsa). (c) Immunofluorescent staining of B-lymphocyte surface immunoglobulin (Ig) using fluorescein-conjugated ( ) anti-Ig. Provided the reaction is carried out in the

cold to prevent pinocytosis, the labeled antibody cannot penetrate tothe interior of the viable lymphocytes and reacts only with surfacecomponents. Patches of aggregated surface Ig (sIg) are seen whichare beginning to form a cap in the right-hand lymphocyte. Duringcap formation, submembranous myosin becomes redistributed inassociation with the sIg and induces locomotion of the previouslysessile cell in a direction away from the cap. (d) (upper) Plasma cells.The nucleus is eccentric. The cytoplasm is strongly basophilic due tohigh RNA content. The juxtanuclear lightly stained zone corre-sponds with the Golgi region (May–Grünwald–Giemsa). (lower)Plasma cells stained to show intracellular Ig using a fluorescein-labeled anti-IgG (green) and a rhodamine-conjugated anti-IgM(red). (Material for (a) was kindly supplied by Mr M. Watts, (b) and(c) by Professor P. Lydyard, and (d) by Professor C. Grossi.)

Figure 2.5 Plasma cell (¥10000). Prominent rough-surfaced endo-plasmic reticulum associated with the synthesis and secretion of im-munoglobulin (Ig).

between the two organisms. The basis for this lies ofcourse in the ability of the recognition sites of the anti-body molecules to distinguish between antigens.

Discrimination between self and nonself

This ability to recognize one antigen and distinguish itfrom another goes even further. The individual mustalso recognize what is foreign, i.e. what is “nonself.”The failure to discriminate between self and nonselfcould lead to the synthesis of antibodies directedagainst components of the subject’s own body (autoantibodies), resulting in autoimmune disease.The body must therefore develop mechanisms where-by “self” and “nonself” can be distinguished. As weshall see later, those circulating body components thatare able to reach the developing lymphoid system inthe perinatal period will thereafter be regarded as“self.” A permanent unresponsiveness or tolerance tothe antigens on these tissues is then created, so that as immunologic maturity is reached, self-reacting lym-phocytes are suppressed or tolerized. It should be


ANTIGEN

ANTIBODY

EFFECTOR CELLS GIVING IMMUNE RESPONSE MEMORY CELLS

Maturation

Clonalproliferation

Selectiveactivationof virgin

lymphocyte

Figure 2.6 The cellular basis for thegeneration of effector and memory cellsby clonal selection after primary contactwith antigen. The cell selected by antigenundergoes many divisions during the clon-al proliferation and the progeny mature togive an expanded population of antibody-forming cells. A fraction of the progeny ofthe original antigen-reactive lymphocytesbecomes memory cells. Others mature intoeffector cells of either humoral, i.e. anti-body-mediated, or cell-mediated immuni-ty. Memory cells require fewer cycles beforethey develop into effectors and this short-ens the reaction time for the secondary re-sponse. The expanded clone of cells withmemory for the original antigen providesthe basis for the greater secondary responserelative to the primary immune response.

response then is characterized by a more rapid andmore abundant production of antibody resulting from the “tuning up” or priming of the antibody-formingsystem.

The higher response given by a primed lymphocytepopulation can be ascribed mainly to an expansion ofthe numbers of cells capable of being stimulated by theantigen, although we shall see later that there are alsosome qualitative differences in these memory cells.

ACQUIRED IMMUNITY HAS ANTIGEN SPECIFICITY

Discrimination between different antigens

The establishment of memory or immunity to one organism does not confer protection against anotherunrelated organism. After an attack of measles we are immune to further infection but are susceptible to other agents such as the polio or mumps viruses. Acquired immunity therefore shows specificity andthe immune system can differentiate specifically

pointed out at this stage that self-tolerance is not absolute and potentially harmful anti-self lympho-cytes do exist in all of us.

VACCINATION DEPENDS ON ACQUIRED MEMORY

Nearly 200 years ago, Edward Jenner carried out theremarkable studies which mark the beginning of immunology as a systematic subject. Noting the prettypox-free skin of the milkmaids, he reasoned that delib-erate exposure to the poxvirus of the cow, which is notvirulent for the human, might confer protectionagainst the related human smallpox organism. Accordingly, he inoculated a small boy with cowpoxand was delighted — and presumably relieved — to observe that the child was now protected against asubsequent exposure to smallpox. By injecting a harm-less form of a disease organism, Jenner had utilized thespecificity and memory of the acquired immune response to lay the foundations for modern vaccination (Latin vacca, cow).

The essential strategy is to prepare an innocuousform of the infectious organism or its toxins, which stillsubstantially retains the antigens responsible for

establishing protective immunity. This has been doneby using killed or live attenuated organisms, purifiedmicrobial components or chemically modified antigens (figure 2.7).

CELL-MEDIATED IMMUNITY PROTECTSAGAINST INTRACELLULAR ORGANISMS

Many microorganisms live inside host cells where it isimpossible for humoral antibody to reach them. Oblig-ate intracellular parasites such as viruses have to repli-cate inside cells. Facultative intracellular parasitessuch as Mycobacteria and Leishmania can replicate with-in cells, particularly macrophages, but do not have to;they like the intracellular life because of the protectionit affords. A totally separate acquired immunity sys-tem has evolved to deal with intracellular organisms. Itis based on a distinct lymphocyte subpopulationcalled T-cells (figure 2.4a), designated thus because,unlike the B-lymphocytes, they differentiate withinthe thymus gland. Because they are specialized to operate against cells bearing intracellular organisms,T-cells only recognize antigen derived from these microbes when it is on the surface of a cell. According-ly, the T-cell surface receptors, which are similar (butnot identical) to the antibody molecules used by B-lymphocytes, recognize antigen plus a surface markerthat informs the T-lymphocyte that it is making contactwith another cell. These cell markers belong to an im-portant group of molecules known as the major histo-compatibility complex (MHC), identified originallythrough their ability to evoke powerful transplanta-tion reactions in other members of the same species.

Cytokine-producing T-cells help macrophages tokill intracellular parasites

Intracellular organisms only survive insidemacrophages through their ability to subvert the innate killing mechanisms of these cells. Nonetheless,they cannot prevent the macrophage from processingsmall antigenic fragments (possibly of organisms thathave spontaneously died) and moving them to theircell surface. A subpopulation of T-lymphocytes calledT-helper cells, if primed to that antigen, will recognizeand bind to the combination of antigen with so-calledclass II MHC molecules on the macrophage surfaceand produce a variety of soluble factors termed cytokines, which include the interleukins (inter-leukin-2, etc; see p. 84). Different cytokines can bemade by various cell types and generally act at a shortrange on neighboring cells. Some T-cell cytokines help B-cells to make antibodies while others such as


0 10 20 30 60 70 80 90Days

Primaryresponse

Seru

m a

ntib

ody

conc

entra

tion

NATURALINFECTION

SecondaryresponseVACCINATION

Generationof memory

Specificacquiredimmunity

TOXOID TOXIN

Figure 2.7 The basis of vaccination is illustrated by the responseto tetanus toxoid. The toxoid is formed by treatment of the bacterialtoxin with formaldehyde which destroys its toxicity (associatedwith ) but retains antigenicity. The antibody response on the sec-ond contact with antigen is more rapid and more intense. Thus, ex-posure to toxin in a subsequent natural infection boosts the memorycells, rapidly producing high levels of neutralizing protective antibody.

interferon g (IFNg) act as macrophage activating fac-tors, which switch on the previously subverted micro-bicidal mechanisms of the macrophage and bringabout the death of the intracellular microorganisms(figure 2.8).

Virally infected cells can be killed by antibody-dependent cellular cytotoxicity (ADCC) and by cytotoxic T-cells

We have already discussed the advantage to the host ofkilling virally infected cells before the virus begins toreplicate and have seen that natural killer (NK) cellscan subserve a cytotoxic function. However, NK cellshave a limited range of specificities using their lectin-like and other activating receptors and in order to improve their efficacy, this range needs to be expanded.

One way in which this can be achieved is by coatingthe target cell with antibodies specific for the virallycoded surface antigens because NK cells have Fcg re-ceptors for the constant part of the antibody molecule,rather like phagocytic cells. Thus antibodies will bringthe NK cell very close to the target by forming a bridge,and the NK cell being activated by the complexed anti-body molecules is able to kill the virally infected cell byits extracellular mechanisms (figure 2.9). This systemis termed antibody-dependent cellular cytotoxicity(ADCC).

Virally infected cells can also be controlled by a sub-set of T-lymphocytes called cytotoxic T-cells. These,like the T-helpers, have a very wide range of antigenspecificities because they clonally express a large num-ber of different T-cell receptors. Again, each lympho-cyte is programmed to make only one receptor and,

again like the T-helper cell, recognizes antigen only inassociation with a cell marker, in this case the class IMHC molecule (figure 2.9). Through this recognitionof surface antigen, the cytotoxic cell comes into inti-mate contact with its target and administers the “kissof apoptotic death.”


Infection byintracellular facultative organisms Death of intracellular microbes

1 2 3

MHC II

IFNg

Macrophage activation

Th Th

Figure 2.8 Intracellular killing of microorganisms by macrophages. (1) Surface antigen ( ) derived from the intra-cellular microbes is complexed with class IImajor histocompatibility complex (MHC)molecules ( ). (2) The T-helper binds tothis surface complex and is triggered to re-lease the cytokine interferon g (IFNg). Thisactivates microbicidal mechanisms in themacrophage. (3) The infectious agent meetsa timely death.

VIRUSINFECTEDCELL

NK

Tc

Specific T-cell cytotoxicity

Nonspecific killing

NK

ADCC

ANTIBODY

MHC I

Figure 2.9 Killing virally infected cells. The nonspecific killingmechanism of the natural killer (NK) cell can be focused on the targetby antibody to produce antibody-dependent cellular cytotoxicity(ADCC). The cytotoxic T-cell homes onto its target specificallythrough receptor recognition of surface antigen in association withclass I major histocompatibility complex (MHC) molecules.

In an entirely analogous fashion to the B-cell, T-cellsare selected and activated by combination with anti-gen. They are then expanded by clonal proliferationand mature to give T-helper and T-cytotoxic cells, together with an enlarged population of memory cells. Thus both T- and B-cells provide specific ac-quired immunity which extends the range of effec-tiveness of innate immunity and confers the valuableadvantage of memory, i.e. a first infection prepares us to withstand further contact with the same microorganism.

IMMUNOPATHOLOGY

The immune system is clearly “a good thing,” butunder certain circumstances it can cause damage to thehost. Thus, where there is an especially heightened response or persistent exposure to exogenous

antigens, tissue-damaging or hypersensitivity reac-tions may result. Examples are an allergy to grass pol-lens, blood dyscrasias associated with certain drugs,immune complex glomerulonephritis occurring afterstreptococcal infection, and chronic granulomas pro-duced during tuberculosis or schistosomiasis.

In other cases, hypersensitivity to autoantigens mayarise through a breakdown in the mechanisms whichcontrol self-tolerance resulting in a wide variety of autoimmune diseases such as Graves’ disease, myasthenia gravis and many of the rheumatologic disorders.

Another immunopathologic reaction of some conse-quence is transplant rejection, where the MHC anti-gens on the donor graft may well provoke a fiercereaction. Lastly, one should consider the by no meansinfrequent occurrence of inadequate functioning of theimmune system — immunodeficiency.


REVISION

Antibody—the specific adapter• The antibody molecule evolved as a specific adapter toattach to microorganisms which either fail to activate thealternative complement pathway or prevent activation ofthe phagocytic cells.• The antibody fixes to the antigen by its specific recogni-tion site and its constant structure regions activate comple-ment through the classical pathway (binding C1 andgenerating a C4b2a

——; convertase to split C3) and phago-

cytes through their antibody receptors.

Cellular basis of antibody production• Antibodies are made by plasma cells derived from B-lymphocytes; each of which is programmed to make onlyone antibody specificity, which is placed on the cell surfaceas a receptor.• Antigen binds to the cell with a complementary anti-body, causing cell activation, clonal proliferation and fi-nally maturation to antibody-forming cells and memorycells. Thus, the antigen brings about clonal selection of thecells making antibody to itself.

Acquired memory and vaccination• The increase in memory cells after priming means thatthe acquired secondary response is faster and greater, pro-viding the basis for vaccination using a harmless form ofthe infective agent for the initial immunization.

Acquired immunity has antigen specificity• Antibodies differentiate between antigens becauserecognition is based on molecular shape complementarity.Thus, memory induced by one antigen will not extend toanother unrelated antigen.• The immune system differentiates self componentsfrom foreign antigens by making immature self-reactinglymphocytes unresponsive through contact with hostmolecules; lymphocytes reacting with foreign antigens areunaffected since they only make contact after reaching maturity.

Cell-mediated immunity protects against intracellular organisms• Another class of lymphocyte, the T-cell, is concernedwith control of intracellular infections. Like the B-cell, eachT-cell has its individual antigen receptor (although it dif-fers structurally from antibody) which recognizes antigenand the cells undergo clonal expansion to form effectorand memory cells providing specific acquired immunity.• The T-cell recognizes cell surface antigens in associationwith molecules of the major histocompatibility complex(MHC).• T-helper cells that see antigen with class II MHC on thesurface of macrophages release cytokines, which in somecases can help B-cells to make antibody and in other casesactivate macrophages and enable them to kill intracellularparasites.


• Cytotoxic T-cells have the ability to recognize specificantigen plus class I MHC on the surface of virally infectedcells, which are killed before the virus replicates. They alsorelease interferon g, which can make surrounding cells resistant to viral spread (figure 2.10).• Natural killer (NK) cells have lectin-like “non-specific”receptors for cells infected by viruses but do not have anti-gen-specific receptors; however, they can recognize anti-body-coated virally infected cells through their Fcgreceptors and kill the target by antibody-dependent cellular cytotoxicity (ADCC).• Although the innate mechanisms do not improve withrepeated exposure to infection as do the acquired, theyplay a vital role since they are intimately linked to the acquired systems by two different pathways which all butencapsulate the whole of immunology. Antibody, com-plement and polymorphs give protection against most extracellular organisms, while T-cells, soluble cytokines,

macrophages and NK cells deal with intracellular infections (figure 2.11).

Immunopathology• Immunopathologically mediated tissue damage to thehost can occur as a result of:

(a) inappropriate hypersensitivity reactions to exoge-nous antigens;

(b) loss of tolerance to self giving autoimmune disease;(c) reaction to foreign grafts.

• Immunodeficiency leaves the individual susceptible toinfection.

See the accompanying website (www.roitt.com) for multiplechoice questions

Tc

INTRACELLULARPARASITES

Th

NK

VIRUS-INFECTEDCELL

UNINFECTEDCELL

I

Macrophageactivation andchemotaxis

Intracellularkilling

CYTOKINES INTERFERON

Extracellularkilling Viral

resistance

GIVES RISE TO ACTIVATES INHIBITS

II

Mf HelpFigure 2.10 T-cells link with the innateimmune system to resist intracellularinfection. Class I ( ) and class II ( )major histocompatibility complex(MHC) molecules are important for T-cell recognition of surface antigen. TheT-helper (Th) cells cooperate in the development of cytotoxic T-cells (Tc)from precursors. The macrophage (Mf)microbicidal mechanisms are switchedon by macrophage-activating cytokines.Interferon inhibits viral replication and stimulates natural killer (NK) cellswhich together with Tc kill virus-infected cells.

INNATE

ACQUIRED

Humoral immunity

EXTRACELLULAR INFECTIONS

COMPLEMENTPOLYMORPH

ANTIBODY B-CELL

Cell-mediated immunity

INTRACELLULAR INFECTIONS

MACROPHAGECYTOKINES

NK-CELL

T-CELL

Figure 2.11 The two pathways linkinginnate and acquired immunity whichprovide the basis for humoral and cell-mediated immunity, respectively.

FURTHER READING

Alt, F. & Marrack, P. (eds) Current Opinion in Immunology. CurrentScience, London. (Six issues published a year.)

Mackay, I. & Rosen, F.S. (eds). Series of articles entitled Advances inimmunology. New England Journal of Medicine (2000) 343, 37–49,108–17, 702–9, 1020–34, 1313–24, 1703–14; (2001) 344, 30–7, 350–62,655–64; 345, 340–50.

Roitt, I.M. & Delves, P.J. (eds) (1998) Encyclopedia of Immunology, 2nd edn. Academic Press, London. (Covers virtually all aspects of the subject and describes immune responses to most infections.)

Schwaeble, W.J. & Reid, K.B.M. (1999) Does properdin crosslink thecellular and the humoral immune response? Immunology Today,20, 17–21.

The Immunologist. Hogrefe & Huber, Seattle. (Official organ of the International Union of Immunological Society — IUIS. Excellent,didactic and compact articles on current trends in immunology.)

Trends in Immunology. Elsevier Science, Amsterdam. (The immunol-ogist’s “newspaper”: Excellent.)

Website www.roitt.com (linked to Roitt’s Essential Immunology andthis book). Animated immunology tutorials, over 400 multiple-choice questions with teaching comments on every answer, fur-ther reading, downloadable figures.


27

THE BASIC STRUCTURE IS A FOUR-PEPTIDE UNIT

The antibody molecule is made up of two identicalheavy and two identical light chains held together byinterchain disulfide bonds. These chains can be sepa-rated by reduction of the S–S bonds and acidification.In the most abundant type of antibody, immunoglo-bulin G (IgG), the exposed hinge region is extended instructure due to the high proline content and is there-fore vulnerable to proteolytic attack. Therefore themolecule is split by papain to yield two identical Fabfragments, each with a single combining site for anti-gen, and a third fragment, Fc, which lacks the ability tobind antigen. Pepsin strikes at a different point andcleaves the Fc from the remainder of the molecule toleave a large fragment, which is formulated as F(ab¢)2since it is still divalent with respect to antigen bindingjust like the parent antibody (figure 3.1).

AMINO ACID SEQUENCES REVEALVARIATIONS IN IMMUNOGLOBULIN (Ig)STRUCTURE

For good reasons, the antibody population in anygiven individual is incredibly heterogeneous, withmaybe 109 or more different Ig molecules in normal

serum. This meant that determination of amino acidsequences was utterly useless until it proved possibleto obtain the homogeneous product of a single clone.The opportunity to do this first came from the study ofmyeloma proteins.

In the human disease known as multiple myeloma,one cell making one particular individual Ig dividesover and over again in the uncontrolled way a cancercell does, without regard for the overall requirement ofthe host. The patient then possesses enormous num-bers of identical cells derived as a clone from the origi-nal cell and they all synthesize the same Ig — themyeloma protein, or M-protein , which appears in theserum, sometimes in very high concentrations. By pu-rification of the myeloma protein we can obtain apreparation of an Ig with a unique structure. Mono-clonal antibodies can also be obtained by fusing indi-vidual antibody-forming cells with a B-cell tumor toproduce a constantly dividing clone of cells dedicatedto making the one antibody.

The sequencing of a number of such proteins has revealed that the N-terminal portions of both heavy andlight chains show considerable variability, whereas theremaining parts of the chains are relatively constant,being grouped into a restricted number of structures. Itis conventional to speak of variable and constant regions of both heavy and light chains (figure 3.2).

CHAPTER 3

Antibodies

The basic structure is a four-peptide unit, 27Amino acid sequences reveal variations in

immunoglobulin (Ig) structure, 27Immunoglobulin genes, 28

Immunoglobulins are encoded by multiple gene segments, 28

A special mechanism effects VDJ recombination, 29Structural variants of the basic lg molecule, 29

Isotypes, 29Allotypes, 30Idiotypes, 30

Immunoglobulins are folded into globular domainswhich subserve dif ferent functions, 30Immunoglobulin domains have a characteristic

structure, 30

The variable domain binds antigen, 30Constant region domains determine secondary biological

function, 31Immunoglobulin classes and subclasses, 31

Immunoglobulin G has major but varied roles in extracellulardefenses, 33

Immunoglobulin A guards the mucosal surfaces, 34Immunoglobulin M provides a defense against bacteremia, 35Immunoglobulin D is a cell surface receptor, 35Immunoglobulin E triggers inflammatory reactions, 35Immunoglobulins are further subdivided into subclasses, 36

Making antibodies to order, 37The monoclonal antibody revolution, 37Engineering antibodies, 37

Certain sequences in the variable regions show quiteremarkable diversity, and systematic analysis localizesthese hypervariable sequences to three segments onthe light chain and three on the heavy chain (figure 3.3).

IMMUNOGLOBULIN GENES

Immunoglobulins are encoded by multiple gene segments

Clusters of genes on three different chromosomes codefor k light chains, l light chains and heavy chains re-spectively. Since a wide range of antibodies with dif-fering amino acid sequences can be produced, theremust be corresponding nucleotide sequences to en-code them. However, the complete gene encodingeach heavy and light chain is not present as such in thegerm line DNA, but is created during early develop-ment of the B-cell by the joining together of mini seg-ments of the gene. Take the human k light chain, forexample; the variable region is encoded by two genesegments, a large Vk and a small Jk , while a single geneencodes the constant region (figure 3.4). There is a clus-

ter of some 40 or more variable (V) genes and just fivejoining (J) genes. In the immature B-cell, a transloca-tion event leads to the joining of one of the Vk genes toone of the Jk segments. Each V segment has its ownleader sequence and a number of upstream promotersites including a characteristic octamer sequence, towhich transcription factors bind. When the im-munoglobulin gene is transcribed, splicing of the nu-clear RNA brings the Vk Jk sequence into contiguitywith the constant region Ck sequence, the whole being read off as a continuous k chain peptide withinthe endoplasmic reticulum.

The same general principles apply to the arrange-ment of l light chains with about 30 V gene segmentsand four J segments, and the heavy chain genes. The latter constellation shows additional features: thedifferent constant region genes form a single clusterand there is a group of around 25 highly variable D seg-ments located between the 50 or so V genes and six Jgenes (figure 3.5). The D segment, together with itsjunctions to the V and J segments, encodes almost theentire third hypervariable region, the first two beingencoded entirely within the V sequence.

PART 2—The recognition of antigen28

SS

SS

SS

IMMUNOGLOBULIN

SS

SS

SS

IMMUNOGLOBULIN

SS

SS

SS

IMMUNOGLOBULIN

Antigencombining

site

PAPAIN PEPSINREDUCE AND ACIDIFY

SS

SS

SS

Fab

Fab

Fc

SS

SS

SS pFc’

F(ab’)2

PEPSIN FRAGMENTS PAPAIN FRAGMENTSISOLATED CHAINS

SH

SH

SH

SH

SHSH

a b c

Light chain

Heavy chain

Figure 3.1 The antibody basic unit consisting of two identicalheavy and two identical light chains held together by interchaindisulfide bonds, can be broken down into its constituent peptidechains and to proteolytic fragments, the pepsin F(ab¢)2 retaining twobinding sites for antigen and the papain Fab with one. After papain

digestion the Fc fragment representing the C-terminal half of the Igmolecule is formed and is held together by disulphide and noncova-lent bonds. The portion of the heavy chain in the Fab fragment isgiven the symbol Fd. The N-terminal residue is on the left for eachchain.

A special mechanism effects VDJ recombination

In essence, the translocation involves the mutualrecognition of conserved heptamer–spacer–nonamerrecombination signal sequences (RSS) which flankeach germ line V, D and J segment. The products of therecombination activating genes RAG-1 and RAG-2catalyse the introduction of double-strand breaks be-tween the elements to be joined and their respectiveflanking sequences. At this stage, nucleotides may either be deleted or inserted between the VD, DJ or VJjoining elements before they are ultimately ligated.

STRUCTURAL VARIANTS OF THE BASIC Ig MOLECULE

Isotypes

Based upon the structure of their heavy chain constantregions, Igs are divided into major groups termedclasses, which may be further subdivided into sub-classes. In the human, there are five classes: IgG, IgA,IgM, IgD and IgE. Since all the heavy chain constant re-

gion (CH) structures that give rise to classes and sub-classes are expressed together in the serum of a normalsubject, they are termed isotypic variants (table 3.1).Likewise, the light chain constant regions (CL) exist inisotypic forms known as k and l which are associatedwith all heavy chain isotypes. Because the light chainsin a given antibody are identical, Igs are either k or lbut never mixed (unless specially engineered in the

CHAPTER 3—Antibodies 29

CH

SS

SS

S

S

CL

VL

VH

Hypervariableregions

Constant

Variable

Figure 3.2 Amino acid sequence variability in the antibody mole-cule. The terms “V region” and “C region” are used to designate thevariable and constant regions respectively; “VL” and “CL” are gener-ic terms for these regions on the light chain and “VH” and “CH” spec-ify variable and constant regions on the heavy chain. Certainsegments of the variable region are hypervariable but adjacentframework regions are more conserved. As stressed previously, eachpair of heavy chains is identical, as is each pair of light chains.

HEAVY CHAINS

LIGHT CHAINS

Position

Position

0 20 40 60 80 100 120

0 25 50 75 100

50

100

150

50

100

150

Varia

bilit

yVa

riabi

lity

a

b

CDR1 CDR2 CDR3

CDR1 CDR2 CDR3 Fr4Fr3Fr2Fr1

Fr4Fr3Fr2Fr1

Figure 3.3 Wu and Kabat plot of amino acid variability in the vari-able region of immunoglobulin (Ig) heavy and light chains. The se-quences of chains from a large number of myeloma monoclonalproteins are compared and variability at each position is computedas the number of different amino acids found divided by the fre-quency of the most common amino acid. Obviously, the higher thenumber the greater the variability; for a residue at which all 20 aminoacids occur randomly, the number will be 400 (20/0.05) and at a com-pletely invariant residue, the figure will be 1 (1/1). The three hypervariable regions (darker blue) in the (a) heavy and (b) lightchains, usually referred to as complementarity-determining regions(CDRs), are clearly defined. The intervening peptide sequences(gray) are termed framework regions (Fr1–4). (Courtesy of ProfessorE.A. Kabat.)

laboratory). Thus IgG exists as IgGk or IgGl, IgM asIgMk or IgMl, and so on.

Allotypes

This type of variation depends upon the existence of al-lelic forms (encoded by alleles or alternative genes at asingle locus) which therefore provide genetic markers(table 3.1). In somewhat the same way as the red cells ingenetically different individuals can differ in terms ofthe blood group antigen system ABO, so the Ig heavychains differ in the expression of their allotypic groups.Typical allotypes are the Gm specificities on IgG (Gm= marker on IgG). Allotypic differences at a given Gmlocus usually involve one or two amino acids in the

peptide chain. Take, for example, the G1m(a) locus onIgG1. An individual with this allotype would have thepeptide sequence Asp . Glu . Leu . Thr . Lys on each of their IgG1 molecules. Another person whose IgG1 was a-negative would have the sequenceGlu .Glu .Met .Thr .Lys, i.e. two amino acids different.To date, 25 Gm groups have been found on the g-heavy chains and a further three on the k constant region.

Idiotypes

It is possible to obtain antibodies that recognize the isotypic and allotypic variants described above. Similarly one can also raise antisera that are specific forindividual antibody molecules and discriminate between one monoclonal antibody and another independently of isotypic or allotypic structures. Suchantisera define the individual determinants character-istic of each antibody, collectively termed the idiotype.Not surprisingly, it turns out that the idiotypic deter-minants are located in the variable part of the antibodyassociated with the hypervariable regions. The readerwill (or should) be startled to learn that it is possible toraise autoanti-idiotypic sera since this means that indi-viduals can make antibodies to their own idiotypes.Some T-cell receptors are also capable of recognizingidiotypes.

IMMUNOGLOBULINS ARE FOLDED INTOGLOBULAR DOMAINS WHICH SUBSERVEDIFFERENT FUNCTIONS

Immunoglobulin domains have a characteristic structure

In addition to the interchain disulfide bonds, whichbridge heavy and light chains, there are internal, intra-chain disulfide links, which form loops in the peptidechain. These loops are compactly folded to form globu-lar domains which have a characteristic b-pleatedsheet protein structure.

Significantly, the hypervariable sequences appear atone end of the variable domain where they form partsof the b-turn loops and are clustered close to each otherin space (figure 3.6).

The variable domain binds antigen

The clustering of the hypervariable loops at the tips ofthe variable regions where the antigen binding site islocalized makes them the obvious candidates to sub-serve the function of antigen recognition (figures 3.6 &


VARIABLE REGIONCONST ANTREGION

GERM-LINEDNA

B-CELL

B-CELL DNA

CYTOPLASMIC mRNA

k LIGHT CHAIN PEPTIDE

Randomtranslocation

Vk J

EMBRYO - HUMAN k GENE CLUSTER

L Vk J Ck

Transcription and splicing of RNA

L Vk J Ck

Protein synthesis

V J Ckk

J1 2 3 4 5

Vk1 40

Ck

Figure 3.4 Genetic basis for synthesis of human k chains. The Vkgenes are arranged in a series of families or sets of a closely related se-quence. Each Vk gene has its own leader sequence (L). As the cell be-comes immunocompetent, the variable region is formed by therandom combination of a Vk gene with a joining segment J, a translo-cation process facilitated by base sequences in the intron followingthe 3¢ end of the Vk segment pairing up with sequences in the intron5¢ to J. The final joining occurs when the intervening intron sequenceis spliced out of the RNA transcript. By convention, the genes arerepresented in italics and the antigens they encode in normal type.

3.7). X-ray crystallographic analysis of complexesformed between the Fab fragments of monoclonal an-tibodies and their respective antigens has confirmedthis. The sequence heterogeneity of the three heavyand three light chain hypervariable loops ensurestremendous diversity in combining specificity for anti-gen through variation in the shape and nature of thesurface they create. Thus, each hypervariable regionmay be looked upon as an independent structure contributing to the complementarity of the bindingsite for antigen, and one speaks of complementarity-determining regions (CDRs).

Constant region domains determine secondarybiological function

The classes of antibody differ from each other in manyrespects: in their half-life, their distribution through-out the body, their ability to fix complement and their

binding to cell surface Fc receptors. Since the classes allhave the same k and l light chains, and heavy and lightvariable region domains, these differences must lie inthe heavy chain constant regions.

A model of the IgG molecule is presented in figure3.8. It shows the spatial disposition and interaction ofthe domains in IgG and ascribes the various biologicalfunctions to the relevant structures. In principle, the V-region domains form the recognition unit and the C-region domains mediate the secondary biologicalfunctions.

IMMUNOGLOBULIN CLASSES ANDSUBCLASSES

The physical and biological characteristics of the fivemajor Ig classes in the human are summarized in tables3.2 and 3.3. The following comments are intended tosupplement this information.


VH/V L

ISOTYPE ISOTYPEIDIOTYPE

Hypervariable(Ag combining site)

ALLOTYPE

TYPE OF VARIA TION

ISOTYPIC

ALLOTYPIC

IDIOTYPIC

All variants presentin serum of a normalindividual

Alternative forms:genetically controlledso not present in allindividuals

Individually specificto each immuno-globulin molecule

ClassesSubclassesTypesSubgroupsSubgroups

Allotypes

Idiotypes

CHCHCLCLVH

Mainly CH/CLsometimesVH/ VL

Variableregions

IgM, IgEIgA1, IgA2k,llOz

+, lOz–

VkI VkII VkIIIVHI VHII VHIII

Gm groups (human)b4, b5, b6, b9 (rabbit light chains)Igh-1a, Igh-1b (mouse g

2a heavy chains)

Probably one or more hypervariableregions forming the antigen-combining site

DISTRIBUTION VARIANT LOCA TION EXAMPLES

/VL

CONSTANT

Table 3.1 Summary of immunoglobulin (Ig) variants.

J2

HUMAN H CHAIN GENES

VARIABLE REGION CONSTANT REGION

50VH

1 25D

1 6J

1

D3V33g2

GERM-LINEGENES

B-CELLDNA

e.g.Example of gene encoding anIgG2 heavy chain withspecificity defined by thevariable region sequenceV33 D3 J2

Translocation

m d g3 g1 a1 g2 g4 e a2

Figure 3.5 Human V-region genes shuf-fled by translocation to generate the singleheavy chain specificity characteristic ofeach B-cell. Note the additional D (diversi-ty)-segment minigenes.


F

VARIABLE DOMAIN (VL) CONSTANT DOMAIN (CL)

C-TERMINUSAB

CE

D

G

F

A

G

N-TERMINUSCDR3 96

26

53

CDR1

CDR2

C

E

DB

Figure 3.6 Immunoglobulin (Ig) domain structure. Structure of theglobular domains of a light chain (from X-ray crystallographic stud-ies of a Bence-Jones protein by Schiffer, M., Girling, R.L., Ely, R.R. et al. (1973) Biochemistry 12, 4620. Copyright American Chemical So-ciety.). One surface of each domain is composed essentially of fourchains (light blue arrows) arranged in an antiparallel b-pleatedstructure stabilized by interchain H bonds between the amide CO·and NH· groups running along the peptide backbone, and the other

surface of three such strands (darker blue arrows); the black bar rep-resents the intrachain disulfide bond. This overall structure is char-acteristic of all Ig domains. Of particular interest is the location of thehypervariable regions ( ) in three separate loops which areclosely disposed relative to each other and form the light chain con-tribution to the antigen binding site. One numbered residue fromeach complementarity determinant is identified.

Figure 3.7 The binding site. A simulated combining site for the re-active surface of a single antigenic epitope (such as a hapten, cf. fig-ure 5.1) formed by apposing the three middle fingers of each hand,each finger representing a hypervariable loop. With protein epitopesthe area of contact is usually greater and tends to involve more su-perficial residues (cf. figure 5.3). (Photograph by B.N.A. Rice; in-spired by A. Munro!)

CL

VL

C�1VH

C�2

C1q fixation

Control catabolic rateBind to Fc receptors

C�3

Antigen binding C4b binding

Figure 3.8 Location of biological function. The combined Cg2 andCg3 domains bind to Fc receptors on phagocytic cells, natural killer(NK) cells and placental syncytiotrophoblast, and also to staphylo-coccal protein A. (Note the immunoglobulin G (IgG) heavy chain isdesignated g and the constant region domains Cg1, Cg2 and Cg3.)

Immunoglobulin G has major but varied roles inextracellular defenses

Its relative abundance, its ability to develop high-affin-ity binding for antigen and its wide spectrum of sec-ondary biological properties make IgG appear as theprime workhorse of the Ig stable. During the sec-ondary response IgG is probably the major Ig to be syn-thesized. Immunoglobulin G diffuses more readily

than the other Igs into the extravascular body spaceswhere, as the predominant species, it carries the majorburden of neutralizing bacterial toxins and of bindingto microorganisms to enhance their phagocytosis.

Activation of the classical complement pathway

Complexes of bacteria or other antigens with IgG anti-body trigger the C1 complex when a minimum of two


Molecular formula†

7S,9S,11S*

1,2*

(a2k2)1-2(a2l2)1-2(a2k2)2S*(a2l2)2S*

17-450 ng/ml‡

DESIGNATION

SedimentationcoefficientMolecular weight

Number of basicfour-peptide unitsHeavy chains

Light chains

Valency forantigen bindingConcentration rangein normal serum% Totalimmunoglobin% Carbohydratecontent

IgG IgA IgM IgD IgE

7S

150 000

1

gk or l

g 2k2,g 2l2

2

8-16 mg/ml

75

3

160 000 anddimer

�

k or l

2,4

1.4-4 mg/ml

15

8

19S

970 000

5

mk or l(m2k2)5(m2l2)5

5(10)

0.5-2 mg/ml

5-10

12

7S

175 000

1

dk or ld2k2d2l2?

2

0.003-0.04 mg/ml

0-1

9

8S

190 000

1

ek or l

e2k2,e2l2

2

0.002

12

IgG IgA IgM IgD IgE

Majorcharacteristics

Most abundantIg of internalbody fluidsparticularlyextravascularwhere itcombats micro-organisms andtheir toxins

Major Ig inseromucoussecretionswhere itdefendsexternal bodysurfaces

Very effectiveagglutinator;produced earlyin immuneresponse –effective first-line defenceagainstbacteremia

Mostlypresent onlymphocytesurface

Protection ofexternal bodysurfaces.Recruits anti-microbialagents.Raised inparasiticinfections.Responsible forsymptoms ofatopic allergy

Complementfixation Classical Alternative

Cross placenta

Sensitizesmast cellsand basophils

Binding tomacrophagesand polymorphs

++

++–

–

+++

–

–

–

+

–

+++–

–

–

–

––

–

–

–

+++

––

+

+

Table 3.2 Physical properties of majorhuman immunoglobulin (Ig) classes.

Table 3.3 Biological properties of majorimmunoglobulin (Ig) classes in thehuman.

*7S monomer, 9S dimer, and 11S dimer in external secretions which carries the secretory component (S).†IgAdimer and IgM contain J chain.‡ng = 10-9 g.

Fcg regions in the complex bind C1q. Activation of thenext component, C4, produces attachment of C4b tothe Cg1 domain. Thereafter, one observes C3 conver-tase formation, covalent coupling of C3b to the bacte-ria and release of C3a and C5a leading to thechemotactic attraction of our friendly polymorphonu-clear phagocytic cells. These adhere to the bacteriathrough surface receptors for complement and the Fcportion of IgG (Fcg) and then ingest the microorgan-isms through phagocytosis. In a similar way, the extra-cellular killing of target cells coated with IgG antibodyis mediated largely through recognition of the surfaceFcg by natural killer (NK) cells bearing the appropriatereceptors. The thesis that the biological individualityof different Ig classes is dependent on the heavychain constant regions, particularly the Fc, is amplyborne out in the activities of IgG. Functions such as itsability to cross the placenta, transport across the intes-tine in the newborn, complement fixation and bindingto various cell types have been shown to be mediatedby the Fc part of the molecule.

Opsonization by IgG

All phagocytic cells have the ability to engulf microor-ganisms, a process which can be considerably en-hanced by coating the invaders with IgG antibody. TheIgG binds to specific epitopes on the microbe via its Fabfragment and to Fcg receptors on the phagocytic cellsby its Fc portion, allowing the phagocytic cell to attachitself firmly to the microbe and provoking muchgreater phagocytic activity.

Transplacental passage of IgG

Alone of the Ig classes, IgG possesses the crucially im-portant ability to cross the human placenta so that itcan provide a major line of defense for the first fewweeks of a baby’s life. This may be further reinforcedby the transfer of colostral IgG across the gut mucosa inthe neonate. These transport processes involvetranslocation of IgG across the cell barrier by complex-ing to an Fcg receptor called FcRn (see below).

The diversity of Fcg receptors

Since a wide variety of interactions between IgG com-plexes and different effector cells have been identified,we really should spend a little time looking at themembrane receptors for Fcg that mediate these phe-nomena.

Specific Fc receptors may exist for all the classes of

antibody, although receptors for IgD and IgM are notparticularly well characterized. For IgG a quite exten-sive family of Fc receptors has emerged, the nineknown genes encoding four major groups of receptorsFcgRI, FcgRII, FcgRIII and FcgRn (or, more simply,FcRn). Within these groups there are a number of splicevariants and inherited polymorphic forms.

FcgRI (CD64) is the major Fcg receptor constitutive-ly present on monocytes, macrophages and dendriticcells, and induced on neutrophils following their acti-vation by the cytokines interferon g (IFNg) and granu-locyte colony-stimulating factor (G-CSF). Conversely,FcgRI can be downregulated in response to inter-leukin-4 (IL-4) and IL-13. Its main roles are in facilitat-ing phagocytosis and antigen presentation and inmediating extracellular killing of target cells coatedwith IgG antibody, a process referred to as antibody-dependent cellular cytotoxicity (ADCC; p. 23).

FcgRII (CD32) is present on the surface of most typesof leukocytes and other cells including endothelialcells. These receptors bind insignificant amounts ofmonomeric IgG but immune complexes bind reallywell and are selectively adsorbed to the cell surface.

The two FcgRIII (CD16) genes encode the isoformsFcgRIIIA and FcgRIIIB found on most types of leuko-cytes. With respect to their functions, FcgRIIIAis large-ly responsible for mediating ADCC by NK cells andthe clearance of immune complexes from the circula-tion by macrophages. FcgRIIIB cross-linking stimu-lates the production of superoxide by neutrophils.

FcRn is the fourth major type of FcgR. It is not pre-sent on leukocytes but instead is found on epithelialcells. It is referred to as FcRn due to its original descrip-tion as a neonatal receptor, although it is also present inadults. It is involved in transferring maternal circulat-ing IgG across the placenta to the fetus (figure 3.9).

Immunoglobulin A guards the mucosal surfaces

Immunoglobulin A appears selectively in the seromu-cous secretions such as saliva, tears, nasal fluids,sweat, colostrum, and secretions of the lung, genitouri-nary and gastrointestinal tracts, where it has the job of defending the exposed external surfaces of the body against attack by microorganisms. Im-munoglobulin A is synthesized locally by plasma cellsand is dimerized intracellularly together with a cys-teine-rich polypeptide called the J chain. The dimericIgA binds strongly through its J chain to a receptor forpolymeric Ig present in the membrane of mucosal epithelial cells. The complex is then actively endocy-tosed, transported across the cytoplasm and secreted


into the external body fluids after cleavage of the poly-Ig receptor peptide chain. The fragment of the receptorremaining bound to the IgA is termed secretory com-ponent, and the whole molecule is called secretory IgA(figure 3.10).

The function of the secretory component may be toprotect the IgA hinge from bacterial proteases. It mayalso serve to anchor IgAto the soluble mucus acting asmolecular Teflon to inhibit the adherence of coated mi-croorganisms to the surface of mucosal cells, therebypreventing entry into the body tissues. The secretoryIgAwill also combine with the numerous soluble anti-gens of dietary and microbial origin to block their ac-cess to the body. Aggregated IgA binds to neutrophilsand can also activate the alternative complement path-way. Plasma IgA is predominantly monomeric and,since this form is a relatively poor activator of comple-ment, it seems likely that the body uses it for the directneutralization of any antigens which breach the epithelial barrier to enter the circulation. However, ithas additional functions which are mediated throughthe FcaR (CD89) for IgA present on monocytes,macrophages, neutrophils, activated eosinophils andon subpopulations of both T- and B-lymphocytes. Fol-lowing cross-linking, the receptor can activate endocy-

tosis, phagocytosis, inflammatory mediator releaseand ADCC. Expression of the FcaR on monocytes isstrongly upregulated by bacterial lipopolysaccharide.

Immunoglobulin M provides a defense against bacteremia

Often referred to as the macroglobulin antibodies be-cause of their high molecular weight, IgM moleculesare polymers of five four-peptide subunits each bear-ing an extra CH domain. As with IgA, a single J chain isincorporated into the pentamer. Immunoglobulin Mantibodies tend to be of relatively low affinity as mea-sured against single determinants (haptens) but, be-cause of their high valency, they bind with quiterespectable avidity to antigens with multiple repeat-ing epitopes. For the same reason, these antibodies areextremely efficient agglutinating and cytolytic agentsand, since they appear early in the response to infec-tion and are largely confined to the bloodstream, it islikely that they play a role of particular importance incases of bacteremia. The isohemagglutinins (anti-A,anti-B) and many of the “natural” antibodies to microorganisms are usually IgM.

Monomeric IgM (i.e. a single four-peptide unit) anchored in the cell membrane is the major antibodyreceptor used by B-lymphocytes to recognize antigen.

Immunoglobulin D is a cell surface receptor

This class was recognized through the discovery of amyeloma protein which did not have the antigenicspecificity of IgG, IgA or IgM, although it reacted withantibodies to Ig light chains and had the basic four-peptide structure. The hinge region is particularly ex-tended and, although protected to some degree bycarbohydrate, it may be this feature that makes IgD,among the different Ig classes, uniquely susceptible toproteolytic degradation and accounts for its short half-life in plasma (2.8 days). Nearly all the IgD is presenttogether with IgM on the surface of a proportion of B-lymphocytes, where it seems likely that the two Igs may operate as mutually interacting antigen re-ceptors for the control of lymphocyte activation andsuppression.

Immunoglobulin E triggers inflammatory reactions

Only very low concentrations of IgE are present inserum, and only a very small proportion of the plasmacells in the body are synthesizing this Ig. It is not sur-


FcRn

MaternalIgG

Releasedmaternal

IgG

Maternalcirculation

FetalcirculationPLACENTA

FETAL ACQUISITION OF MATERNAL IgG

Figure 3.9 The epithelial cell surface receptor for immunoglobu-lin G (IgG) Fc regions. The FcRn receptor is present in the placentawhere it fulfills the important task of transferring maternal IgG to thefetal circulation. This will provide important protection prior to thegeneration of immunocompetence in the fetus. Furthermore, it isself-evident that any infectious agent which might reach the fetus inutero will have had to have passed through the mother first, and thefetus will rely upon the mother’s immune system to have producedIgG with appropriate binding specificities. This maternal IgG alsoprovides protection for the neonate, because it takes some weeks fol-lowing birth before the transferred IgG is eventually all catabolized.

prising, therefore, that so far only a handful of IgEmyelomas have been recognized compared with tensof thousands of IgG paraproteinemias. Immunoglobu-lin E antibodies remain firmly fixed for an extendedperiod when they are bound with high affinity to theFceRI receptor on mast cells. Contact with antigenleads to degranulation of the mast cells with release ofpreformed vasoactive amines and cytokines, and thesynthesis of a variety of inflammatory mediators de-rived from arachidonic acid. This process is responsi-ble for the symptoms of hay fever and of extrinsicasthma when patients with atopic allergy come intocontact with the allergen, for example grass pollen.

The main physiological role of IgE would appear to beprotection of anatomical sites susceptible to traumaand pathogen entry, by local recruitment of plasma fac-tors and effector cells through triggering an acute in-flammatory reaction. Infectious agents penetratingthe IgA defenses would combine with specific IgE onthe mast cell surface and trigger the release of vasoac-tive agents and factors chemotactic for granulocytes,so leading to an influx of plasma IgG, complement,neutrophils and eosinophils.

Another IgE receptor called the low affinity receptor

FceRII or CD23 is present on many different types ofhematopoietic cells. Its primary function is to regulateIgE synthesis by B-cells, with a stimulatory role at lowconcentrations of IgE and an inhibitory role at highconcentrations.

Immunoglobulins are further subdivided into subclasses

Antigenic analysis of IgG myelomas revealed furthervariation and showed that they could be grouped intofour isotypic subclasses now termed IgG1, IgG2, IgG3and IgG4. The differences all lie in the heavy chains,which have been labeled g1, g2, g3 and g4, respectively.These heavy chains show considerable homology andhave certain structures in common with each other —the ones that react with specific anti-IgG antisera — buteach has one or more additional structures characteris-tic of its own subclass arising from differences in pri-mary amino acid composition and in interchaindisulfide bridging. These give rise to differences in bio-logical behavior, which are summarized in table 3.4.

Two subclasses of IgA have also been found, ofwhich IgA1 constitutes 80–90% of the total.


a bBASAL

SURFACEMUCOSAL(APICAL)SURFACE

GLANDULAR EPITHELIAL CELL

POLY IgRECEPTOR

SECRETEDIgA

CLEAVAGE

ENDOCYTICVACUOLE

SECRETORYPIECE

J CHAIN J

VH VL

CL

Ca1

Ca2

DIMERIgA

Ca3

Figure 3.10 Secretory immunoglobulin A (IgA). (a) The mecha-nism of IgA secretion at the mucosal surface. The mucosal cell synthesizes a receptor for polymeric immunoglobulin which is in-serted into the basal membrane. Dimeric IgA binds to this receptor

and is transported via an endocytic vacuole to the apical surface.Cleavage of the receptor releases secretory IgA still attached to partof the receptor termed the secretory piece. (b) Schematic view of thestructure of secreted IgA.

MAKING ANTIBODIES TO ORDER

The monoclonal antibody revolution

First in rodents

A fantastic technological breakthrough was achievedby Milstein and Köhler who devised a technique forthe production of “immortal” clones of cells makingsingle antibody specificities by fusing normal anti-body-forming cells with an appropriate B-cell tumorline. These so-called “hybridomas” can be grown upeither in the ascitic form in mice, when quite prodi-gious titers of monoclonal antibody can be attained, orpropagated in large-scale culture. A major advance ofthe monoclonal antibody as a reagent is that it providesa single standard material for all laboratories through-out the world and can be used in many different appli-cations. They include the enumeration and separationof individual cell types with specific surface markers(lymphocyte subpopulations, neural cells, etc.), celldepletion, cancer diagnosis, imaging, immunoassay,purification of antigen from complex mixtures,serotyping of microorganisms, immunologic inter-vention with passive antibody, therapeutic neutraliza-tion of inflammatory cytokines and “magic bullet”therapy with cytotoxic agents coupled to antitumor-specific antibody.

Human monoclonals can be made

Mouse monoclonals injected into human subjects fortherapeutic purposes are highly immunogenic andmay lead to immune-complex mediated disease. Itwould be useful to remove the xenogeneic (foreign)portions of the monoclonal antibody and replace thoseportions with human Ig structures using recombinant

DNAtechnology. Chimeric constructs, in which the VHand VL mouse domains are spliced onto human CH andCL genes (figure 3.11a), are far less immunogenic in humans. Such antibodies can now be produced in “hu-manized” mice immunized by conventional antigens.One initiative involved the replacement of the mouseCg1 gene by its human counterpart in embryonic stemcells. The resulting mutant mice were crossed with ani-mals expressing human in place of murine Ck chainsand mice homozygous for both mutants produced humanized IgG1k antibodies

Yet another approach is to graft the six CDRs of ahigh-affinity rodent monoclonal onto a completelyhuman Ig framework without loss of specific reactivity(figure 3.11b). This is not a trivial exercise, however,and the objective of fusing human B-cells to make hy-bridomas is still appealing. A major restriction arisesbecause the peripheral blood B-cells, which are theonly B-cells readily available in the human, are not nor-mally regarded as a good source of antibody-formingcells. Using the various approaches described above,large numbers of human monoclonals have been es-tablished and several are undergoing clinical trials;one can cite an antibody to the TRAIL (tumor necrosisfactor-related apoptosis-inducing ligand) receptor ontumor cells as an anti-cancer agent, the TrabioTM anti-body to transforming growth factor-b aimed at pre-venting scarring following eye surgery, the ABthraxTM

antibody against a Bacillus anthracis protective antigenfor the prevention and treatment of anthrax infection,and highly potent monoclonals for protection againstvaricella zoster and cytomegalovirus.

Engineering antibodies

There are other ways around the problems associatedwith the production of human monoclonals which ex-


Serum concentration (mg/ml)

% Total IgG in normal serum

Serum half-life (days)

Complement activation (classicalpathway)

Binding to monocyte/macrophage Fcreceptors

IgG1 IgG4IgG2 IgG3

9 0.53 1

67 422 7

23 2323 8

+++ ++++

Binding to staphyloccal protein A

Binding to staphyloccal protein G

Gm allotypes

+++

+++ +++

a,z,f,x 4a,4bn b0,b1,b3,g,s,t, etc.

Ability to cross placenta

Spontaneous aggregation +++

+++ ++++++

_+

++ + +++

_+

_+

––

+++ _+ +++

+++ +++

Table 3.4 Comparison of human im-munoglobulin G (IgG) subclasses.

ploit the wiles of modern molecular biology. Referencehas already been made to the “humanizing” of rodentantibodies (figure 3.11), but an important new strategybased upon bacteriophage expression and selectionhas achieved a prominent position. In essence, mes-senger RNA (mRNA) isolated from the B-cells of nonimmunized or preferably immunized donors isconverted to complementary DNA (cDNA) and theantibody genes, or fragments therefrom, expanded bythe polymerase chain reaction (PCR). Single constructsare then made in which the light- and heavy-chaingenes are allowed to combine randomly in tandemwith the bacteriophage pIII gene. This combinatoriallibrary containing most random pairings of heavy-and light-chain genes encodes a huge repertoire of an-

tibodies (or their fragments) expressed as fusion pro-teins with the filamentous coat protein pIII on the bac-teriophage surface. The extremely high number ofphages can now be panned on solid-phase antigen toselect those bearing the highest affinity antibodies at-tached to their surface. Because the genes that encodethese antibodies are already present within the select-ed phage, they can readily be cloned and the antibodyexpressed in bulk. It should be recognized that this selection procedure has an enormous advantage over techniques which employ screening because thenumber of phages that can be examined is several logshigher.

Although a “test-tube” operation, this approach tothe generation of specific antibodies does resemble theaffinity maturation of the immune response in vivo inthe sense that antigen is the determining factor in selecting out the highest affinity responders.

Fields of antibodies

Not only can the products of genes for a monoclonalantibody be obtained in bulk in the milk of lactatingtransgenic animals but plants can also be exploited forthis purpose. So-called “plantibodies” have been ex-pressed in bananas, potatoes and tobacco plants. Onecan imagine a high-tech farmer drawing the attentionof a bemused visitor to one field growing anti-tetanustoxoid, another anti-meningococcal polysaccharide,and so on. Multifunctional plants might be quite prof-itable with, say, the root being harvested as a food cropand the leaves expressing some desirable gene prod-uct. At this rate there may not be much left for sciencefiction authors to write about!


RAT CDRs

a CHIMERIC ANTIBODY

MOUSE

HUMAN

VL

CL

VH

CH

GRAFTED CDRsb

HUMANFRAMEWORK

Figure 3.11 Genetically engineering rodent antibody specificitiesinto the human. (a) Chimeric antibody with mouse variable regionsfused to human immunoglobulin (Ig) constant region. (b) “Human-ized” rat monoclonal in which gene segments coding for all six com-plementarity-determining regions (CDRs) are grafted onto a humanIg framework.

REVISION

The basic immunoglobulin (Ig) structure is a four-peptide unit• Immunoglobulins have a basic four-peptide structureof two identical heavy and two identical light chainsjoined by interchain disulfide links.• Papain splits the molecule at the exposed flexible hingeregion to give two identical univalent antigen-bindingfragments (Fab) and a further fragment (Fc). Pepsin prote-olysis gives a divalent antigen-binding fragment F(ab¢)2lacking the Fc.

Amino acid sequences reveal variations in Ig structure• There are perhaps 108 or more different Ig molecules innormal serum.

• Analysis of myeloma proteins, which are homogeneousIg produced by single clones of malignant plasma cells,has shown the N-terminal region of heavy and light chainsto have a variable amino acid structure and the remainderto be relatively constant in structure.

Immunoglobulin genes• Clusters of genes on three different chromosomes en-code k, l and heavy Ig chains respectively. In each clusterthere is a pool of 30–50 variable region (V) genes andaround five small J minisegments. Heavy chain clusters inaddition contain of the order of 25 D minigenes. There is asingle gene encoding each constant region.


tions, where it is the major Ig concerned in the defense ofthe external body surfaces, it is present as a dimer linked toa secretory component.• Immunoglobulin A acts by inhibiting the adherence ofcoated organisms to mucosal cells.• Phagocytic cells possess an FcaR and can be activatedwhen the receptor is engaged by cross-linked IgA.

The other Igs have various functions in the immune response• Immunoglobulin M is a pentameric molecule, essential-ly intravascular and produced early in the immune response. Because of its high valency it is a very effectivebacterial agglutinator and mediator of complement-dependent cytolysis and is therefore a powerful first-linedefense against bacteremia.• Immunoglobulin D is largely present on the lympho-cyte and functions as an antigen receptor.• Immunoglobulin E binds firmly to mast cells and con-tact with antigen leads to local recruitment of antimicro-bial agents through degranulation of the mast cells andrelease of inflammatory mediators. Immunoglobulin E isof importance in certain parasitic infections and is respon-sible for the symptoms of atopic allergy.• Further diversity of function is possible through thesubdivision of classes into subclasses based on structuraldifferences in heavy chains all present in each normal individual.

Making antibodies to order• Immortal hybridoma cell lines making monoclonal an-tibodies provide powerful immunologic reagents and in-sights into the immune response. Applications includeenumeration of lymphocyte subpopulations, cell deple-tion, immunoassay, cancer diagnosis and imaging, andpurification of antigen.• Mouse monoclonal antibodies are immunogenic in hu-mans. Chimeric constructs can be produced by splicingmouse V genes onto human C genes.• Genetically engineered human antibody fragments canbe derived by expanding the VH and VL genes from unim-munized or preferably immunized donors and expressingthem as completely randomized combinatorial librarieson the surface of bacteriophage.


• A special splicing mechanism involving mutual recog-nition of 5¢ and 3¢ flanking sequences, catalysed by recom-binase enzymes, effects the VD, VJ and DJ translocations.

Structural variants of the basic Ig molecule• Isotypes are Ig variants based on different heavy chainconstant structures, all of which are present in each indi-vidual; examples are the Ig classes, IgG, IgA, etc.• Allotypes are heavy chain variants encoded by allelic(alternative) genes at single loci and are therefore geneti-cally distributed; examples are Gm groups found on IgGmolecules.• An idiotype is the collection of antigenic determinantson an antibody, usually associated with the hypervariableregions, recognized by other antigen-specific receptors, either antibody (the anti-idiotype) or T-cell receptors.

Immunoglobulin domains serve different functions• The variable region binds antigen, and three hypervari-able loops on the heavy chain, termed complementarity-determining regions (CDRs), and three on the light chain,form the antigen-binding site.• The constant region domains of the heavy chain (partic-ularly the Fc) carry out secondary biological functionsafter the binding of antigen, e.g. complement fixation andbinding to phagocytes.

Immunoglobulin classes and subclasses• In the human there are five major types of heavy chaingiving five classes of Ig. Immunoglobulin G is the mostabundant Ig, particularly in the extravascular fluids whereit neutralizes toxins and combats microorganisms by acti-vating complement via the C1 pathway, and facilitatingthe binding to phagocytic cells by receptors for C3b andFcg. It crosses the placenta in late pregnancy and the intes-tine in the neonate.

Fcg receptors• There is an extensive family of receptors for IgG includ-ing FcgRI, FcgRII, FcgRIII and FcRn. By binding IgG, thesereceptors are crucial for many antibody functions such as phagocytosis, opsonization and antibody-dependentcellular cytotoxicity (ADCC). FcRn transfers circulatingIgG across the placenta to the fetus.

Immunoglobulin A guards the mucosal surfaces• Immunoglobulin A exists mainly as a monomer (basicfour-peptide unit) in plasma. In the seromucous secre-

FURTHER READING

Cedar, H. & Bergman, Y. (1999) Developmental regulation of im-mune system gene rearrangement. Current Opinion inImmunology, 11, 64–9.

Delves, P.J. & Roitt, I.M. (eds) (1998) Encyclopedia of Immunology, 2ndedn. Academic Press, London. (Articles on IgG, IgA, IgM, IgD andIgE and immunoglobulin function and domains.)

Grawunder, U. & Harfst, E. (2001) How to make ends meet in V(D)Jrecombination. Current Opinion in Immunology, 13, 186–94.

Grawunder, U., West, R.B. & Lieber, M.R. (1998) Antigen receptorgene rearrangement. Current Opinion in Immunology, 10, 172–80.

Kinet, J-P. (1999) The high-affinity IgE receptor (FceRI): from physi-ology to pathology. Annual Review of Immunology, 17, 931–72.

Ravetch, J.V. & Bolland, S. (2001) IgG Fc receptors. Annual Review ofImmunology, 19, 275–90.


41

THE B-CELL SURFACE RECEPTOR FOR ANTIGEN

The B-cell inserts a transmembraneimmunoglobulin (Ig) into its surface

In Chapter 2 we discussed the cunning system bywhich an antigen can be led inexorably to its doom byselecting the lymphocytes capable of making antibod-ies complementary in shape to itself through its abilityto combine with a copy of the antibody molecule on thelymphocyte surface. It will be recalled that combina-tion with the surface receptor activates the cell to proliferate and mature into a clone of plasma cells se-creting antibody specific for the inciting antigen (cf.figure 2.6)

Immunofluorescent staining of live B-cells with la-beled anti-Ig (e.g. figure 2.4c) reveals the earliest mem-brane Ig to be of the IgM class. The cell is committed tothe production of just one antibody specificity and sotranscribes its individual rearranged VJCk (or l) andVDJCm genes. The solution to the problem of secretingantibody with the same specificity as that present onthe cell surface as a membrane Ig, is found in a differ-ential splicing mechanism. The initial nuclear m chainRNA transcript includes sequences coding for hy-drophobic transmembrane regions which enable theIgM to sit in the membrane as the B-cell receptor (BCR)but, if these are spliced out, the antibody molecules canbe secreted in a soluble form (figure 4.1).

As the B-cell matures, it co-expresses a BCR utilizing

surface IgD of the same specificity. This surface com-bined IgM + IgD surface phenotype is abundant in themantle zone lymphocytes of secondary lymphoid fol-licles (cf. figure 6.7c & d) and is achieved by differentialsplicing of a single transcript containing VDJ, Cm andCd segments producing either membrane IgM or IgD.As the B-cell matures further, other isotypes such asIgG may be utilized in the BCR.

The surface immunoglobulin (sIg) is complexedwith associated membrane proteins

The cytoplasmic tail of the surface IgM is only threeamino acids long. In no way could this accommodatethe structural motifs required for interaction with intracellular protein tyrosine kinases or G proteinswhich mediate the activation of signal transductioncascades. However, the sIg gets around this problemby transducing signals through a pair of associatedmembrane glycoproteins called Ig-a (CD79a) and Ig-b(CD79b) which become phosphorylated.

THE T-CELL SURFACE RECEPTOR FOR ANTIGEN

The receptor for antigen is a transmembraneheterodimer

The antigen-specific T-cell receptor (TCR) is a membrane-bound molecule composed of two disulfide-linked chains, a and b. Each chain folds into

CHAPTER 4

Membrane receptors for antigen

The B-cell surface receptor for antigen, 41The B-cell inserts a transmembrane immunoglobulin (Ig) into

its surface, 41The surface immunoglobulin (sIg) is complexed with

associated membrane proteins, 41The T-cell surface receptor for antigen, 41

The receptor for antigen is a transmembrane heterodimer, 41There are two classes of TCRs, 42The encoding of TCRs is similar to that of Igs, 42The CD3 complex is an integral part of the TCR, 42

The generation of diversity for antigen recognition, 43

Intrachain amplification of diversity, 43Interchain amplification, 44Somatic hypermutation, 44

Recognition of antigen by natural kil ler (NK) cells, 45

The major histocompatibil i ty complex (MHC), 45Class I and class II molecules are membrane-bound

heterodimers, 45The genes of the MHC display remarkable polymorphism, 46The tissue distribution of MHC molecules, 48Major histocompatibility complex functions, 48

two Ig-like domains, one having a relatively invariantstructure, the other exhibiting a high degree of vari-ability—rather like an Ig Fab fragment.

There are two classes of TCRs

Not long after the breakthrough in identifying the TCRab came reports of the existence of a second type of receptor composed of g and d chains. This TCR gdappears earlier in thymic ontogeny than the TCR ab.

In the human, gd cells make up only 0.5–15.0% of the T-cells in peripheral blood, but they show greaterdominance in the intestinal epithelium and in skin.

The encoding of TCRs is similar to that of Igs

The gene segments encoding the TCR b chains follow asimilar arrangement of V, D, J and C segments to thosedescribed for the Igs (figure 4.2). As an immunocompe-tent T-cell is formed, rearrangement of V, D and J genesoccurs to form a continuous VDJ sequence. The firmestevidence that B- and T-cells use similar recombinationmechanisms comes from mice with severe combinedimmunodeficiency (SCID) due to a single autosomalrecessive defect preventing successful linkage of V, Dand J segments. Homozygous mutants fail to developimmunocompetent B- and T-cells and identical se-quence defects in VDJ joint formation are seen in bothpre-B- and pre-T-cell lines.

Looking first at the b-chain cluster, one of the two Dbgenes rearranges next to one of the Jb genes. Note thatthe first Db gene, Db1, can utilize any of the 13 Jb genes,but Db2 can only choose from the seven Jb2 genes. Next,one of the 50 or so Vb genes is rearranged to the pre-

formed DbJb segment. Variability in junction forma-tion and the random insertion of nucleotides to createN-region diversity either side of the D segment, mirrorthe same phenomenon seen with Ig gene rearrange-ments. Sequence analysis emphasizes the analogywith the antibody molecule; each V segment containstwo hypervariable regions, while the DJ sequence pro-vides a very hypervariable CDR3 structure, making atotal of six potential complementarity-determining regions for antigen binding in each TCR. As in the synthesis of antibody, the intron between VDJ and C is spliced out of the messenger RNA (mRNA) beforetranslation with the restriction that rearrangements involving genes in the Db2Jb2 cluster can only link to Cb2.

All the other chains are encoded by genes formedthrough similar translocations. The a-chain gene poollacks D segments but more than makes up for it with aprodigious number of J segments. The number of Vgand Vd genes is very small in comparison with Va andVb. Like the a-chain pool, the g chain cluster has no Dsegments.

The CD3 complex is an integral part of the TCR

The T-cell antigen recognition complex and its B-cellcounterpart can be likened to specialized army pla-toons whose job is to send out a signal when the enemyhas been sighted. When the TCR “sights the enemy,”i.e. ligates antigen, it relays a signal through an associ-ated complex of transmembrane polypeptides (CD3)to the interior of the T-lymphocyte, instructing it toawaken from its slumbering G0 state and do some-thing useful —like becoming an effector cell. In all im-


PROTEIN

Translation

DNA

Cm1-4V D J Cm1-4 MM

Secreted IgM

V D J

V D J

mRNA

Cm1 M MCm2 Cm3 Cm4

Membrane IgM

Transcription and splicing

Figure 4.1 Splicing mechanism for theswitch from the membrane to the secretedform of immunoglobulin M (IgM). Thehydrophobic sequence encoded by theexons M–M, which anchors the receptorIgM to the membrane, is spliced out in thesecreted form. For simplicity, the leader sequence has been omitted. , introns.

munocompetent T-cells, the antigen receptor is nonco-valently but still intimately linked with CD3 in a com-plex which contains two heterodimeric TCR ab or gdrecognition units closely apposed to the invariant CD3peptide chains g and d, two molecules of CD3e plus thedisulfide-linked z–z dimer. The total complex has thestructure TCR2CD3gde2z2 (figure 4.3). The relationshipof the CD3 molecules to the TCR heterodimer is there-fore directly comparable to that of the Ig-a and Ig-bmolecules to the sIg in the B-cell.

THE GENERATION OF DIVERSITY FORANTIGEN RECOGNITION

We know that the immune system has to be capable ofrecognizing virtually any pathogen that has arisen ormight arise. The extravagant genetic solution to thisproblem of anticipating an unpredictable future in-volves the generation of millions of different specificantigen receptors, probably vastly more than the life-time needs of the individual. Since this is likely to exceed the number of genes in the body, there must besome clever ways to generate all this diversity, particu-larly since the number of genes coding for antibodiesand TCRs is only of the order of 400. We can now prof-itably examine the mechanisms which have evolved togenerate tremendous diversity from such limited genepools.

Intrachain amplification of diversity

Random VDJ combination increases diversitygeometrically

Just as we can use a relatively small number of differ-ent building units in a child’s construction set to createa rich variety of architectural masterpieces, so the indi-vidual receptor gene segments can be viewed as build-ing blocks to fashion a multiplicity of antigen-specificreceptors for both B- and T-cells.

Take, for example, the Ig heavy-chain genes (table4.1). Although the precise number of gene segmentsvaries from one individual to another, there are typi-cally around 25 D and six J functional segments. If therewere entirely random joining of any one D to any one Jsegment (cf. figure 3.5), we would have the possibilityof generating 150 DJ combinations (25 ¥ 6). Let us go tothe next stage. Since each of these 150 DJ segmentscould join with any one of the approximately 50 VHfunctional sequences, the net potential repertoire of VDJ genes encoding the heavy-chain variable region would be 50 ¥ 150 = 7500. In other words, justtaking our V, D and J genes, which in this example addup arithmetically to 81, we have produced a range ofsome 7500 different variable regions by geometric recombination of the basic elements. But that is onlythe beginning.

Playing with the junctions

Another ploy to squeeze more variation out of thegerm line repertoire involves variable boundary re-

CHAPTER 4—Membrane receptors for antigen 43

Jb21 71 50Vb Db1 Jb11 6 Cb1 Db2 Cb2

ab TCR

1 75Va d genes Ja1 60 Ca

1 ~8Vd Cd

gd TCR

1 12Vg

Dd1 3 Jd1 3 Vd

Jg11 3 Cg1 1 2 Cg 2Jg2

HUMAN TCR GENES

g

N

L V J C TM

d

N

L V D J TMD C

N

a

N

L J C TMV

b

N

L J C TM

N

DV

Figure 4.2 Genes encoding ab and gd T-cell receptors (TCRs).Genes encoding the d chains lie between the Va and Ja clusters andsome V segments in this region can be used in either d or a chains, i.e.

as either Va or Vd. T-cell receptor genes rearrange in a manner analo-gous to that seen with immunoglobulin genes.

combinations of V, D and J to produce different junc-tional sequences.

Further diversity arises from the insertion of nu-cleotides between the V, D and J segments, a processreferred to as N-region diversity and associated withthe expression of terminal deoxynucleotidyl trans-ferase. This maneuver greatly increases the repertoire,especially for the TCR g and d genes which are other-wise rather limited in number.

Yet additional mechanisms relate specifically to theD-region sequence: particularly in the case of the TCRd genes, where the D segment can be read in three different reading frames and two D segments can jointogether giving a DD combination.

Receptor editing

Recent observations have established that lympho-cytes are not necessarily stuck with the antigen recep-tor they initially make; if they do not like it, they canchange it. The replacement of an undesired receptorwith one that has more acceptable characteristics is referred to as receptor editing. This process has beendescribed for both Igs and for TCR, allowing the re-placement of either nonfunctional or autoreactive rearrangements. Furthermore, receptor editing in theperiphery may rescue low affinity B-cells from apop-totic cell death by replacing a low affinity receptor witha selectable one of higher affinity. The process involvesreactivation of RAG genes followed by production of anew light or heavy chain which will change the Ig orTCR. Receptor editing occurs more readily in Ig lightchains and TCR a chains than in Ig heavy chains andTCR b chains. Indeed, it has been suggested that theTCR a chain may undergo a series of rearrangementscontinuously deleting previously rearranged VJ seg-ments until a selectable TCR is produced.

Interchain amplification

The immune system took an ingenious step forwardwhen two different types of chain were utilized for therecognition molecules because the combination pro-duces not only a larger combining site with potentiallygreater affinity but also new variability. Thus, whenone heavy chain is paired with different light chainsthe specificity of the final antibody is altered.

This random association between TCR g and dchains, TCR a and b chains, and Ig heavy and lightchains yields a further geometric increase in diversity.From table 4.1 it can be seen that approximately 230functional TCR and 160 functional Ig germ line segments can give rise to 4.5 million and 2.4 million different combinations respectively, by straightfor-ward associations without taking into account all of thefancy junctional mechanisms mechanisms describedabove.

Somatic hypermutation

There is inescapable evidence that Ig V-region genescan undergo significant somatic mutation.

A number of features of this somatic diversificationphenomenon deserve mention. The mutations are theresult of single nucleotide substitutions, they are restricted to the variable as distinct from the constantregion, and occur in both framework and hypervari-able regions. The mutation rate is remarkably high,


-S–S-

CD3 COMPLEX

_ + +

e

+ _ _

yy

yy

yyyy

yy

yy

yy

yy

yy

g z2

ITAM

CDRs

Ag recognition

(D)J

_+

yy

CD3e CD3g CD3dCb(d)

Ca(g)

Vb(d)

Va(g)

d

Figure 4.3 The T-cell receptor (TCR)/CD3 complex. The TCR re-sembles the immunoglobulin (Ig) Fab antigen-binding fragment instructure. The variable and constant segments of the TCR a and bchains (VaCa/VbCb), and of the corresponding g and d chains of thegd TCR, belong structurally to the Ig-type domain family. The cyto-plasmic domains of the CD3 peptide chains, shown in black, contactprotein tyrosine kinases which activate cellular transcription factors.

between 2 and 4% for VH genes as compared with avalue of < 0.0001% for a nonimmunologic lymphocytegene. The mutational mechanism is bound up in someway with class switch since hypermutation is more fre-quent in IgG and IgAthan in IgM antibodies. It is likelythat somatic mutation does not add to the repertoireavailable in the early phases of the primary response,but occurs during the generation of memory and isprobably responsible for tuning the response towardshigher affinity.

T-cell receptor genes, on the other hand, do not appear to undergo somatic mutation. This is fortunatesince, because TCRs already partly recognize selfmajor histocompatibility complex (MHC) molecules,mutations could readily encourage the emergence ofhigh affinity autoreactive receptors and resulting autoimmunity.

RECOGNITION OF ANTIGEN BY NATURALKILLER (NK) CELLS

As was described in Chapter 1, one of the main func-tions of NK cells is to patrol the body looking for malig-nant or infected cells which have lost expression of thenormally ubiquitously present MHC class I molecules.Activating receptors recognize molecules collectivelypresent on all cell surfaces and inhibitory receptors recognize MHC class I molecules. Any nucleated celllacking MHC class I will not engage the inhibitory re-ceptors and will only trigger the activating receptors,resulting in its execution by the NK cell. Unlike theTCR on T-cells and the BCR on B-cells, this recognitionsystem is not antigen specific. However, a second

mode of killing which NK cells employ is antibody-dependent cellular cytotoxicity (ADCC, cf. p. 23) forwhich they are equipped with FcgRIII receptors inorder to recognize antibody-coated target cells (figure4.4). In this case the antibody alerts the NK cell to thepresence of specific antigen.

THE MAJOR HISTOCOMPATIBILITYCOMPLEX (MHC)

Molecules expressed by genes which constitute thiscomplex chromosomal region were originally definedby their ability to provoke vigorous rejection of graftsexchanged between different members of a species. InChapter 2, brief mention was made of the necessity forcell-surface antigens to be associated with class I orclass II MHC molecules so that they can be recognizedby T-lymphocytes. The intention now is to give moreinsight into the nature of these molecules.

Class I and class II molecules are membrane-bound heterodimers

Major histocompatibility complex class I

Class I molecules consist of a heavy polypeptide chainof 44 kDa noncovalently linked to a smaller 12 kDapeptide called b2-microglobulin. The largest part of theheavy chain is organized into three globular domains(a1, a2 and a3; figure 4.5a) which protrude from the cellsurface; a hydrophobic section anchors the molecule in the membrane and a short hydrophilic sequence carries the C-terminus into the cytoplasm.


g d a b

12 ~8- 3

3,2 3V J V D J

12 x 5 8 x 3 x 360 72

60 x 724.3 x 103

75 50- 1,1

60 6,7V J V D J

75 x 60 50(13+7)4500 1000

4500 x 10004.5 x 106

Ig

kH

lL

50256

V D J50 x 25 x 6

7500

40-5

V J40 x 5200

30-4

V J30 x 4

1207500 x 200 7500 x 120

gene segments

gene segments gene segments

Random combinatorial joining(without junctional diversity)

TotalCombinatorial heterodimers

Total (rounded)

Other mechanisms: D's in 3 reading frames,junctional diversity, N region insertion; x103

– +++ +++–Somatic mutation

1.5 x 106

1.5 x 109 0.9 x 109

0.9 x 106

x x x x x x x x x x

VD J

gdTCR (TCR1) abTCR (TCR2)

4.3 x 106 4.5 x 109

Table 4.1 Calculations of human V gene diversity. The minimum number of specificities generated by straightforward random combination ofgerm line segments are calculated. These will be increased by the further mechanisms listed.

X-ray analysis of crystals of a human class I moleculeprovided an exciting leap forward in our understand-ing of MHC function. Both b2-microglobulin and the a3region resemble classic Ig domains in their folding pat-tern. However, the a1 and a2 domains, which are mostdistal to the membrane, form two extended a-helicesabove a floor created by peptide strands held togetherin a b-pleated sheet, the whole forming an undeniablecavity (figure 4.5b,c). Another curious featureemerged. A linear molecule, now known to be a pep-tide, which had co-crystallized with the class I protein,occupied the cavity. The significance of these unique

findings for T-cell recognition of antigen will be revealed in Chapter 5.

Major histocompatibility complex class II

Class II MHC molecules are also transmembrane gly-coproteins, in this case consisting of a and b polypep-tide chains of molecular weight 34 kDa and 29 kDarespectively.

There is considerable sequence homology with class I. Structural studies have shown that the a2 and b2 domains, the ones nearest to the cell membrane, assume the characteristic Ig fold, while the a1 and b1domains mimic the class I a1 and a2 in forming agroove bounded by two a-helices and a b-pleatedsheet floor (figure 4.5a).

Several immune response related genes contribute tothe class III region of the MHC

A variety of other genes that congregate within theMHC chromosome region are grouped under theheading of class III. Broadly, one could say that manyare directly or indirectly related to immune defensefunctions. A notable cluster are the genes coding for complement components including C2, two C4 isotypes and factor B. The cytokines tumor necrosisfactor (TNF) and lymphotoxin are also encoded underthe class III umbrella.

Gene map of the MHC

The complete sequence of a human MHC was pub-lished at the very end of the last millennium. The se-quence comprises 224 gene loci. An overall view of themain clusters of class I, II and III genes in the humanMHC (human leukocyte antigen, HLA, system) maybe gained from figure 4.6. A number of silent orpseudogenes have been omitted from this gene map inthe interest of simplicity.

The genes of the MHC display remarkablepolymorphism

Unlike the Ig and TCR systems where, as we have seen,variability is achieved in each individual by rearrange-ment of multiple gene segments, the MHC has evolvedin terms of variability between individuals with ahighly polymorphic (literally “many-shaped”) sys-tem based on multiple alleles (i.e. alternative genes ateach locus). A given MHC gene complex is referred toas a “haplotype” and is usually inherited en bloc as asingle Mendelian trait. The class I and class II genes are


Inhibitory receptor

+

+

–

Normalcell

MHC class I

Abnormal celllacking MHC

class I

Inhibitory receptor

Nosignal

NK cell

FcgR

IgG

Antibody-coatedtarget cell

(a) (b)

(c)

Activating ligand

Activatingreceptor

Figure 4.4 Natural killer (NK) cells. Natural killer cells possessboth activating and inhibitory receptors. Engagement of an activat-ing receptor by its ligand sends a stimulatory signal into the NK cellbut this is normally subverted by a signal transmitted upon recogni-tion of major histocompatibility complex (MHC) class I molecules by an inhibitory receptor (a). Any nucleated cell lacking class I isdeemed abnormal and is killed following unimpeded transmissionof the activation signal (b). Natural killer cells usually possess seve-ral different inhibitory and activating receptors and it is the balanceof signals from these that determines whether the cell becomes activated. Like several other cell types, NK cells can utilize their Fcgreceptors to mediate antibody-dependent cellular cytotoxicity(ADCC) on target cells coated with antibody (c).


MEMBRANE

COOH

CLASS I CLASS II

b

a1 a2

a3

a-HELIX CLEFT

�-PLEATEDSHEET

b2-MICROGLOBULIN

a1

a2

b1

b2

a

a

b c

PEPTIDE BINDING CLEFT

N

N

C

C

a1

b2m

a2

a3

Figure 4.5 Class I and class II MHC molecules. (a) Diagram show-ing domains and transmembrane segments; the a-helices and b-sheets are viewed end on. (b) Schematic bird’s eye representation ofthe top surface of a human class I molecule (HLA-A2) based on X-raycrystallographic structure. The strands making the b-pleated sheetare shown as thick gray arrows in the amino to carboxy direction; a-helices are represented as dark-red helical ribbons. The inside-facingsurfaces of the two helices and the upper surface of the b-sheet form acleft which binds and presents the peptide antigen to the T-cell. Thetwo black spheres represent an intrachain disulfide bond. (c) Sideview of the same molecule clearly showing the anatomy of the cleftand the typical immunoglobulin folding of the a3- and b2-microglobulin domains (four antiparallel b-strands on one face and three on the other). (Reproduced from P.J. Bjorkman et al. (1987)Nature, 329, 506–12, with permission.)

the most polymorphic genes in the human genome.For some of these genes, over 200 allelic variants havebeen identified. The amino acid changes responsiblefor these polymorphisms are restricted to the a1 and a2domains of class I and to the a1 and b1 domains of classII. It is of enormous significance that they occur essen-tially in the b-sheet floor and the inner surfaces of the a-helices that line the central cavity (figure 4.5) and alsoon the upper surface of the helices.

The tissue distribution of MHC molecules

Essentially, all nucleated cells carry classical class Imolecules. These are abundantly expressed on lym-phoid cells, less so on liver, lung and kidney, and only

sparsely on brain and skeletal muscle. In the human,the surface of the villous trophoblast lacks HLA-Aand-B and bears HLA-G, which does not appear on anyother body cell. Class II molecules are also restricted in their expression, being present only on antigen-presenting cells (APCs) such as B-cells, dendritic cellsand macrophages and on thymic epithelium. When activated by agents such as interferon g, capillary en-dothelia and many epithelial cells in tissues other thanthe thymus, they can develop surface class II and in-creased expression of class I.

Major histocompatibility complex functions

Although originally discovered through transplanta-tion reactions, the MHC molecules are utilized for vitalbiological functions by the host. Their function as cellsurface markers enabling infected cells to signal cyto-toxic and helper T-cells will be explored in depth insubsequent chapters. There is no doubt that this role inimmune responsiveness is immensely important, andin this respect the rich polymorphism of the MHC re-gion would represent a species response to maximizeprotection against diverse microorganisms.


MHC class

HLA

The major histocompatibility complex

III III

DP LMP TA P DQ DR C4 FB C2 HSP70 TNF B C E A G&

Figure 4.6 Main genetic regions of the human major histocompat-ibility complex (MHC). HLA, human leukocyte antigen. Note that,although originally identified on leukocytes, HLA class I moleculesare present on virtually all nucleated cells.

REVISION

The B-cell surface receptor for antigen• The B-cell inserts its immunoglobulin (Ig) gene productcontaining a transmembrane segment into its surfacewhere it acts as a specific receptor for antigen.• The surface immunoglobulin (sIg) is complexed withthe membrane proteins Ig-a and Ig-b which become phos-phorylated on cell activation and transduce signals received through the Ig antigen receptor.

The T-cell surface receptor for antigen• The receptor for antigen is a transmembrane dimer,each chain consisting of two Ig-like domains.• The outer domains are variable in structure, the innerones constant, rather like a membrane-bound Fab.• Most T-cells express a receptor (T-cell receptor, TCR)with a and b chains. A separate lineage bearing gd recep-tors is fairly abundant during early thymic ontogeny but isassociated mainly with epithelial tissues in the adult.• The encoding of the TCR is similar to that of Igs. Thevariable region coding sequence in the differentiating T-cell is formed by random rearrangement from clusters of

V, D (for b and d chains) and J segments to form a single re-combinant V(D)J sequence for each chain.• Like the Ig chains, each variable region has three hyper-variable sequences which function in antigen recognition.• The CD3 complex, composed of g, d, e2 and z2, forms anintimate part of the receptor and has a signal transducingrole following ligand binding by the TCR.

The generation of antibody diversity for antigen recognition• The individual receptor gene segments can be viewedas building blocks to produce very large numbers of anti-gen-specific TCRs or B-cell receptors (BCRs).• Further diversity is introduced at the junctions betweenV, D and J segments by variable recombination as they arespliced together by recombinase enzymes.• In addition, after a primary response, B-cells, but not T-cells, undergo high rate somatic mutation affecting the Vregions.

Major histocompatibility complex (MHC)• Each vertebrate species has a MHC identified originally

FURTHER READING

Campbell, K.S. (1999) Signal transduction from the B-cell antigen-re-ceptor. Current Opinion in Immunology, 11, 256–64.

Campbell, R.D. & Trowsdale, J. (1997) Map of the human major his-tocompatibility complex. Immunology Today, 18 (1), pullout.

Campbell, R.D. & Trowsdale, J. (1993) Map of the human MHC. Immunology Today, 14, 349–52.

Howard, J.C. (1991) Disease and evolution. Nature, 352, 565–7.Hughes, A.L., Yeager, M., Ten Elshof, A.E. & Chorney, M.J. (1999) A

new taxonomy of mammalian MHC class I molecules. Immunolo-gy Today, 20, 22–6.


through its ability to evoke very powerful transplantationrejection.• Each contains three classes of genes. Class I encodes apolypeptide chain associated at the cell surface with b2-microglobulin. Class II molecules are transmembrane het-erodimers. Class III products are heterogeneous butinclude complement components and tumor necrosis fac-tor (TNF).• The genes display remarkable polymorphism. The par-ticular set of MHC genes inherited by an individual is referred to as the haplotype.• Classical class I molecules are present on virtually allnucleated cells in the body and signal cytotoxic T-cells.• Class II molecules are particularly associated with B-cells, dendritic cells, macrophages and thymic epithelium,but can be induced on capillary endothelial cells and many

epithelial cells by interferon g. They signal T-helpers for B-cells and macrophages.• Structural analysis indicates that the two domains distalto the cell membrane form a cavity bounded by two paral-lel a-helices sitting on a floor of b-sheet peptide strands;the walls and floor of the cavity and the upper surface ofthe helices are the sites of maximum polymorphic aminoacid substitutions.• Many class III gene products are associated with immune defense mechanisms, including complementproteins and TNF.


50

WHAT IS AN ANTIGEN?

Aman cannot be a husband without a wife and a mole-cule cannot be an antigen without a corresponding an-tiserum or antibody or T-cell receptor (TCR). The termantigen is used in two senses, the first to describe amolecule which generates an immune response (alsocalled an immunogen), and the second a moleculewhich reacts with antibodies or primed T-cells. Hap-tens are small well-defined chemical groupings, suchas dinitrophenyl (DNP) or m-aminobenzene sulfonate,which are not immunogenic on their own but will reactwith preformed antibodies induced by injection of thehapten linked to a “carrier” molecule which is itself animmunogen (figure 5.1).

The part of the hypervariable regions on the anti-body which contacts the antigen is termed theparatope, and the part of the antigen which is in con-tact with the paratope is designated the epitope orantigenic determinant. It is important to be aware thateach antigen usually bears several determinants on itssurface, which may well be structurally distinct fromeach other; thus a monoclonal antibody reacting withone determinant will usually not react with any otherdeterminants on the same antigen (figure 5.2). To getsome idea of size, if the antigen is a linear peptide orcarbohydrate, the combining site can usually accom-

modate up to five or six amino acid residues or hexoseunits. With a globular protein as many as 16 or soamino acid side-chains may be in contact with an anti-body (figure 5.3).

The structure of antigens

In general, large proteins, because they have more po-tential determinants, are better antigens than smallones. Furthermore, the more foreign an antigen, that isthe less similar to self configurations, the more effec-tive it is in provoking an immune response. Althoughantibodies can be produced to almost any part of a for-eign protein, certain areas of the antigen are more immunogenic and are called the immunodominant re-gions of the antigen. These sites of high epitope densityare often those parts of the peptide chains that pro-trude significantly from the globular surface.

ANTIGEN AND ANTIBODY INTERACTIONS

Antigen and antibody interact due to complement-arity in shape over a wide area of contact (figure 5.3),not so much as inflexible entities which fit togetherprecisely in a “lock and key” manner but rather as mutually deformable surfaces — more like clouds thanrocks. The interaction depends upon weak van der

CHAPTER 5

The primary interaction with antigen

What is an antigen?, 50The structure of antigens, 50

Antigen and antibody interactions, 50The specificity of antigen recognition by antibody

is not absolute, 52In vitro antigen–antibody reactions, 52

Numerous techniques are available to detect the presence andthe titer of an antibody, 53

Identification and measurement of antigen, 53What the T-cell sees, 54

Haplotype restriction reveals the need for MHC participation, 54

T-cells recognize a linear peptide sequence from the antigen, 54

Processing of intracellular antigen for presentationby class I MHC, 54

Processing of antigen for class I I MHC presentationfollows a dif ferent pathway, 55Binding to MHC class I, 57Binding to MHC class II, 57

T-cells with a dif ferent outlook, 57The family of CD1 non-MHC class I-like molecules can present

exotic antigens, 57Superantigens stimulate whole families of

lymphocyte receptors, 58Bacterial toxins represent one major group of T-cell

superantigens, 58

CHAPTER 5—The primary interaction with antigen 51

NH2 N

N

OVALBUMIN

m-AMINOBENZENESULFONATE

SO3

HAPTENConjugate

to ovalbumin(carrier)

NO ANTIBODY ANTI-CARRIERANTI-HAPTEN

Reactin vitro

Inject Inject

SO3

Figure 5.1 A hapten on its own will not induce antibodies. How-ever, it will react in vitro with antibodies formed to a conjugate of thehapten with an immunogenic carrier.

EPITOPE CLUSTER (DETERMINANT)

INDIVIDUAL ANTIBODY PARATOPES IN POLYCLONAL ANTISERUM

ANTIGEN EPITOPE

PARATOPE

50 150 50 20

Figure 5.2 A globular protein antigen usually bears a mosaic ofdeterminants (dominant epitope clusters) on its surface, definedby the heterogeneous population of antibody molecules in a givenantiserum. This highly idealized diagram illustrates the idea that individual antibodies in a polyclonal antiserum with different combining sites (paratopes) can react with overlapping epitopesforming a determinant on the surface of the antigen. The numbersrefer to the imagined relative frequency of each antibody specificity.

12918

25124

1921

22

2324

27 121116

117118

119120

49

102

10099

323130

5354

52

93

92

3050

32

91101

a

b

c

2918

25124

1921

22

2324

27 121116

117118

119120

49

102

10099

323130

5354

52

93

92

3050

32

91101

Figure 5.3 Structure of the contact regions between a monoclonalFab antilysozyme and lysozyme. (a) Space-filling model showingFab and lysozyme molecules fitting snugly together. Antibodyheavy chain, blue; light chain, yellow; lysozyme, green with its glutamine-121 in red. (b) Fab and lysozyme models pulled apart toshow how the protuberances and depressions of each are comple-mentary to each other. (c) End-on views of antibody combining site(left) and the lysozyme epitope (right) obtained from (b) by rotatingeach molecule 90° about a vertical axis. (Reproduced with permis-sion from A. Amit et al. (1986) Science, 233, 747–53. Copyright 1986 bythe AAAS.)

Waals, electrostatic, hydrophobic and hydrogen bond-ing forces which only become of significant magnitudewhen the interacting molecules are very close together.Thus, the more snugly the paratope and epitope fit to-gether, the stronger the strength of binding, termed theaffinity. This is defined as the equilibrium constant(Ka) of the reversible association of antibody with a sin-gle epitope (e.g. a hapten, figure 5.1), represented bythe equation:

Ab + Hp ∫AbHp

Obviously a high affinity betokens strong binding.Usually, we are concerned with the interaction of anantiserum, i.e. the serum from an immunized indi-vidual with a multivalent antigen where the binding is geometrically increased relative to a monovalent hapten or single antigenic epitope. The term employedto express this binding is avidity, which being a mea-sure of the functional affinity of an antiserum for thewhole antigen is of obvious relevance to the reactionwith antigen in the body. High avidity is superior tolow for a wide variety of functions in vivo, includingimmune elimination of antigen, virus neutralizationand the protective role against bacteria and other organisms.

THE SPECIFICITY OF ANTIGENRECOGNITION BY ANTIBODY IS NOT ABSOLUTE

The strength of an antigen–antibody reaction can bequantified by the affinity or avidity of the antibody tothat specific antigen. In so far as we recognize that anantiserum may have a relatively greater avidity for oneantigen than another, by the same token we are sayingthat the antiserum is displaying relative rather than ab-solute specificity; in practice we speak of degrees ofcross-reactivity. An antiserum raised against a givenantigen can cross-react with a partially related antigenwhich bears one or more identical or similar determi-nants. In figure 5.4 it can be seen that an antiserum toantigen-1 (Ag1) will react less strongly with Ag2, whichbears just one identical determinant, because only cer-tain of the antibodies in the serum can bind. Antigen-3,which possesses a similar but not identical determi-nant, will not fit as well with the antibody, and hencethe binding is even weaker. Antigen-4, which has nostructural similarity at all, will not react significantlywith the antibody. Thus, based upon stereochemicalconsiderations, we can see why the avidity of the anti-

serum for Ag2 and Ag3 is less than for the homologousantigen, while for the unrelated Ag4 it is negligible. Itwould be customary to describe the antiserum as beinghighly specific for Ag1 in relation to Ag4 but cross-reacting with Ag2 and Ag3 to different extents. Theseprinciples have significance in human autoimmunediseases since an antibody may react not only with theantigen which stimulated its production but also withsome quite unrelated molecules.

IN VITRO ANTIGEN–ANTIBODY REACTIONS

The specificity of antigen–antibody reactions enablesus to detect the presence of specific antibody in serumor other fluid by combining it with the known antigenunder strict laboratory conditions. Not only will posi-tive reactions tell us that specific antibodies are presentbut by testing a series of antibody dilutions we can getan idea of the quantity, or titer, of specific antibody inthe fluid. To take an example, a serum might be diluted10000 times and still just give a positive reaction. Thistiter of 1 : 10000 enables comparison to be made withanother much weaker serum which has a titer of, say,1 :100, i.e. it can only be diluted out 100 times before thetest becomes negative. The titer is of crucial impor-tance in interpreting the significance of antibodies inpatients with clinical disease. Many normal individu-als, for example, may have low titer antibodies to cytomegalovirus resulting from previous, perhapssubclinical, infection. In active infection titers may behigh and increasing.


Ag2 Ag3 Ag4

Originalantigen

One identicaldeterminant

Similardeterminant

No structuralsimilarity

CROSS-REACTION NO REACTION

Ag1

Figure 5.4 Specificity and cross-reaction. The avidity of the serumantibodies and for antigen-1 (Ag1) > Ag2 > Ag3 >> Ag4.

Numerous techniques are available to detect thepresence and the titer of an antibody

Precipitation

When an antigen solution is added progressively to apotent antiserum, antigen–antibody precipitates areformed which can be detected by a variety of laborat-ory techniques. This precipitation can be enhanced bycountercurrent immunoelectrophoresis. For example,antigen and antiserum can be placed in wells punchedin an agar gel and a current applied to the system. Theantigen migrates steadily into the antibody zone andforms a precipitin line where the complex forms. Thisprovides a fairly sensitive and rapid test that has beenapplied to the detection of many clinically significantantibodies and, by the same means, antigen.

Nonprecipitating antibodies can be detected by nephelometry

The small aggregates formed when dilute solutions of antigen and antibody are mixed create a cloudinessor turbidity that can be measured by forward anglescattering of an incident light source (nephelometry).Greater sensitivity can be obtained by using mono-chromatic light from a laser.

Agglutination of antigen-coated particles

Whereas the cross-linking of multivalent protein anti-gens by antibody leads to precipitation, cross-linkingof cells or large particles by antibody directed againstsurface antigens leads to agglutination.

Agglutination reactions are used to identify bacteriaand to type red cells. They have been observed withleukocytes and platelets and even with spermatozoa incases of male infertility due to sperm antibodies.

Immunoassay for antibody using solid-phaseantigen

The antibody content of a serum can be assessed by itsability to bind to antigen that has been attached to aplastic tube or micro-agglutination tray with multiplewells. Immunoglobulin (Ig) binding to the antigenmay then be detected by addition of a labeled sec-ondary antibody, i.e. an anti-Ig raised in anotherspecies (figure 5.5). Consider, for example, the deter-mination of DNA autoantibodies in systemic lupuserythematosus (SLE). When a patient’s serum is addedto a microwell coated with antigen (in this case DNA),the autoantibodies will bind to the immobilized DNAand remaining serum proteins can be readily washedaway. Bound antibody can now be estimated by addi-tion of enzyme-labeled rabbit anti-human IgG. Afterrinsing out excess unbound reagent, the enzyme activ-ity of the tube will clearly be a measure of the autoanti-body content of the patient’s serum. The distributionof antibody in different classes can be determined byusing specific antisera. For example, to detect IgE anti-bodies in allergic individuals the allergen such aspollen extract is covalently coupled to a paper disc orplastic well to which is added patient serum. Theamount of specific IgE bound to antigen can be esti-mated by the addition of enzyme-labeled anti-IgE. Enzymes such as horseradish peroxidase and alkalinephosphatase, which give a colored soluble reactionproduct, are widely employed in these enzyme-linkedimmunosorbent assays (ELISAs).

Identification and measurement of antigen

Antigens can be readily identified and quantitated invitro, provided that specific antibody is available in thereaction mixture. Laboratory techniques similar tothose employed to detect antibodies can be used to


MEASURELABEL

Plastic tube

wash wash wash

Add Ag Add patient's serum Add labeled anti-Ig

Figure 5.5 Solid-phase immunoassay forantibody. The anti-immunoglobulin (anti-Ig) may be labeled by radioactive iodine or,more usually, by an enzyme which gives asoluble colored or chemiluminescent reac-tion product. Ag, antigen.

detect antigens. For example, precipitation reactionscan be carried out in gels to detect an abnormal proteinin serum or urine secreted by a B-cell or plasma-celltumor. The monoclonal paraprotein localizes as adense compact “M” band of defined electrophoreticmobility, and its antigenic identity is then revealed by immunofixation with specific precipitating anti-serums applied in paper strips overlying parallel lanesin the electrophoresis gel.

The nephelometric assay for antigen

If antigen is added to a solution of excess antibody, theamount of complex assessed by forward light scatter ina nephelometer is linearly related to the concentrationof antigen. With the ready availability of a huge rangeof monoclonal antibodies which facilitate the stan-dardization of the method, nephelometry is com-monly used to detect a wide array of serum proteinsincluding Igs, C3, C4, haptoglobin, ceruloplasmin andC-reactive protein (CRP).

Immunoassay on multiple microspots

The field of DNA microarray technology has led to the area of miniaturization of immunoassays. Thisdepends on spotting multiple antigens, or when neces-sary antibodies, in an ordered dense array on a solidsupport such as a glass slide. Patient serum or otherbodily fluid is layered over the slide and the presenceof binding is detected with chemiluminescent orenzyme-labeled secondary antibodies. Expression isrecorded by sophisticated analytical apparatus andstored in a database. Sensitivities compare very favor-ably with the best immunoassays and, with suchminiaturization, arrays of microspots which captureantibodies of different specificities can be placed on asingle chip. This opens the door to multiple analytescreening in a single test, with each analyte being iden-tified by its grid coordinates in the array.

WHAT THE T-CELL SEES

We have on several occasions alluded to the fact thatthe TCR sees antigen on the surface of cells associatedwith a major histocompatibility complex (MHC) class Ior II molecule. Now is the time for us to go into the nutsand bolts of this relationship.

Haplotype restriction reveals the need for MHCparticipation

It has been established that T-cells bearing ab recep-

tors, with some exceptions, only respond when theantigen-presenting cells (APCs) express the sameMHC haplotype as the host from which the T-cellswere derived. This haplotype restriction on T-cellrecognition tells us unequivocally that MHC mole-cules are intimately and necessarily involved in the interaction of the antigen-bearing cell with its corre-sponding antigen-specific T-lymphocyte. We alsolearn that cytotoxic T-cells recognize antigen in thecontext of class I MHC, and helper T-cells respondwhen the antigen is associated with class II moleculeson APCs.

One of the seminal observations which helped toelucidate the role of the MHC was the dramatic NobelPrize-winning revelation by Peter Doherty and RolfZinkernagel that cytotoxic T-cells taken from an indi-vidual recovering from a viral infection will only kill virally infected cells which share an MHC haplo-type with the host. For example, on recovery from in-fluenza, individuals bearing the MHC haplotypeHLA-A2 have CD8+ T-cells which kill HLA-A2 targetcells infected with influenza virus but not cells of a different HLA-Atissue-type specificity.

T-cells recognize a linear peptide sequence from the antigen

With any complex antigen only certain linear peptidescan be recognized by a specific T-cell. When clones of identical specificity are derived from these T-cells,each clone reacts with only one of the peptides; in otherwords, like B-cell clones, each clone is specific for onecorresponding epitope. Therefore, when either cyto-toxic or helper T-cell clones are stimulated by APCs towhich certain peptides derived from the original anti-gen had been added, the clones can be activated. Bysynthesizing a series of such peptides, the T-cell epi-tope can be mapped with some precision.

The conclusion is that the T-cell recognizes bothMHC and peptide, and we now know that the peptidewhich acts as a T-cell epitope lies along the grooveformed by the a-helices and the b-sheet floor of theclass I and class II outermost domains. Just how does itget there?

PROCESSING OF INTRACELLULAR ANTIGEN FOR PRESENTATION BY CLASS I MHC

Within the cytosol lurk proteolytic structures, the proteasomes, involved in the routine turnover and cellular degradation of proteins. Cytosolic proteinsdestined for antigen presentation, including viral pro-


teins, link to a small polypeptide called ubiquitin andare fed into a specialized proteasome, the immunopro-teasome, where they are degraded (figure 5.6). The de-graded peptides now attach to transporter proteinscalled TAP1/TAP2 (transporters associated with anti-gen processing) which moves them into the endoplas-mic reticulum (ER). The immunoproteasome containsthe nonpolymorphic MECL-1 molecule together withtwo MHC-linked low molecular weight proteins,LMP2 and LMP7, which are polymorphic and mayserve to optimize delivery of the peptides to theTAP1/TAP2 (figure 5.7). Now within the lumen of theER, the peptides complex with the membrane-boundclass I MHC molecules thereby releasing peptide fromtheir association with the TAP transporter. Thence, thecomplex traverses the Golgi stack, presumably pick-ing up carbohydrate side-chains en route, and reachesthe surface where it is a sitting target for the cytotoxicT-cell. It is worth pointing out at this stage that the cy-tokine interferon g (IFNg), which is actively producedby T-cells during cell-mediated immune responses, in-creases the production of the proteasomal subunits

LMP2 and LMP7 and thereby amplifies antigen pro-cessing.

PROCESSING OF ANTIGEN FOR CLASS IIMHC PRESENTATION FOLLOWS ADIFFERENT PATHWAY

Class II MHC complexes with antigenic peptide aregenerated by a fundamentally different intracellularmechanism, since the APCs which interact with T-helper cells need to sample the antigen from both the extracellular and intracellular compartments. Inessence, a trans-Golgi vesicle containing class II has tointersect with a late endosome containing exogenousprotein antigen taken into the cell by an endocyticmechanism.

First let us consider the class II molecules them-selves. These are assembled from a and b chains in theER in association with the transmembrane invariantchain (figure 5.8), which has several functions. It actsas a dedicated chaperone to ensure correct folding of the nascent class II molecule and it inhibits the


19S

Cap

20S

Prot

easo

me

19S

Cap

Cytosolic protein

Polyubiquitin

Cleaved peptides

a a

bb

bb

a a

Figure 5.6 Cleavage of cytosolic proteinsby the proteasome. The whole 26S proteasecomplex consisting of the 20S proteasomewith twin 19S caps is displayed as a contourplot derived from electron microscopy and image analysis. The cross-section of theproteasome reveals the sites of peptidaseactivity (o) within the inner core of theseven-membered rings of b-subunits.(Based on J.-M. Peters et al. (1993) Journal ofMolecular Biology, 234, 932–7 and D.M.Rubin & D. Finley (1995) Current Biology, 5,854–8.)

Golgistack

TAP1 TAP2

b2m

Calnexin

Class IH chain

Release ofclass I/peptidecomplex from

associatedmolecules

Endogenousantigen

Class I/peptidecomplex

Antigen Presentation

Cell Surface

Transportthrough Golgi

ER

Transportinto ER and

class I/peptidecomplexformation

Immunoproteasomecleavage

Cytosol

Peptides

Proteinsynthesis

LMP2/LMP7/MECL-1

Figure 5.7 Processing and presentation of endogenous antigen byclass I major histocompatibility complex (MHC). Cytosolic pro-teins are degraded by the proteasome complex into peptides whichare transported into the endoplasmic reticulum (ER). There, theybind to membrane-bound class I MHC formed by b2-microglobulin(b2m)-induced dissociation of nascent class I heavy chains from theircalnexin chaperone. The peptide/MHC complex is now releasedfrom its association with the TAP transporter, traverses the Golgisystem, and appears on the cell surface ready for presentation to theT-cell receptor.

CLASS II MHC + INV. CHAIN

Antigen Presentation

Transport through Golgi

� CHAIN

� CHAIN

INVARIANT CHAIN

Fusion

�� DM

LATE ENDOSOME

Degradation

EARLYENDOSOME

Acidification

MIIC

Inv. chaindegradation

Peptideloading

CLIP

EXOGENOUS ANTIGEN

CELLSURFACE

CLASS II/PEPTIDE COMPLEX

ER

Figure 5.8 Processing and presentation of exogenous antigen byclass II major histocompatibility complex (MHC). Class II mole-cules with invariant chain are assembled in the endoplasmic reticu-lum (ER) and transported through the Golgi to the trans-Golgireticulum. There they are sorted to a late endosomal vesicle withlysosomal characteristics known as MIIC (meaning MHC class II en-riched compartment) containing partially degraded protein derivedfrom the endocytic uptake of exogenous antigen. Degradation of in-variant chains leaves the CLIP (class II-associated invariant chainpeptide) peptides lying in the groove but, under the influence of theMHC-related dimeric molecule (DM), they are replaced by otherpeptides in the vesicle including those derived from exogenous anti-gen, and the complexes are transported to the cell surface for presen-tation to T-helper cells.

precocious binding of peptides in the ER before theclass II reaches the endocytic compartment containingantigen. Additionally, combination of the invariantchain with the ab class II heterodimer inactivates a re-tention signal and allows transport to the Golgi.

Meanwhile, exogenous protein is taken up by endo-cytosis and is subjected to partial degradation as theearly endosome undergoes progressive acidification.The late endosome having many of the characteristicsof a lysosomal granule now fuses with the vacuole con-taining the class II-invariant chain complex. Under theacidic conditions within this MHC class II-enrichedcompartment the invariant chain is degraded and re-placed with other vesicular peptides, after which thecomplexes are transported to the membrane for pre-sentation to T-helper cells.

Processing of antigens for class II presentation is notconfined to soluble proteins taken up from the exteriorbut can also encompass proteins and peptides withinthe ER, and microorganisms whose antigens reach thelysosomal structures, either after direct phagocytosisor prolonged intracellular cohabitation. Conversely,class I-restricted responses can be generated againstexogenous antigens. This may occur either by a TAP-dependent pathway in phagocytic cells where pro-teins are transferred from the phagosome to thecytosol, or by endocytosis of cell surface MHC class Imolecules which then arrive in the class II-enrichedcompartments where peptide exchange occurs withsequences derived from the endocytic processingpathway.

Binding to MHC class I

The peptides binding along the length of the grooveare predominantly 8–9 residues long. Except in thecase of viral infection, the natural class I ligands will beself peptides derived from proteins endogenouslysynthesized by the cell, histones, heat-shock proteins,enzymes, leader signal sequences and so on. It turnsout that 75% or so of these peptides originate in the cytosol.

Binding to MHC class II

The open nature of the class II groove places no con-straint on the length of the peptide, which can danglenonchalantly from each end of the groove, quite unlikethe straitjacket of the class I ligand site (figure 5.9).Thus, each class II molecule binds a collection of pep-tides with a spectrum of lengths ranging from 8 to30 mers.

T-CELLS WITH A DIFFERENT OUTLOOK

The family of CD1 non-MHC class I-likemolecules can present exotic antigens

After MHC class I and class II, the CD1 family repre-sents a third lineage of antigen-presenting moleculesrecognized by T-lymphocytes. The CD1 polypeptidechain also associates with b2-microglobulin and theoverall structure is similar to that of classical class Imolecules. CD1 molecules present lipid and glycolipid


Peptide

Peptide

DRa chain

DRb chain

a2-Helix

a1-Helix

MHC Class I - bound peptide

MHC Class II - bound peptide

a

b

Figure 5.9 Binding of peptides to the major histocompatibilitycomplex (MHC) cleft. T-cell receptor “view” looking down on the a-helices lining the cleft represented in space-filling models. (a) Pep-tide 309–317 from HIV-1 reverse transcriptase bound tightly withinthe class I HLA-A2 cleft. (b) Influenza hemagglutinin 306–318 lyingin the class II HLA-DR1 cleft. In contrast with class I, the peptide extends out of both ends of the binding groove. (Based on D.A.A. Vignali & J.L. Strominger (1994) The Immunologist, 2, 112, with permission of the authors and publisher.)

microbial antigens, especially those derived from Mycobacterium tuberculosis to T-cells. Because antigenrecognition by CD1-restricted T-cells involves clonallydiverse ab and gd TCRs, it is likely that CD1 can presenta broad range of such antigens. Just like their proteina-ceous colleagues, exogenously derived lipid and gly-colipid antigens are delivered to the acidic endosomalcompartment. Antigens derived from endogenouspathogens can also be presented by the CD1 pathway,but in a process that, unlike class I-mediated presenta-tion, is independent of TAP.

Some T-cells have natural killer (NK) markers

Agroup of T-cells which possess various markers char-acteristic of NK cells, together with a TCR, are calledNK-T-cells or NK1.1+ T-cells. These cells are a majorcomponent of the T-cell compartment, accounting for20–30% of T-cells in bone marrow and liver, and up to1% of spleen cells. They are most abundant in the liver,and unique molecular and cellular mechanisms con-trol their migration and/or retention in this organ.

The NK-T-cells rapidly secrete high levels of inter-leukin-4 (IL-4) and IFNg following stimulation andtherefore have important regulatory functions. Al-though substantial numbers of NK-T-cells are CD1-restricted, others are restricted by classical MHC mole-cules indicating that there are subsets within thisgroup.

gd T-cell receptors have some features of antibody

Unlike ab T-cells, gd T-cells recognize antigens directlywithout a requirement for antigen processing. Whilstsome T-cells bearing a gd receptor are capable of recog-nizing MHC molecules, neither the polymorphicresidues associated with peptide binding nor the pep-tide itself are involved. Thus, a gd T-cell clone specificfor the herpes simplex virus glycoprotein-1 can bestimulated by the native protein bound to plastic, sug-gesting that the cells are triggered by cross-linking oftheir receptors by antigen, which they recognize in theintact native state just as antibodies do. The X-ray crys-tallographic structure of a TCR variable domain high-lights that the gd TCR indeed incorporates keystructural elements of both Ig and TCR V regions.

gd T-cells can be stimulated by a variety of antigensincluding heat-killed bacteria, bacterial extracts andboth peptide and non-peptide components of My-cobacterium tuberculosis. Stressed or damaged cells ap-pear to be powerful activators of gd cells, and there isevidence that molecules such as heat-shock proteins

can stimulate gd T-cells. Low molecular weight non-proteinaceous antigens, such as isopentenyl py-rophosphate and ethyl phosphate, which occur in arange of microbial and mammalian cells, have beenidentified as potent stimulators. In fact, responses of gdT-cells are evident in almost every infectious disease.There is now extensive evidence that these cells are important in terminating host immune responses. This is mediated by activated gd T-cells which acquirecytotoxic activity and kill stimulatory macrophages,leading to resolution of inflammatory immune responses and prevention of chronic inflammatorydisease.

The above characteristics provide the gd cells with adistinctive role complementary to that of the ab popu-lation and enable them to function in the recognition of microbial pathogens and damaged or stressed hostcells as well as regulating immune responses.

SUPERANTIGENS STIMULATE WHOLEFAMILIES OF LYMPHOCYTE RECEPTORS

Bacterial toxins represent one major group of T-cell superantigens

Individual peptides complexed to MHC will react onlywith antigen-specific T-cells, which represent a rela-tively small percentage of the T-cell pool. Moleculescalled superantigens stimulate the 5–20% of the total T-cell population, which express the same TCR Vb fami-ly structure irrespective of their antigen specificity.Superantigens include Staphylococcus aureus entero-toxins (staphylococcal enterotoxin A, SEA; staphylo-coccal enterotoxin B, SEB; and several others), whichare single-chain proteins responsible for food poison-ing. They are strongly mitogenic for T-cells expressingparticular Vb families in the presence of MHC class IIaccessory cells. Superantigens are not processed by theAPC, but cross-link the class II and Vb independentlyof direct interaction between MHC and TCR molecules(figure 5.10). Staphylococcal enterotoxin Ais one of themost potent T-cell mitogens known, causing markedT-cell proliferation with release of copious amounts of cytokines, including IL-2 and lymphotoxin. Super-antigens can also cause the release of mast cellleukotrienes, and this probably forms the basis of toxicshock syndrome. This is a multisystem febrile illnesscaused by staphylococci or Group A streptococci seenmore often in menstruating women using tampons. Itis due to bacterial toxins acting as superantigens, in-ducing sustained release of inflammatory mediatorsfrom T-cells, macrophages and mast cells.



Figure 5.10 Interaction of superantigen with major histocompati-bility complex (MHC) and T-cell receptor (TCR). In this compositemodel, the interaction with the superantigen staphylococcal entero-toxin B (SEB) involves SEB wedging itself between the TCR Vb chainand the MHC, effectively preventing interaction between the TCR

and the peptide in the groove, and between the TCR b chain and theMHC. Thus, direct contact between the TCR and the MHC is limitedto Va amino acid residues. (Reproduced from H. Li et al. (1999) Annual Review of Immunology 17, 435–66, with permission.)

REVISION

The nature of antigens• An antigen is defined by its antibody. The contact areawith an antibody is called an epitope and the correspond-ing area on an antibody is a paratope.• Antisera recognize a series of dominant epitope clusterson the surface of an antigen; each cluster is called a deter-minant.

Antigens and antibodies interact by spatial complementarity,not by covalent binding• The forces of interaction include electrostatic, hydro-gen-bonding, hydrophobic and van der Waals.• Antigen–antibody bonds are readily reversible.• Antigens and antibodies are mutually deformable.• The strength of binding to a single antibody combiningsite is measured by the affinity.• The reaction of multivalent antigens with the heteroge-

neous mixture of antibodies in an antiserum is defined byavidity (functional affinity).• Specificity of antibodies is not absolute and they maycross-react with other antigens to different extents measured by their relative avidities.

In vitro antigen–antibody reactions• Antibody can be detected in serum or other fluids by itsability to bind to specific antigens.• The level of antibodies in a solution is called the anti-body titer.

Numerous techniques are available for the estimation of antibody• Antibody in solution can be assayed by the formation offrank precipitates which can be enhanced by countercur-rent electrophoresis in gels.

(continued)


• Nonprecipitating antibodies can be measured by lasernephelometry.• Antibodies can also be detected by macroscopic agglutination of antigen-coated particles, and by enzyme-linked immunosorbent assays (ELISA), a two-stage proce-dure in which antibody bound to solid-phase antigen is detected by an enzyme-linked anti-immunoglobulin(anti-Ig).

Identification and measurement of antigen• Antigens can be quantified by their reaction in gels withantibody using single radial immunodiffusion.• Higher concentrations of antigens are frequently esti-mated by nephelometry.• Spotting multiple antigens or antibodies onto glassslides or chips can miniaturize immunoassays. This willallow multiple analyte screening in a single test.

T-cell recognition• ab T-cells see antigen in association with major histo-compatibility complex (MHC) molecules.• They are restricted to the haplotype of the cell that firstprimed the T-cell.• Protein antigens are processed by antigen-presentingcells (APCs) to form small linear peptides which associatewith the MHC molecules, binding to the central grooveformed by the a-helices and the b-sheet floor.

Processing of antigen for presentation by class I MHC• Endogenous cytosolic antigens such as viral proteinslink to ubiquitin and are cleaved by proteasomes. The pep-tides so-formed are transported to the endoplasmic reticu-lum (ER) by the TAP1/TAP2 system.• The peptide then dissociates and forms a stable het-erotrimer with newly synthesized class I MHC heavychain and b2-microglobulin.• This peptide–MHC complex is then transported to thesurface for presentation to cytotoxic T-cells.

Processing of antigen for presentation by class II MHC• The ab class II molecule is synthesized in the ER andcomplexes with membrane-bound invariant chain.• This facilitates transport of the vesicles containing classII across the Golgi and directs it to the late endosomal com-partment.

• The antigen is degraded to peptides which bind to classII now free of invariant chain.• The class II-peptide complex now appears on the cellsurface for presentation to T-helper cells.

The nature of the peptide• Class I peptides are held in extended conformationwithin the MHC groove.• They are usually 8–9 residues in length.• Class II peptides are between 8 and 30 residues long andextend beyond the groove.

T-cells with a different outlook• CD1 molecules present lipid and glycolipid antigens toT-cells.• Both exogenous and endogenous lipids can be pres-ented by CD1.• Agroup of T-cells, which in addition to a T-cell receptor(TCR) also have natural killer (NK) cell markers, are calledNK-T-cells.• NK-T-cells produce cytokines such as interleukin-4 (IL-4) and interferon g (IFNg) which are important in regulat-ing immune responses.

gd T-cells• This subpopulation of T-cells can recognize antigen inits native, unprocessed state.• These cells are activated by stressed or damaged cellsand by numerous bacteria• gd T-cells play an important regulatory role in terminat-ing immune responses by killing activated macrophages.

Superantigens• These are potent mitogens that stimulate whole T-cellsubpopulations sharing the same TCR Vb family indepen-dently of antigen specificity.• Staphylococcus aureus enterotoxins are powerful humansuperantigens that cause food poisoning and toxic shocksyndrome.• They are not processed but cross-link MHC class II andTCR Vb independently of their direct interaction.


FURTHER READING

Carding, S.R., & Egan, P.J. (2000) The importance of gd T cells in theresolution of pathogen-induced inflammatory immune response.Immunological Reviews, 173, 98–108.

Eisen, H.N. (2001) Specificity and degeneracy in antigen recogni-tion: Yin and yang in the immune system. Annual Review of Im-munology, 19, 1–21.

van den Elsen, P.J. & Rudensky, A. (eds) (2004) Section on antigen

processing and recognition. Current Opinion in Immunology, 16,63–125.

Hennecke, J. & Wiley, D.C. (2001) T cell receptor–MHC interactionsup close. Cell, 104, 1–4.

Moss, D.J. & Khanna, R. (1999) Major histocompatibility complex:From genes to function. Immunology Today, 20, 165–7.

Van den Eynde, B.J. & Morel, S. (2001) Differential processing of classI-restricted epitopes by the standard proteasome and the im-munoproteasome. Current Opinion in Immunology, 13, 147–53.


62

THE SURFACE MARKERS OF CELLS IN THEIMMUNE SYSTEM

In order to discuss the events that occur in the opera-tion of the immune system as a whole, it is imperativeto establish a nomenclature which identifies the sur-face markers on the cells involved. These are usuallyfunctional molecules reflecting the state of cellular dif-ferentiation. The nomenclature system is establishedas follows. Immunologists from the far corners of theworld who have produced monoclonal antibodies di-rected to surface molecules on cells involved in the im-mune response get together every so often to comparethe specificities of their reagents in international work-shops. Where a cluster of monoclonals are found toreact with the same polypeptide, they clearly representa series of reagents defining a given marker and welabel it with a CD (cluster of differentiation) number.There are now over 250 CD specificities assigned andsome of them are shown in table 6.1.

Detection of surface markers

Because fluorescent dyes such as fluorescein and rho-damine can be coupled to antibodies without destroy-ing their specificity, the conjugates can combine withantigen present in a tissue section and be visualized inthe fluorescence microscope (figure 6.1a). In this waythe distribution of antigen throughout a tissue andwithin cells can be demonstrated.

In place of fluorescent markers, other workers haveevolved methods in which enzymes such as alkalinephosphatase or horseradish peroxidase are coupled

to antibodies and these can be visualized by conven-tional histochemical methods at the level of both the light microscope (figure 6.1b) and the electron microscope.

When enumerating cells in suspensions such as inblood or other fluids, surface antigens can be detectedby the use of labeled antibodies. It is possible to stainsingle cells with several different fluorochromes andanalyse the cells in individual droplets as they flowpast a laser beam in a flow cytometer. The laser light ex-cites the fluorochrome and the intensity of the fluores-cence is measured by detectors (fig 6.2). With theimpressive number of monoclonal antibodies to hand,highly detailed phenotypic analysis of single-cell pop-ulations is now a practical proposition. Of the ever-growing number of applications, the contribution to diagnosis and classification of leukemia and lym-phoma is quite notable.

THE NEED FOR ORGANIZED LYMPHOID TISSUE

For an effective immune response an intricate series of cellular events must occur. Antigen must bind and if necessary be processed by antigen-presenting cells(APCs), which must then make contact with and acti-vate T- and B-cells. In addition T-helper cells must as-sist certain B-cells and cytotoxic T-cell precursors toperform their functions, which include amplificationof the numbers of potential effector cells by prolifera-tion and the generation of the mediators of humoraland cellular immunity. In addition, memory cells forsecondary responses must be formed and the whole

CHAPTER 6

The anatomy of the immune response

The surface markers of cells in the immune system, 62Detection of surface markers, 62

The need for organized lymphoid t issue, 62Lymphocytes traff ic between lymphoid

t issues, 63Lymphocytes home to their specific tissues, 63Transmigration, 65

Encapsulated lymph nodes, 66B-cell areas, 66T-cell areas, 68

Spleen, 68Mucosa-associated lymphoid t issue (MALT), 69The enjoyment of privileged sites, 70The handling of antigen, 71

Macrophages are general APCs, 71Dendritic cells (DCs) are professional APCs, 71Interdigitating DCs present antigen to T-lymphocytes, 71Follicular dendritic cells (FDCs) stimulate B-cells in germinal

centers, 71M-cells provide the gateway to the mucosal lymphoid

system, 72

CHAPTER 6—The anatomy of the immune response 63

B, Mo, FDC

*T, *B, *Mo, *Mf

T, *B

Progenitors

B, Mf, IDC, FDC

Mo, Mf, DC

B

CD Expression Functions

CD1

CD2

CD3

CD4

CD5

CD8

CD14

CD16

CD19

CD20

CD21

IDC, B subset

T, NK

T

T-helper, Mo, Mf

T, B subset

T-cytotoxic

G, Mo, Mf

G, NK, B, Mf, IDC

B, FDC

B

B, FDC

Mo, Mf, IDC,FDC, G, NK, B,

Resting/Naive T-cells, B, G, Mo, NK

Activated/MemoryT-cells, Mo, DC

*B, *T, Mf, DC

Widespread

Presents glycolipid and other non-peptide antigens to T-cells

Receptor for CD58 (LFA-3)costimulator. Binds sheep rbc

Transducing elements of T-cell receptor

MHC class II. HIV receptor

Involved in antigen receptor signaling

MHC class I receptor

LPS/LBP complex receptor

FcgRIII (medium affinity IgG receptor)

Part of B-cell antigen receptor complex

Unknown, but able to provideintracellular signals

CR2. Receptor for C3d and Epstein–Barr virus. Part of B-cell antigenreceptor complex

FceRII (low affinity IgE receptor)

IL-2 receptor a chain

Receptor for CD80/CD86 (B7.1 andB7.2) costimulators

FcgRII (low affinity IgG receptor)

Adhesion molecule. Stem cell marker

Receptor for CD40L costimulator

Phosphatase, cell activation

Phosphatase, cell activation

FcgRI (high affinity IgG receptor)

Transducing elements of B-cell receptor

B7.1 receptor for CD28 costimulatorand for CTLA4 inhibitory signal

B7.2 receptor for CD28 costimulatorand for CTLA4 inhibitory signal

Fas. Receptor for FasL. Transmitsapoptotic signals

CD23

CD25

CD28

CD32

CD34

CD40

CD45RA

CD45RO

CD64

CD79a/CD79b

CD80

CD86

CD95

B, IDC, Mo

Table 6.1 Some of the major clusters of differentiation (CD) mark-ers on human cells.

response controlled so that it is adequate but not exces-sive and is appropriate to the type of infection beingdealt with. The integration of the complex cellular interactions which form the basis of the immune re-sponse takes place within the organized architecture ofperipheral, or secondary, lymphoid tissue, which in-cludes the lymph nodes, spleen and unencapsulatedtissue lining the respiratory, gastrointestinal and geni-tourinary tracts.

These tissues become populated by cells of reticularorigin and by macrophages and lymphocytes derivedfrom bone marrow stem cells; the T-cells first dif-ferentiating into immunocompetent cells by a high-intensity training period in the thymus, the B-cells undergoing their education in the bone marrow itself(figure 6.3). In essence, the lymph nodes filter off and, ifnecessary, respond to foreign material draining bodytissues, the spleen monitors the blood, and the unen-capsulated lymphoid tissue is strategically integratedinto mucosal surfaces of the body as a forward defen-sive system based on immunoglobulin A (IgA) secre-tion. The bone marrow also contributes substantiallyto antibody production.

Communication between these tissues and the restof the body is maintained by a pool of recirculatinglymphocytes which pass from the blood into thelymph nodes, spleen and other tissues and back to theblood by the major lymphatic channels such as the tho-racic duct (figure 6.4).

LYMPHOCYTES TRAFFIC BETWEENLYMPHOID TISSUES

This traffic of lymphocytes between the tissues, thebloodstream and the lymph nodes enables antigen-sensitive cells to seek the antigen and to be recruited to sites at which a response is occurring, while the dissemination of memory cells enables a more wide-spread response to be organized throughout the lym-phoid system. Thus, antigen-reactive cells aredepleted from the circulating pool of lymphocyteswithin 24h of antigen first localizing in the lymphnodes or spleen; several days later, after proliferationat the site of antigen localization, a peak of activatedcells appears in the thoracic duct.

Lymphocytes home to their specific tissues

Naive lymphocytes enter a lymph node through the af-ferent lymphatics and by guided passage across thespecialized high-walled endothelium of the postcap-illary venules (HEVs) (figure 6.5). Comparable HEVsoffer the transit of cells concerned with mucosal immu-

*, activated; B, B-lymphocytes; FDC, follicular dendritic cells; G,granulocytes; IDC, interdigitating dendritic cells; Mf, macrophages;Mo, monocytes; NK, natural killer cells; T, T-lymphocytes.

PART 3—The acquired immune response64

nity to Peyer’s patches. In other cases involving migra-tion into normal and inflamed tissues, the lym-phocytes bind to and cross nonspecialized flatterendothelia in response to locally produced mediators.

This highly organized traffic is orchestrated by di-

recting the relevant lymphocytes to different parts ofthe lymphoid system and the various other tissues by aseries of homing receptors. These include members ofthe integrin superfamily and also a member of the se-lectin family, L-selectin. These homing receptors rec-ognize their complementary ligands, termed vascularaddressins, on the surface of the appropriate endothe-lial cells of the blood vessels. Endothelium expressingthese addressins acts as a selective gateway which al-lows lymphocytes access to the appropriate tissue.Chemokines presented by vascular endothelium play a key role in triggering lymphocyte arrest, the chemokine receptors on the lymphocyte being involved in the functional upregulation of integrin-homing receptors.

Figure 6.1 Staining of gastric parietal cells by (a) fluo-rescein (white cells) and (b) peroxidase-linked anti-body (black cells). The sections were sequentiallytreated with human parietal cell autoantibodies andthen with the conjugated rabbit anti-human im-munoglobulin G (IgG). The enzyme was visualized bythe peroxidase reaction. (Courtesy of Miss V. Petts.)

100 101 102 103 104100

101

102

103

104

Staining of the V�6 T-cell family

Anti-CD3 fluorescence

Anti-

V�6

fluor

esce

nce

0.3222.6

3.5473.5

SECONDARYLYMPHOID ORGAN

Encapsulated Unencapsulated

MALT

To antigens atmucosal surfaces

To antigensin blood

To antigensin tissues

IMMUNERESPONSE

SpleenLymph node

THYMUS BONE MARROWPRIMARY

LYMPHOID ORGAN

EDUCATION T SC B

Figure 6.2 Cytofluorometric analysis of human peripheral bloodlymphocytes. Cells stained with fluoresceinated anti-T-cell receptor(TCR) Vb6 and phycoerythrin conjugated anti-CD3. Each dot repre-sents an individual lymphocyte and the numbers refer to the per-centage of lymphocytes lying within the four quadrants formed bythe two gating levels arbitrarily used to segregate positive from neg-ative values. Virtually no lymphocytes bearing the TCRs belongingto the Vb6 family lack CD3, while 4.6% (3.5 out of 77.0) of the matureT-cells express Vb6. (Data kindly provided by D. Morrison.)

Figure 6.3 The functional organization of lymphoid tissue. Stemcells (SC) arising in the bone marrow differentiate into immunocom-petent T- and B-cells in the primary lymphoid organs and then colo-nize the secondary lymphoid tissues where immune responses areorganized. The mucosa-associated lymphoid tissue (MALT) pro-duces the antibodies for mucosal secretions.

(a) (b)


Transmigration

Lymphocytes normally travel in the fast lane down thecentre of the blood vessels. However, for the lympho-cyte to become attached to the endothelial cell it has toovercome the shear forces that this creates. This is ef-fected by a force of attraction between homing recep-tors on the lymphocytes and their ligands on the vesselwall. After this tethering process, the lymphocyte rolls

along the endothelial cell, partly through the selectininteractions but now increasingly through the bindingof various integrins to their respective ligands.

This process leads to activation and recruitment ofmembers of the b2-integrin family to the surface of thelymphocyte. In particular, molecules such as LFA-1(lymphocyte function-associated antigen-1) will be-come activated and will bind very strongly to ICAM-1and 2 (intercellular adhesion molecule-1 and 2) on the

SPLEEN

BLOOD

LYMPHATIC

INFLAMEDTISSUE

HEV HEV

LYMPHNODE

EFFERENT

THO

RACIC DUCT

AFFERENTFigure 6.4 Traffic and recirculation of lym-

phocytes through encapsulated lymphoidtissue and sites of inflammation. Blood-borne lymphocytes enter the tissues andlymph nodes passing through the high-walled endothelium of the postcapillaryvenules (HEV) and leave via the draininglymphatics. The efferent lymphatics, finallyemerging from the last node in each chain,join to form the thoracic duct, which returnsthe lymphocytes to the bloodstream. In thespleen, lymphocytes enter the lymphoidarea (white pulp) from the arterioles, pass tothe sinusoids of the erythroid area (redpulp) and leave by the splenic vein. Trafficthrough the mucosal immune system iselaborated in figure 6.10.

Figure 6.5 Lymphocyte association with postcapillary venules. (a)High-walled endothelial cells (HEC) of postcapillary venules in ratcervical lymph nodes showing intimate association with lympho-cytes (Ly). (b) Flattened capillary endothelial cell (EC) for compari-

son. (c) Lymphocytes adhering to HEC (scanning electron micro-graph). ((a) and (b) kindly provided by Professor Ann Ager and (c)by Dr W. van Ewijk.)

(a) (b)

(c)


STEP 1

Tethering

STEP 2

Rolling

STEP 3

LFA-1 activation

STEP 4

Flattening

STEP 5

Diapedesis

Fast flow(~4000 mm/s)

Shear force

Slow flow(rolling at~40 mm/s)

Microvillus

Lymphocyte

Chemotacticgradient

Blood vessel

Endothelial vessel wall

1

2 3 4 5

Figure 6.6 Homing and transmigration of lymphocytes. Fast-moving lymphocytes are tethered (Step 1) to the vessel walls of thetissue they are being guided to enter through an interaction betweenspecific homing receptors and their ligands. After rolling along the

surface of the endothelial cells making up the vessel wall, activationof b2 integrins occurs (Step 3), which leads to firm binding, cell flat-tening and (Step 5) migration of the lymphocyte between adjacentendothelial cells.

endothelial cell. This intimate contact will cause thelymphocyte to flatten (figure 6.6, step 4) and it maynow elbow its way between the endothelial cells andinto the tissue (figure 6.6, step 5). Similar homingmechanisms enable lymphocytes bearing receptors formucosa-associated lymphoid tissue (MALT) to circu-late within and between the collections of lymphoidtissue guarding the external body surfaces (see figure6.10).

ENCAPSULATED LYMPH NODES

The encapsulated tissue of the lymph node contains ameshwork of reticular cells and their fibers organizedinto sinuses. These act as a filter for lymph draining thebody tissues and possibly bearing foreign antigens;this lymph enters the subcapsular sinus by the afferentvessels and diffuses past the lymphocytes in the cortexto reach the macrophages of the medullary sinuses(figure 6.7a,c) and thence the efferent lymphatics.What is so striking about the organization of the lymph node is that the T- and B-lymphocytes are verylargely separated into different anatomical com-partments.

B-cell areas

The follicular aggregations of B-lymphocytes are aprominent feature of the outer cortex. In unstimulated

nodes they are present as spherical collections of cellstermed primary follicles (figure 6.7f), which are com-posed of a mesh of follicular dendritic cells (FDCs).Spaces within this meshwork are filled with recirculat-ing but resting small B-lymphocytes. After antigenicchallenge they form secondary follicles which consistof a corona or mantle of concentrically packed, resting,small B-lymphocytes possessing both IgM and IgD ontheir surface surrounding a pale-staining germinalcenter (figure 6.7b,c). This contains large, usually pro-liferating, B-blasts and a tight network of specializedFDCs. Germinal centers are greatly enlarged in sec-ondary antibody responses (figure 6.7d), and they areregarded as important sites of B-cell maturation andthe generation of B-cell memory.

A proportion of the B-cells that are shunted downthe memory cell pathway take up residence in themantle zone population, the remainder joining the re-circulating B-cell pool. Other cells differentiate intoplasmablasts, with a well-defined endoplasmic reticu-lum, prominent Golgi apparatus and cytoplasmic Ig;these migrate to become plasma cells in the medullarycords of lymphoid cells which project between themedullary sinuses. This maturation of antibody-forming cells at a site distant from that at which anti-gen triggering has occurred is also seen in the spleen,where plasma cells are found predominantly in themarginal zone. The remainder of the outer cortex isalso essentially a B-cell area with scattered T-cells.

M.S.

M.C.

LYMPH FLOWAFFERENT

LYMPHATICS VALVESUBCAPSULAR

MARGINAL SINUSCONNECTIVE

TISSUE CAPSULE

HILUM L. NODEVEIN

L. NODEARTERY

SECONDARYFOLLICLE WITHMANTLE OFSMALLB-LYMPHOCYTESAND GERMINALCENTER

OUTER CORTEX(B-CELL AREA)

PARACORTICAL(T-CELL) AREA

M.C. MEDULLARY CORDS M.S. MEDULLARY SINUSES

a

SECONDARY B-BLASTS

CENTROCYTES

CENTROBLASTS

FDC

FDC

FDC

bPRIMARY B-BLASTS

MEMORY B-CELLSPLASMA CELLS

MANTLEZONE

DARKZONE

BASALLIGHTZONE

APICALLIGHTZONE

Mf

(c)

(d) (e) (f)

Figure 6.7 Lymph node. (a) Diagrammatic representation of sectionthrough a whole node. (b) Diagram showing differentiation of B-cells during passage through different regions of an active germinalcenter. ¥, apoptotic B-cell; FDC, follicular dendritic cell; Mf,macrophage. (c) Human lymph node, low-power view. GC, germi-nal center; LM, lymphocyte mantle of SF; MC, medullary cords; MS,medullary sinus; PA, paracortical area; SF, secondary follicle; SS,subcapsular sinus. (d) Secondary lymphoid follicle showing germi-nal center in a mouse immunized with the thymus-independentantigen, pneumococcus polysaccharide SIII, revealing prominentstimulation of secondary follicles with germinal centers. GC, germi-

nal center; LM, lymphocyte mantle of secondary follicle. (e) MethylGreen/Pyronin stain of lymph node draining site of skin paintedwith the contact sensitizer oxazolone, highlighting the generalizedexpansion and activation of the paracortical T-cells, the T-blastsbeing strongly basophilic. PA, paracortical area. (f) The same studyin a neonatally thymectomized mouse shows a lonely primary nod-ule (follicle) with complete lack of cellular response in the paracorti-cal area. PA, paracortical area; PN, primary nodule. ((c) Courtesy ofProfessor P.M. Lydyard and (d–f) courtesy of Dr M. de Sousa andProfessor D.M.V. Parrott.)


T-cell areas

T-cells are mainly confined to a region referred to as theparacortical (or thymus-dependent) area (figure 6.7a);in nodes taken from children with selective T-cell defi-ciency (cf. figure 13.4) or neonatally thymectomizedmice the paracortical region is seen to be virtually de-void of lymphocytes (figure 6.7f). Furthermore, whena T-cell-mediated response is elicited, say by a skingraft or by exposure to poison ivy which induces con-tact hypersensitivity, there is a marked proliferation ofcells in the thymus-dependent area and typical lym-phoblasts are evident (figure 6.7e). In contrast, stimu-

lation of antibody formation by thymus-independentantigens leads to proliferation in the cortical lymphoidfollicles with development of germinal centers, whilethe paracortical region remains inactive (figure 6.7d).As expected, nodes taken from children with congeni-tal hypogammaglobulinemia associated with failureof B-cell development conspicuously lack primary andsecondary follicles.

SPLEEN

On a fresh section of spleen, the lymphoid tissue form-ing the white pulp is seen as circular or elongated gray

SPLEEN MANTLEMARGINAL ZONE(B-CELL AREA)

T-CELL AREA

SPLENIC CORDS

VENOUS SINUSOIDS

ARTERY

VEIN

SPLENIC ARTERIOLE

SECONDARYFOLLICLE

MZ

PALS

AGCM

RP

Figure 6.8 Spleen. (a) Diagrammatic representation. (b) Low-power view showing lymphoid white pulp (WP) and red pulp (RP).(c) High-power view of germinal center (GC) and lymphocyte man-tle (M) surrounded by marginal zone (MZ) and red pulp (RP). Adja-cent to the follicle, an arteriole (A) is surrounded by the periarteriolar

lymphoid sheath (PALS) predominantly consisting of T-cells. Notethat the marginal zone is only present above the secondary follicle.((b) Photographs by Professor P.M. Lydyard and (c) by Professor N.Milicevic.)

(b) (c)

(a)


areas (figure 6.8b,c) within the erythrocyte-filled red pulp, which consists of splenic cords lined withmacrophages and venous sinusoids. As in the lymphnode, T- and B-cell areas are segregated (figure 6.8a).The spleen is a very effective blood filter, removing ef-fete red and white cells and responding actively toblood-borne antigens, the more so if they are particu-late. Plasmablasts and mature plasma cells are presentin the marginal zone extending into the red pulp (figure 6.8c).

MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT)

The respiratory, gastrointestinal and genitourinarytracts are guarded immunologically by subepithelialaccumulations of lymphoid tissue which are not con-strained by a connective tissue capsule (figure 6.9).These may occur as diffuse collections of lymphocytes,plasma cells and phagocytes throughout the lung andthe lamina propria of the intestinal wall (figure 6.9a,b)

LY

LY

LP PC

ME

lgA

SF

PP

(a) (b)

(c) (d)

Figure 6.9 The immunoglobulin A (IgA) secretory immune sys-tem (mucosa-associated lymphoid tissue, MALT). (a) Section oflung showing a diffuse accumulation of lymphocytes (LY) in thebronchial wall. (b) Section of human jejunum showing lymphoidcells (LY) stained green by a fluorescent anti-leukocyte monoclonalantibody, in the mucosal epithelium (ME) and in the lamina propria(LP). A red fluorescent anti-IgA conjugate stains the cytoplasm ofplasma cells (PC) in the lamina propria and detects IgAin the surface

mucus, altogether a super picture! (c) Low-power view of humantonsil showing the MALT with numerous secondary follicles (SF)containing germinal centers. (d) Peyer’s patches (PP) in mouseileum. The T-cell areas are stained brown by a peroxidase-labeledmonoclonal antibody to Thy 1. ((a) Kindly provided by ProfessorP.M. Lydyard, (b) by Professor G. Jannosy, (c) by Mr C. Symes and (d)by Dr E. Andrew.)


or as more clearly organized tissue with well-formedfollicles. The latter includes tonsils (figure 6.9c), thesmall intestinal Peyer’s patches (figure 6.9d) and theappendix. Mucosa-associated lymphoid tissue forms a separate interconnected secretory system withinwhich cells committed to IgA or IgE synthesis may circulate.

In the gut, antigen enters the Peyer’s patches (figure6.9d) across specialized epithelial cells called M-cells(cf. figure 6.12) and stimulates the antigen-sensitivelymphocytes. After activation these drain into thelymph and, after a journey through the mesentericlymph nodes and the thoracic duct, pass from thebloodstream into the lamina propria (figure 6.10). Herethey become IgA-forming cells which protect a widearea of the bowel with protective antibody. The cellsalso appear in the lymphoid tissue of the lung and inother mucosal sites guided by the interactions of specific homing receptors with appropriate HEV addressins as discussed earlier.

Intestinal lymphocytes

The intestinal lamina propria is home to a predomi-nantly activated T-cell population. These T-cells bear aphenotype comparable to that of peripheral blood

lymphocytes: viz., > 95% T-cell receptor (TCR) ab anda CD4 : CD8 ratio of 7 : 3. There is also a generoussprinkling of activated B-blasts and plasma cells se-creting IgA for transport by the poly-Ig receptor to theintestinal lumen. Intestinal intraepithelial lympho-cytes, however, are different. They are also mostly T-cells but 10–40% are TCR gd+ cells. This relatively highproportion of TCR gd cells is unusual.

THE ENJOYMENT OF PRIVILEGED SITES

Certain selected parts of the body, brain, anteriorchamber of the eye and testis have been designatedprivileged immunologic sites, in the sense that anti-gens located within them do not provoke reactionsagainst themselves. It has long been known, for exam-ple, that foreign corneal grafts are not usually rejectedeven without immunosuppressive therapy.

Generally speaking, privileged sites are protectedby rather strong blood–tissue barriers and low perme-ability to hydrophilic compounds. Functionally in-significant levels of complement reduce the threat ofacute inflammatory reactions and unusually high concentrations of immunomodulators, such as inter-leukin-10 (IL-10) and transforming growth factor-b(TGFb) endow macrophages with an immunosup-

IgA IgA IgA

LACTATINGMAMMARY

GLAND

GENITO-URINARYTRACT

LUNG

IgA IgA SMALL INTESTINE

BLOODMESENTERIC

LYMPHNODE

THO

RACIC DUCT

PEYER’SPATCHLAMINA

PROPRIA

SALIVARY ANDLACRYMALGLANDS

Ag

Figure 6.10 Circulation of lymphocyteswithin the mucosal-associated lymphoidsystem. Antigen-stimulated cells movefrom Peyer’s patches (and probably lungand maybe all the mucosal member sites) tocolonize the lamina propria and the othermucosal surfaces ( ).


pressive capacity. It has also been shown that tissues inimmune privileged sites may constitutively expressFasL (Fas-ligand), which by attaching to Fas on infiltrating leukocytes could result in their death byapoptosis. However, inflammatory reactions at theblood–tissue barrier can open the gates to invasion by immunologic marauders —witness the inability ofcorneal grafts to take in the face of a local pre-existinginflammation.

THE HANDLING OF ANTIGEN

Where does antigen go when it enters the body? If itpenetrates the tissues, it will tend to finish up in thedraining lymph nodes. Antigens that are encounteredin the upper respiratory tract or intestine are trappedby local MALT, whereas antigens in the blood provokea reaction in the spleen.

Macrophages are general APCs

Antigens draining into lymphoid tissue are taken upby macrophages. They are then partially, if not com-pletely, broken down in the lysosomes; some may es-cape from the cell in a soluble form to be taken up byother APCs and a fraction may reappear at the surface,as a processed peptide associated with class II majorhistocompatibility molecules. Some antigens, such as polymeric carbohydrates, cannot be degraded be-cause the macrophages lack the enzymes required; inthese instances specialized macrophages in the mar-ginal zone of the spleen or the lymph node subcapsularsinus trap and present the antigen to B-cells directly,apparently without any processing or interventionfrom T-cells.

Dendritic cells (DCs) are professional APCs

Although macrophages are important APCs there isone function where they are seemingly deficient,namely the priming of naive lymphocytes. This func-tion is performed by DCs of which there are at least twopopulations. The first is derived from myeloid cellsand includes Langerhans’ cells in the skin and inter-digitating DCs (IDCs) found in all other tissues. Thesecond consist of plasmacytoid DCs that, on exposureto viruses, secrete large amounts of type 1 interferon, acytokine with potent antiviral activity. In peripheraltissues DCs are in an immature form and express a variety of pattern recognition receptors (PRRs), in-cluding Toll-like receptors (TLRs), that can recognizemolecular patterns expressed by pathogens. Once acti-vated by contact with microbial products or by proin-

flammatory cytokines such as tumor necrosis factor(TNF), interferon g (IFNg) or IL-1 they undergo matu-ration into competent APCs, bearing high levels ofmajor histocompatibility complex (MHC), costimula-tory and adhesion molecules.

In addition to their antigen-presenting function,DCs are important producers of chemokines, especi-ally T-cell attracting chemokines. Once these cells get together, there are a number of receptor-ligand interactions involved in DC activation of T-cells aside from MHC–peptide recognition by the TCR.These include B7–CD28 and CD40–CD40L (CD40-ligand).

Interdigitating DCs present antigen to T-lymphocytes

The scenario for T-cell priming appears to be as fol-lows. Peripheral immature DCs such as the Langer-hans’ cells can pick up and process antigen. As theirmaturation proceeds, they upregulate expression ofthe chemokine receptor CCR7 and travel as “veiled”cells in the lymph to the paracortical T-cell zone of thedraining lymph node. There, its maturation complete,the DC delivers the antigen with costimulatory signalsto naive specific T-cells which take advantage of thelarge surface area to bind to the MHC–peptide com-plex on the membrane of the DC (figure 6.11). Sites ofchronic T-cell inflammation seem to attract these cells,since abnormally high numbers are found closely ad-hering to activated T-lymphocytes in synovial tissuefrom patients with ongoing rheumatoid arthritis andin the glands of subjects with chronic autoimmune thy-roiditis lesions.

Follicular dendritic cells (FDCs) stimulate B-cells in germinal centers

Another type of cell with dendritic morphology, butthis time of mesenchymal origin, is the FDC. These arenonphagocytic and lack lysosomes but have very elon-gated processes which can make contact with numer-ous lymphocytes present in the germinal centers ofsecondary follicles. Their surface receptors for IgG Fcand for C3b enable them to trap complexed antigenvery efficiently and hold the antigen on their surfacefor extended periods, in keeping with the memoryfunction of secondary follicles. This would explainhow secondary antibody responses can be boosted byquite small amounts of immunogen. These complexwith circulating antibody and fix C3 so that they local-ize very effectively on the surface of the FDCs withinthe germinal centers of secondary follicles.


M-cells provide the gateway to the mucosallymphoid system

The mucosal surface is in the front line facing a veryunfriendly sea of microbes and, for the most part, anti-gens are excluded by the epithelium with its tight junc-tions and mucous layer. Gut lymphoid tissue, such asPeyer’s patches, is separated from the lumen by a sin-

gle layer of columnar epithelium interspersed with M-cells; these are specialized antigen-transporting cells.They overlay intraepithelial cells and macrophages(figure 6.12). A diverse array of foreign material in-cluding bacteria is taken up by M-cells (figure 6.12) andpassed on to the underlying APCs which, in turn, migrate to the local lymphoid tissue to stir the appro-priate lymphocytes into action.

Antigenuptake Nonlymphoid tissue

LymphBlood

Lymphoid tissueMHC/peptide

TCR

B7 CD28

Antigen presentation

NaiveT-cell

ActivatedT-cell

IDC precursor

Bonemarrowstem cell

Veiled cell

Immature IDC

Mature IDC

Figure 6.11 Migration and maturation ofinterdigitating dendritic cells (IDCs). Theprecursors of the IDCs are derived frombone marrow stem cells. They travel via theblood to nonlymphoid tissues. These im-mature IDCs, e.g. Langerhans’ cells in skin,are specialized for antigen uptake. Subse-quently they travel via the afferent lym-phatics as veiled cells to take up residencewithin secondary lymphoid tissues wherethey express high levels of major histocom-patibility complex (MHC) class II and co-stimulatory molecules such as B7. Thesecells are highly specialized for the activa-tion of naive T-cells.

ANTIGEN

M

EE L L

L

Mf Mf

a b c

Figure 6.12 M-cell within Peyer’s patch epithelium. (a) Scanningelectron micrograph of the surface of the Peyer’s patch epithelium.The antigen-sampling M-cell in the center is surrounded by absorp-tive enterocytes covered by closely packed, regular microvilli. Notethe irregular and short microfolds of the M-cell. (b) After uptake andtranscellular transport by the M-cell (M), antigen is processed bymacrophages and thence by dendritic cells, which present antigen toT-cells in Peyer’s patches and mesenteric lymph nodes. E, entero-cyte; L, lymphocyte; Mf, macrophage. (c) Electron photomicro-

graph of an M-cell (M in nucleus) with adjacent lymphocyte (Lin nu-cleus). Note the flanking epithelial cells are both absorptive entero-cytes with a typical brush border. (Lead citrate and uranyl acetate,¥ 1600.) ((a) Reproduced with permission of the authors and pub-lishers from T. Kato & R.L. Owen (1999) in R. Ogra et al. (eds) MucosalImmunology, 2nd edn. Academic Press, San Diego, pp. 115–32; (b)Based on T. Sminia & G. Kraal (1998) in P.J. Delves & I.M. Roitt (eds)Encyclopedia of Immunology, 2nd edn. Academic Press, London, p. 188.)


REVISION

The surface markers of cells in the immune system• Individual surface molecules are assigned a cluster ofdifferentiation (CD) number defined by a cluster of mono-clonal antibodies reacting with that molecule.• Antigens can be localized if stained by fluorescent anti-bodies and viewed in an appropriate microscope.• Antibodies can be labeled by enzymes for histochemicaldefinition of antigens.• Cells in suspension can be labeled with fluorescent anti-bodies and analysed in a flow cytometer.

Organized lymphoid tissue• The complexity of immune responses is catered for by asophisticated structural organization.• Lymph nodes filter and screen lymph flowing from thebody tissues while spleen filters the blood.• B- and T-cell areas are separated. B-cell structures ap-pear in the lymph node cortex as primary follicles whichbecome secondary follicles with germinal centers afterantigen stimulation.• Germinal centers with their meshwork of follicular den-dritic cells (FDCs) expand B-cell blasts produced by sec-ondary antigen challenge and direct their differentiationinto memory cells and antibody-forming plasma cells.• A pool of recirculating lymphocytes moves from theblood into lymphoid tissue and back again via the majorlymphatic channels.

Lymphocyte traffic• Lymphocyte recirculation between the blood and lym-phoid tissues is guided by specialized homing receptorson the surface of the high-walled endothelium of the post-capillary venules (HEV).• Lymphocytes are tethered and then roll along the sur-face of the selected endothelial cells through interactionsbetween selectins and integrins and their respective lig-ands. Flattening of the lymphocyte and transmigrationacross the endothelial cell follow LFA-1 activation.• Entry of memory T-cells into sites of inflammation is facilitated by upregulation of integrin molecules on thelymphocyte and corresponding binding ligands on thevascular endothelium.

Lymph nodes and spleen• Lymphoid follicles consist predominantly of B-cells andFDCs. B-cells may mature into plasma cells.

• T-cells reside in the paracortical areas of the lymphnodes.

Mucosal-associated lymphoid tissue• Lymphoid tissue guarding the gastrointestinal tract is unencapsulated and somewhat structured (tonsils,Peyer’s patches, appendix) or present as diffuse cellularcollections in the lamina propria.• Together with the subepithelial accumulations of cellslining the mucosal surfaces of the respiratory and geni-tourinary tracts, this lymphoid tissue forms the “secretoryimmune system” which bathes the surface with protectiveimmunoglobulin A(IgA) antibodies.

Other sites• Bone marrow is a major site of antibody production.• The brain, anterior chamber of the eye and testis are privileged sites in which antigens can be safely sequestered.

The handling of antigen• Macrophages are general antigen-presenting cells(APCs) for primed lymphocytes but cannot stimulatenaive T-cells.• Immature dendritic cells (DCs) are situated in periph-eral tissues and recognize and respond to antigen. Onceactivated they mature, upregulate chemokine receptorCCR7 and migrate to draining lymph nodes where theysettle down as interdigitating dendritic cells (IDCs) whichpowerfully initiate primary T-cell responses.• Follicular DCs in germinal centers bind immune com-plexes to their surface through Ig and C3b receptors. Thecomplexes are long-lived and provide a sustained sourceof antigenic stimulation for B-cells.• Specialized antigen-transporting M-cells in the gut pro-vide the gateway for antigens to the mucosal lymphoid tissue.



FURTHER READINGBradley, L.M. & Watson, S.R. (1996) Lymphocyte migration into tis-

sue: The paradigm derived from CD4 subsets. Current Opinion inImmunology, 8, 312–20.

Brandtzaaeg, P., Farstad, I.N. & Haraldsen, G. (1999) Regional spe-cialization in the mucosal immune system: Primed cells do not always home along the same track. Immunology Today, 20, 267–77.

Cavanagh, L.L. & Von Andrian, U.H. (2002) Travellers in many gui-ses: The origins and destinations of dendritic cells. Immunologyand Cell Biology, 80, 448–62

Lane, P.J.L. & Brocker, T. (1999) Developmental regulation of den-dritic cell function. Current Opinion in Immunology, 11, 308–13.

Peters, J.H., Gieseler, R., Thiele, B. & Steinbach, F. (1996) Dendriticcells: From ontogenetic orphans to myelomonocytic descendants.Immunology Today 17, 273–8.

75

IMMUNOCOMPETENT T- AND B-CELLSDIFFER IN MANY RESPECTS

The differences between immunocompetent T- and B-cells are sharply demarcated at the cell surface (table7.1). The most clear-cut discrimination is establishedby reagents which recognize anti-CD3 for T-cells andanti-CD19 or anti-CD20 for B-cells. In laboratory prac-tice these are the markers most often used to enumer-ate the two lymphocyte populations. B-cells alsopossess surface membrane immunoglobulin (Ig)whilst T-cells bear the T-cell receptor (TCR).

Differences in the cluster of differentiation (CD)markers determined by monoclonal antibodies reflectdisparate functional properties and in particular de-fine specialized T-cell subsets. CD4 is, generally speak-ing, a marker of T-helper cell populations, whichpromote activation and maturation of B-cells and cyto-toxic T-cells, and control antigen-specific chronic inflammatory reactions through stimulation ofmacrophages. CD4 forms subsidiary links with class IImajor histocompatibility complex (MHC) moleculeson antigen-presenting cells (APCs). Similarly, the CD8molecules on cytotoxic T-cells associate with MHCclass I (figure 7.1).

T-LYMPHOCYTES AND APCs INTERACTTHROUGH SEVERAL PAIRS OF ACCESSORY MOLECULES

The affinity of an individual TCR for its specificMHC–antigen peptide complex is relatively low and asufficiently stable association with the APC can only be

achieved by the interaction of complementary pairs ofaccessory molecules such as LFA-1/ICAM-1 (lympho-cyte function-associated antigen-1/intercellular adhesion molecule-1) and CD2/LFA-3 (figure 7.2).However, these molecular couplings are not neces-sarily concerned just with intercellular adhesion.

THE ACTIVATION OF T-CELLS REQUIRESTWO SIGNALS

It has been known for some time that two signals are re-quired to induce RNA and protein synthesis in a rest-ing T-cell (figure 7.2) and to move it from G0 into the G1phase of the mitotic cycle. Antigen in association withMHC class II on the surface of APCs is clearly capableof providing one of these signals for the CD4+ T-cellpopulation. Complex formation between the TCR,antigen and MHC thus provides signal 1 through thereceptor–CD3 complex, and this is greatly enhancedby the coupling of CD4 with the MHC. The T-cell isnow exposed to a costimulatory signal 2 from the APCin the form of two related molecules called B7.1 (CD80)and B7.2 (CD86). These bind to CD28 on the T-helpercell, and also to a receptor called CTLA-4 (cytotoxic T-lymphocyte antigen-4) on activated T-cells. Thus, anti-bodies to the B7 molecules can block activation ofresting T-cells. Surprisingly, this renders the T-cell an-ergic, i.e. unresponsive to any further stimulation byantigen. As we shall see in later chapters, the principlethat two signals activate but one may induce anergy inan antigen-specific cell provides a potential for target-ed immunosuppressive therapy. A further costimula-tory interaction is between CD40 on the APC and

CHAPTER 7

Lymphocyte activation

Immunocompetent T- and B-cells dif fer in manyrespects, 75

T-lymphocytes and APCs interact through severalpairs of accessory molecules, 75

The activation of T-cells requires two signals, 75Protein tyrosine phosphorylation is an early event

in T-cell signaling, 76Downstream events following TCR signaling, 77

Control of interleukin-2 (IL-2) gene transcription, 77Damping T-cell enthusiasm, 77

The nature of B-cell activation, 79B-cells are stimulated by cross-linking surface

immunoglobulin (sIg), 79B-cells respond to T-independent and T-dependent

antigens, 79


CD40L (CD40-ligand) on the T-cell. Although this in-teraction does not activate the T-cells directly, it in-creases the production of the B7 molecules by the APCsand also stimulates these cells to release cytokines thatenhance T-cell activity.

Adhesion molecules such as ICAM-1, VCAM-1(vascular cell adhesion molecule-1) and LFA-3 are notthemselves costimulatory but augment the effect ofother signals — an important distinction.

Activation of CD8+ T-cells requires an interaction ofthe TCR with peptides within the groove of class IMHC molecules, and costimulatory adhesion mole-cules also strengthen this interaction. The CD4 andCD8 responses, however, are not separate entities anda variety of cytokines produced by CD4+ cells can acti-vate CD8+ cells, almost acting as a second signal.

PROTEIN TYROSINE PHOSPHORYLATION ISAN EARLY EVENT IN T-CELL SIGNALING

The major docking forces that conjugate the APC andits T-lymphocyte counterpart come from the comple-mentary accessory molecules such as ICAM-1/LFA-1and LFA-3/CD2, rather than through the relativelylow-affinity TCR–MHC/peptide links. Nonetheless,cognate antigen recognition by the TCR remains a sinequa non for T-cell activation. Interaction of the TCRwith peptide bound to MHC molecules triggers a re-markable cascade of signaling events that culminatesin cell cycle progression and cytokine production.

The initial signal for T-cell activation through theTCR is greatly enhanced by bringing the CD4-associat-ed protein tyrosine kinase (PTK), Lck, close to the CD3-

g

% in peripheral blood 65–80 8–15

CELL SURFACE MOLECULES

ANTIGEN RECOGNITION

T-cells B-cells

Processed Native

Antigen receptorMHC class IMHC class IICD2CD4

CD5

CD8

CD19CD20CD21 (CR2: C3d and EBV receptorCD23 (FceRII)CD32 (Fc RII)

TCR/CD3+

only after activation+

MHC class II-restricted(helper)

+

MHC class I-restricted(cytotoxic)

–––

––

Surface Ig++––

only on B1aminor subset

–

+++

++

Anti-IgEpstein-Barr virus

Anti-CD3Phytohemagglutinin

POLYCLONAL ACTIVATION

Table 7.1 Comparison of human T- and B-cells.

APCCD4

MHC IITCR

TARGETCELL

CD8

TCR

MHC IDENDRITICCELLMACROPHAGEB-CELL

(e.g.VIRALLYINFECTED)

HELPERT-CELL

CYTOTOXICT-CELL

Th Tc

Figure 7.1 Helper and cytotoxic T-cell subsets are restricted bymajor histocompatibility complex (MHC) class. CD4 on helperscontacts MHC class II; CD8 on cytotoxic cells associates with class I.

STIMULATECD28

TCR

CD4

SIGNAL SIGNALS +

TCR

LFA-3 CD2

VLA-4VCAM-1

ICAM-1/2

LFA-1

B7

? HSA

APCAPC

1

2

Th

ANERGY ACTIVATION &PROLIFERATION

BLOCK B7

CD4

1

1 1 2

CD28

(CTLA-4)

Th

Figure 7.2 Activation of resting T-cells. Interaction of costimula-tory molecules leads to activation of resting T-lymphocyte by antigen-presenting cell (APC) on engagement of the T-cell receptor(TCR) with its antigen–major histocompatibility complex (MHC).Engagement of the TCR signal 1 without accompanying costimula-tory signal 2 leads to anergy. Note, a cytotoxic rather than a helper T-cell would, of course, involve coupling CD8 to MHC class I.Engagement of the CTLA-4 (cytotoxic T-lymphocyte antigen-4) mol-ecule (CD152) with B7 downregulates signal 1. ICAM-1/2, intercel-lular adhesion molecule-1/2; LFA-1/2, intercellular adhesionmolecule-1/2; VCAM-1, vascular cell adhesion molecule-1; VLA-4,very late integrin antigen-4. (Based on Y. Liu & P.S. Linsley (1992)Current Opinion in Immunology, 4, 265–70.)

CHAPTER 7—Lymphocyte activation 77

associated z-chains. Immunoreceptor tyrosine-basedactivation motifs (ITAMs) on the z-chains becomephosphorylated and bind to ZAP-70, which now be-comes an active PTK (figure 7.3) capable of initiating aseries of downstream biochemical events. It is now be-coming clear that very large numbers of TCRs interactwith only a few peptide–MHC complexes, and it hasbeen suggested that each MHC–peptide complex canserially engage up to 200 TCRs. This they do for a pro-longed period of time during which the TCRs moveacross the surface of the APC. The T-cells thereby undergo a sustained increase in intracellular calciumlevels which is required for the induction of prolifera-tion and cytokine production.

DOWNSTREAM EVENTS FOLLOWING TCR SIGNALING

Following TCR signaling, there is an early increase inthe level of active Ras–GTP (guanosine triphosphate)complexes, which regulate pivotal mitogen-activatedprotein kinases (MAPKs) through sequential kinasecascades. As illustrated in figure 7.4, there are a num-ber of different pathways involved in T-cell activation.

Within 15s of TCR stimulation, phospholipase C activates the phosphatidylinositol pathway, is pho-sphorylated and its catalytic activity increased. Thisultimately triggers the release of Ca2+ into the cytosol.The raised Ca2+ level synergizes with diacylglycerol to activate protein kinase C (PKC) and to act to-gether with calmodulin to increase the activity of calcineurin.

Control of interleukin-2 (IL-2) gene transcription

Transcription of IL-2 is one of the key elements in pre-venting the signaled T-cell from lapsing into anergyand is controlled by multiple receptors for transcrip-tional factors in the promoter region (figure 7.4)

Under the influence of calcineurin, NFkB (nuclearfactor-kappa B) and NFATc (cytoplasmic componentof the nuclear factor of activated T-cells) become acti-vated. The NFkB is released from its inhibitor in the cytoplasm, IkB, and then both NFkB and NFATctranslocates to the nucleus. Here NFATc forms a binarycomplex with NFATn, its partner which is constitutive-ly expressed in the nucleus, and the NFAT complex together with NFkB bind to IL-2 regulatory sites result-ing in cytokine production (figure 7.4). Note here thatthe calcineurin effect is blocked by the anti-T-cell drugscyclosporin and FK506 (see Chapter 15).

We have concentrated on IL-2 transcription as anearly and central consequence of T-cell activation, butmore than 70 genes are newly expressed within 4 h ofT-cell activation, leading to proliferation and the syn-thesis of several cytokines and their receptors.

Damping T-cell enthusiasm

We have frequently reiterated the premise that no self-respecting organism would permit the operation of anexpanding enterprise such as a proliferating T-cellpopulation without some sensible controlling mecha-nisms. Control of T-cell function will be described inChapter 9, but it is worth pointing out that whereas

Activation throughTCR/CD3/CD4/8

complex z chain

Surfa

ce m

embr

ane

PhosphorylationP

P P

ITAM

ITAM YxxL/IxxxxxxxYxxL/I

Immunoreceptor Tyrosine-based Activation Motif

Protein tyrosinekinase

Lck

ZAP-70

SH2

SH2Proteintyrosinekinaseactivity

P

PP

P

Figure 7.3 Signals through the T-cell re-ceptor (TCR)/CD3/CD4/8 complex initiatea tyrosine protein kinase (TPK) cascade.The TPK Lck phosphorylates the tyrosinewithin the immunoreceptor tyrosine-basedactivation motif (ITAM) sequences of CD3z-chains. These bind the z-associated pro-tein (ZAP-70) through its SH2 domains andthis in turn acquires TPK activity for down-stream phosphorylation of later compo-nents in the chain. The other CD3 chainseach bear a single ITAM.

PART 3—The acquired immune response

B7 B7

CD28CTLA-4

NFkB

Calcineurin

NFATc n Jun Fos NFATc n

IKK

Calmodulin

LAT

Shb

LAT

Gads

Nck SLAPVav

ItK

SLP-76

Pak

Cytoskeletalreorganization

MHC-Ag

z-z

TCR CD3 CD4/8 Surfacemembrane

PIP2

DAG

PKC PLCg1

PI3K

IP3

Ca2+

ZAP-70

Lck

Ras

Raf

MEK

ERKJNK

JNKK

Rac

IkB

NFkB

Translocation Translocation

OCT-1

NFkB

NFATn Jun Elk

Fos

Nucleus

Cytosol

IL-2

OCTNFATAP-1

IL-2 Transcription regulatory sites

OCTNFAT

NFATc

Fos

NFATc

NFATn JunNFATc

Grb2SH2SH2

Sos

Extracellularspace

SIGNAL 2 SIGNAL 1

Figure 7.4 T-cell signaling leads to activation. The signals throughthe MHC (major histocompatibility complex)–antigen complex (sig-nal 1) and costimulator B7 (signal 2) initiate a protein kinase cascadeand a rise in intracellular calcium, thereby activating transcriptionfactors which control entry in the cell cycle from G0 and regulate theexpression of interleukin-2 (IL-2) and many other cytokines. Thescheme presented omits several molecules which are thought to playimportant additional roles in signal transduction. DAG, diacylglyc-erol; ERK, extracellular signal regulated kinase; IP3, inositol triphos-phate; JNK, Jun N-terminal kinase; LAT, linker for activated T-cells;

NFkB, nuclear factor-kappa B; NFAT, nuclear factor of activated T-cell; OCT-1, octamer-binding factor; Pak, p21-activated kinase;PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositoldiphosphate; PKC, protein kinase C; PLC, phospholipase C; SH2,Src-homology domain 2; SLAP, SLP-76-associated phosphoprotein;SLP-76, SH2-domain containing leukocyte-specific 76kDa phospho-protein; ZAP-70, z-chain-associated protein kinase; , positivesignal transduction; , negative signal transduction; ,adapter proteins; , guanine nucleotide exchange factors; , ki-nases; , transcription factors; , other molecules.


CD28 is constitutively expressed on T-cells, CTLA-4 isnot found on the resting cell but is rapidly upregulatedfollowing activation. It has a 10–20-fold higher affinityfor both B7.1 and B7.2 and, in contrast to costimulatorysignals generated through CD28, B7 engagement ofCTLA-4 downregulates T-cell activation (figure 7.4).

THE NATURE OF B-CELL ACTIVATION

B-cells are stimulated by cross-linking surfaceimmunoglobulin (sIg)

Cross-linking of B-cell surface receptors, e.g. by thy-mus independent antigens, induces early activationevents. Within 1 min of sIg ligation, Src family kinasesrapidly phosphorylate the Syk kinase, Bruton’s tyro-sine kinase (Btk) and the ITAMs on the sIg receptorchains Ig-a and Ig-b. This is followed by a rise in intra-cellular calcium and activation of phosphokinase C.

B-cells respond to T-independent and T-dependent antigens

Thymus-independent antigens

Certain linear antigens that are not readily degraded inthe body and which have an appropriately spaced,highly repeating determinants such as thePneumococ-cus polysaccharide, are thymus-independent in theirability to stimulate B-cells directly without the need forT-cell involvement. They persist for long periods onthe surface of specialized macrophages located at thesubcapsular sinus of the lymph nodes and the splenic marginal zone. This type of antigen binds to antigen-specific B-cells with great avidity through their multi-valent attachment to the complementary Ig receptorswhich they cross-link (figure 7.5). This cross-linkinginduces early B-cell activation events. In general, thethymus-independent antigens give rise to predomi-nantly low-affinity IgM responses, and relatively poor,if any, memory.

Thymus-dependent antigens

Many antigens are thymus-dependent in that theyprovoke little or no antibody response in animalswhich have been thymectomized at birth. Such anti-gens cannot fulfill the molecular requirements for di-rect stimulation, and if they bind to B-cell receptors,they will sit on the surface just like a hapten and donothing to trigger the B-cell (figure 7.6). Cast yourmind back to the definition of a hapten — a small mole-cule that binds to preformed antibody (e.g. the surfacereceptor of a specific B-cell) but fails to stimulate anti-

body production (i.e. stimulate the B-cell). Rememberalso that haptens become immunogenic when coupledto an appropriate carrier protein. Building on theknowledge that both T- and B-cells are necessary forantibody responses to thymus-dependent antigens,we now know that the carrier functions to stimulate T-helper cells, which cooperate with B-cells to enablethem to respond to the hapten by providing accessorysignals (figure 7.6). It should also be evident from fig-ure 7.6 that while one determinant on a typical proteinantigen is behaving as a hapten in binding to the B-cell,the other determinants subserve a carrier function inrecruiting T-helper cells.

Antigen processing by B-cells

Although we think of B-cells primarily as antibody-producing cells, they in fact play an important role inantigen processing and presentation to T-cells. PrimedB-cells, in fact, work at much lower antigen concentra-tions than conventional APCs because they can focusantigen through their surface receptors. Antigenbound to sIg is internalized in endosomes which thenfuse with vesicles containing MHC class II moleculeswith their invariant chain. Processing of the proteinantigen then occurs as described in Chapter 5 and theresulting antigenic peptide is recycled to the surface inassociation with the class II molecules. There it is avail-able for recognition by the TCR on a carrier-specific T-helper cell (figure 7.7). With the assistance of co-stimulatory signals arising from the interaction ofCD40 with its ligand CD40L, B-cell activation is ensured.

MARGINAL ZONEMACROPHAGE

Thymus-independent antigen withrepeating determinants cross-link lg receptors

B-CELL

Figure 7.5 B-cell recognition of thymus-independent antigens.The complex gives a sustained signal to the B-cell because of the longhalf-life of this type of molecule. , activation signal;

, surface immunoglobulin (sIg) receptor; , cross-linking of receptors.


AgB

HAPTENB

PROTEINANTIGEN

AgB

BCARRIER

NO B-CELL STIMULATION T-HELPER-INDUCED B-CELL STIMULATION

Th

Th

Figure 7.6 T-helper cells cooperate through protein carrier deter-minants to help B-cells respond to hapten or equivalent determi-nants on antigens by providing accessory signals. (For simplicity

CARRIERDETERMINANT

sIgRECEPTOR

B-CELL

1Capture of antigen

B-CELL

Endosome formation & fusion

class +invariant chainB-CELL

B-CELL

H+

B-CELL

2

3Antigen processing

4 Carrier antigenic peptide—MHC IIinteracts on surface with T-cell receptor

TCR

MHC II

CD4CD40L

CD40

II

ACTIVATED T-HELPER

RESTING B-CELL

ACID DEPENDENTHYDROLYTIC ENZYMES

Figure 7.7 B-cell handling of a thymus-dependent antigen. Antigen captured bythe surface immunoglobulin (sIg) receptoris internalized within an endosome,processed and expressed on the surfacewith major histocompatibility complex(MHC) class II (cf. figure 5.8). Costimula-tory signals through the CD40–CD40L in-teraction are required for activation of theresting B-cell by the T-helper. , activation signal.

REVISION

Immunocompetent T- and B-cells differ in many respects• The CD3 cell surface antigen can be used to identify T-cells, whilst CD19 and CD20 are characteristic of B-cells.• The antigen-specific TCR on T-cells and surface im-munoglobulin (sIg) on B-cells also provide a clear distinc-tion between these two cell types.• Differences in CD markers define specialized T-cell subsets.

T-lymphocytes and antigen-presenting cells (APCs) interactthrough pairs of accessory molecules• The docking of T-cells and APCs depends upon strong

mutual interactions between complementary molecularpairs on their surfaces: major histocompatibility complex(MHC) II/CD4; MHC I/CD8; ICAM-1/LFA-1; LFA-3/CD2; B7/CD28 (and CTLA-4).

Activation of T-cells requires two signals• Two signals activate T-cells, but one alone produces unresponsiveness (anergy).• One signal is provided by the low-affinity cognate T-cellreceptor (TCR)/MHC–peptide interaction.• The second costimulatory signal is mediated throughligation of CD28 by B7.1 or B7.2.

we are ignoring the major histocompatibility complex (MHC) com-ponent and epitope processing in T-cell recognition, but we won’tforget it.)


FURTHER READING

Acuto, O. & Cantrell, D. (2000) T cell activation and the cytoskeleton.Annual Reviews in Immunology, 18, 165–84.

Borst, J. & Cope, A. (1999) Turning the immune system on. Immunolo-gy Today, 20, 156–8.

Jenkins, M.K., Khoruts, A., Ingulli, E. et al. (2001) In vivo activation ofantigen-specific CD4 T cells. Annual Review of Immunology 19,23–45.

Myung, P.S., Boerthe, N.J. & Koretzky, G.A. (2000) Adaptor proteinsin lymphocyte antigen-receptor signaling. Current Opinion in Immunology, 12, 256–66.

Protein tyrosine phosphorylation is an early event in T-cell signaling• The TCR signal is transduced and amplified through aprotein tyrosine kinase (PTK) enzymic cascade. This cul-minates in cell division and cytokine production.• Large numbers of TCRs interact with only a few pep-tide–MHC complexes.• As activation proceeds, intracellular calcium levels increase, and protein kinase C (PKC) and calcineurin areactivated.• Under the influence of calcineurin the transcription fac-tors NFAT and NFkB are activated, resulting in transcrip-tion of genes for cytokines such as interleukin-2 (IL-2).• There are a number of mechanisms that control T-cell activity including the association of B7 with CTLA-4.

B-cells respond to two different types of antigen• Thymus-independent antigens are polymeric mole-cules which cross-link many sIg receptors and, because oftheir long half-lives, provide a persistent signal to the B-cell.

• Thymus-dependent antigens require the cooperation ofhelper T-cells to stimulate antibody production by B-cells.• Antigen captured by specific sIg receptors is taken intothe B-cell, processed, and expressed on the surface as apeptide in association with MHC II.• This complex is recognized by the T-helper cell, whichactivates the resting B-cell.

The nature of B-cell activation• Cross-linking of sIg receptors (e.g. thymus-indepen-dent antigens) activates B-cells.• T-helper cells activate resting B-cells through TCRrecognition of MHC II–carrier peptide complexes and co-stimulation through CD40L/CD40 interactions (analo-gous to the B7/CD28 second signal for T-cell activation).


82

A SUCCESSION OF GENES AREUPREGULATED BY T-CELL ACTIVATION

We have dwelt upon the early events in lymphocyte ac-tivation consequent upon the engagement of the T-cellreceptor (TCR) and the provision of an appropriatecostimulatory signal. A complex series of tyrosine andserine/threonine phosphorylation reactions producesthe factors which push the cell into the mitotic cycleand drive clonal proliferation and differentiation to ef-fector cells. Within the first half hour, nuclear tran-scription factors which regulate interleukin-2 (IL-2)expression and the cellular proto-oncogene c-myc areexpressed, but the next few hours see the synthesis of arange of soluble cytokines and their receptors (figure8.1). Much later we see molecules like the transferrinreceptor related to cell division and very late antigenssuch as the adhesion molecule VLA-1.

CYTOKINES ACT AS INTERCELLULARMESSENGERS

In contrast to the initial activation of T-cells and T-dependent B-cells, which involves intimate contactwith the antigen-presenting cells (APCs), subsequentproliferation and maturation of the response is orches-trated by soluble mediators generically termed cy-tokines. These relay information between cells assoluble messengers. A list of some of these protein mediators is shown in table 8.1.

Cytokine action is transient and usually short range

These low molecular weight secreted proteins mediatecell growth, inflammation, immunity, differentiation,migration and repair. They are highly potent, often act-ing at femtomolar (10-15 M) concentrations, combiningwith small numbers of high-affinity cell surface re-ceptors to produce changes in the pattern of RNAand protein synthesis. Unlike endocrine hormones,the majority of cytokines normally act locally in aparacrine or even autocrine fashion. Thus, cytokinesderived from lymphocytes rarely persist in the circula-tion. Nonlymphoid cells such as macrophages canhowever be triggered by bacterial products to releasecytokines, which may be detected in the bloodstream,often to the detriment of the host. Certain cytokines, in-cluding IL-1 and tumor necrosis factor (TNF), alsoexist in membrane forms which could exert their stim-ulatory effects without becoming soluble.

Cytokines act through cell surface receptors

There are six major cytokine receptor structural fami-lies of which the largest is the family of hematopoietinreceptors. These generally consist of one or twopolypeptide chains responsible for cytokine bindingand an additional shared (common or “c”) chain in-volved in signal transduction. The gc-chain is used bythe IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors,

CHAPTER 8

The production of effectors

A succession of genes are upregulated by T-cellactivation, 82

Cytokines act as intercellular messengers, 82Cytokine action is transient and usually short range, 82Cytokines act through cell surface receptors, 82Cytokines often have multiple effects, 83Network interactions, 83

Different CD4 T-cell subsets can make dif ferentcytokine patterns, 85The bipolar Th1/Th2 concept, 85Interactions with cells of the innate immune system biases the

Th1/Th2 response, 86Activated T-cells proli ferate in response to

cytokines, 86

T-cell effectors in cell-mediated immunity, 88Cytokines mediate chronic inflammatory responses, 88Killer T-cells, 90

Proliferation and maturation of B-cell responses aremediated by cytokines, 91

What is going on in the germinal center?, 92Immunoglobulin class-switching occurs in

individual B-cells, 92Memory cells, 93

The memory population is not simply an expansion ofcorresponding naive cells, 94

CHAPTER 8—The production of effectors 83

and the bc-chain by IL-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. Other families include the interferon (IFN)receptors, the TNF receptors, the immunoglobulin superfamily (IgSF) receptors (including the IL-1 recep-tor), the large family of chemokine receptors and thereceptors for transforming growth factors (TGFs) suchas TGFb. The interaction of cytokine with its receptorinitiates cell signalling which generally utilizes eitherthe Janus kinase (JAK)-STAT or the Ras-MAP kinasepathways.

Cytokines often have multiple effects

In general, cytokines are pleiotropic, i.e. exhibit multi-ple effects on growth and differentiation of a variety ofcell types (table 8.1). There is considerable overlappingand redundancy between them with respect to indi-vidual functions, partially accounted for by the shar-ing of receptor components and the utilization ofcommon transcription factors. For example many ofthe biological activities of IL-4 overlap with those of IL-13.

Their roles in the generation of T- and B-cell effec-

tors, and in the regulation of chronic inflammatory reactions (figure 8.2a,b) will be discussed later in thischapter. We should note here the important role of cytokines in the control of hematopoiesis (figure 8.2c).The differentiation of stem cells to become the formedelements of blood within the environment of the bonemarrow is carefully nurtured through the productionof cytokines such as GM-CSF by the stromal cells andby T-cells and macrophages. It is not surprising, there-fore, that during a period of inflammation the cytokines produced recruit new precursors into thehematopoietic differentiation pathway, giving rise tothe leukocytosis so often seen in patients with activeinfection.

Network interactions

The complex and integrated relationships between thedifferent cytokines are mediated through cellularevents. For example, the genes for IL-3, IL- 4, IL-5 andGM-CSF are all tightly linked on chromosome 5 in a region containing genes for macrophage colony-stimulating factor (M-CSF) and its receptor and seve-ral other growth factors and receptors. Interaction may

ACTIVATION

EARLY

0 min

15 min

30 min

cfos/cjun Nuclear binding transcription factor; binds to AP-1

Cellular oncogene; controls G0 G1→

Nur77 Function in TCR-mediated apoptosis inimmature T-cells

MEDIUMTERM

LATE

Severalhours

14h

7-14 days

3-5 days

16h

IL-2/3/4/5/6

IFNγ TGFβ

IL-9/10/13

GM-CSF

Cytokines and their receptors influencing growthand differentiation of myeloid and lymphoid cells,controlling viral growth and mediating chronicinflammatory processes

Transferrinreceptor

VLA-1

Class ΙΙ MHC

myb

Related to cell division

Very late “antigen”; adhesion molecule

Antigen presentation

Cellular oncogene

NFκB

NFAT

Nuclear binding protein; regulates expressionof many genes

Nuclear transcription factor of activated-T;regulates IL-2 gene

IκB-α Inhibitor of NFκB

PAC-1 Nuclear phosphatase which inactivatesERKs

c-

mycc-

Figure 8.1 Sequential gene activation onT-cell stimulation, appearance of messenger RNA (mRNA). AP-1, activatorprotein-1; ERKs, extracellular signal-regulated kinases; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL,interleukin; IFNg, interferon g; MHC, majorhistocompatibility complex; NFkB, nuclearfactor-kappa B; NFAT, nuclear factor of acti-vated T-cell; TCR, T-cell receptor; TGFb,transforming growth factor-b.


CYTOKINE SOURCE EFFECTOR FUNCTION

INTERLEUKINS

COLONY STIMULATING FACTORS

TUMOR NECROSIS FACTORS

INTERFERONS

OTHERS

Costimulates T activation by enhancing production of cytokines including IL-2 and itsreceptor; enhances B proliferation and maturation; NK cytotoxicity; induces IL-1,-6,-8, TNF,GM-CSF and PGE2 by Mf; proinflammatory by inducing chemokines and ICAM-1 andVCAM-1 on endothelium; induces fever, APP, bone resorption by osteoclastsInduces proliferation of activated T- and B-cells; enhances NK cytotoxicity and killing of tumorcells and bacteria by monocytes and MfGrowth and differentiation of hematopoietic precursors; MC growthInduces Th2 cells; stimulates proliferation of activated B, T, MC; upregulates MHC class II onB and Mf, and CD23 on B; downregulates IL-12 production and thereby inhibits Th1differentiation; increases Mf phagocytosis; induces switch to IgG1 and IgEInduces proliferation of eosino and activated B; induces switch to IgADifferentiation of myeloid stem cells and of B into plasma cells; induces APP; enhances T proliferationInduces differentiation of lymphoid stem cells into progenitor T and B; activates mature TMediates chemotaxis and activation of neutrophilsInduces proliferation of thymocytes; enhances MC growth; synergizes with IL-4 in switch toIgG1 and IgEInhibits IFNg secretion by mouse, and IL-2 by human, Th1 cells; downregulates MHC class IIand cytokine (including IL-12) production by mono, Mf and DC, thereby inhibiting Th1differentiation; inhibits T proliferation; enhances B differentiationPromotes differentiation of pro-B and megakaryocytes; induces APPCritical cytokine for Th1 differentiation; induces proliferation and IFNg production by Th1,CD8+ and gd T and NK; enhances NK and CD8+ T cytotoxicityInhibits activation and cytokine secretion by Mf; co-activates B proliferation; upregulatesMHC class II and CD23 on B and mono; induces switch to IgG1 and IgE; induces VCAM-1on endoInduces proliferation of T, NK and activated B and cytokine production and cytotoxicity in NKand CD8+ T; chemotactic for T; stimulates growth of intestinal epitheliumChemoattractant for CD4 T, mono and eosino; induces MHC class IIProinflammatory; stimulates production of cytokines including TNF,IL-1b,-6,-8, G-CSFInduces IFNg production by T; enhances NK cytotoxicityModulation of Th1 activityRegulation of inflammatory responses to skin?Regulation of hematopoiesis; NK differentiation; B activation; T costimulationInhibits IL-4 production by Th2Induces proliferation and IFNg production by Th1; induces proliferation of memory cells

Stimulates growth of progenitors of mono, neutro, eosino and baso; activates MfStimulates growth of neutro progenitorsStimulates growth of mono progenitorsStimulates stem cell division (c-kit ligand)

Tumor cytotoxicity; cachexia (weight loss); induces cytokine secretion; induces E-selectin onendo; activates Mf; antiviralTumor cytotoxicity; enhances phagocytosis by neutro and Mf; involved in lymphoid organdevelopment; antiviral

Inhibits viral replication; enhances MHC class IInhibits viral replication; enhances MHC class IInhibits viral replication; Enhances MHC class I and II; activates Mf; induces switch to IgG2a;antagonizes several IL-4 actions; inhibits proliferation of Th2

Proinflammatory by, e.g., chemoattraction of mono and Mf but also anti-inflammatory by,e.g. inhibiting lymphocyte proliferation; induces switch to IgA; promotes tissue repairInduces APPStimulates IL-12 production and inhibits IL-10 production by MfInduces APP

Mono, Mf, DC, NK, B, Endo, Eosino

Th1, Eosino

T, NK, MC, EosinoTh2, Tc2, NK, NK-T, gd T, MC, Eosino

Th2, MC, EosinoTh2, Mono, Mf, DC, BM stroma, Eosino

BM and thymic stromaMono, Mf, Endo, EosinoTh

Th (Th2 in mouse), Tc, B, Mono, Mf, Eosino

BM stromaMono, Mf, DC, B

Th2, MC, Eosino

T, NK, Mono, Mf, DC, B

Th, Tc, EosinoTMf, DCMonoKeratinocytes?ThTDC

Th, Mf, Fibro, MC, Endo, EosinoFibro, EndoFibro, Endo, EpithBM stroma

Th, Mono, Mf, DC, MC, NK, B, Eosino

Th1, Tc

LeukocytesFibroblastsTh1, Tc1, NK

Th3, B, Mf, MC, Eosino

Thymic epith, BM stromaTT, Mf

IL-1a, IL-1b

IL-2

IL-3IL-4

IL-5IL-6

IL-7IL-8IL-9

IL-10

IL-11IL-12

IL-13

IL-15

IL-16IL-17IL-18IL-19IL-20IL-21IL-22IL-23

GM-CSFG-CSFM-CSFSLF

TNF (TNFa)

Lymphotoxin (TNFb)

IFNaIFNbIFNg

TGFb

LIFEta-1Oncostatin M

Table 8.1 Cytokines: their origin and function. Note that there is not an interleukin-14 (IL-14). This designation was given to an activity that,upon further investigation, could not be unambiguously assigned to a single cytokine. Interleukin-8 is a member of the chemokine family. Thesecytokines are listed separately in table 8.2.

APP, acute phase proteins; B, B-cell; baso, basophil; BM, bone marrow; Endo, endothelium; eosino, eosinophil; Epith, epithelium; Fibro, fibrob-last; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; LIF, leukemia inhibitory factor; Mf, macrophage; MC, mastcell; Mono, monocyte; neutro, neutrophil; NK, natural killer; SLF, steel locus factor; T, T-cell; TGFb , transforming growth factor-b.


occur through a cascade in which one cytokine inducesthe production of another, through transmodulation ofthe receptor for another cytokine and through syner-gism or antagonism of two cytokines acting on thesame cell (figure 8.3). Furthermore, as mentionedabove, many cytokines share the same signaling path-ways and this too may contribute to the redundancy intheir effect.

DIFFERENT CD4 T-CELL SUBSETS CANMAKE DIFFERENT CYTOKINE PATTERNS

The bipolar Th1/Th2 concept

T-helper cells (Th) can be classified into varioussubsets depending on the cytokines they secrete. Th0cells secrete IL-2, IL-3, IL-4, IL-5, IFNg and GM-CSF.These cells can develop into Th1 cells which character-istically secrete IL-2, IFNg and lymphotoxin andpromote cell-mediated immunity, especially cytotoxicand delayed hypersensitivity reactions andmacrophage activation. Alternatively the Th0 cells candifferentiate into Th2 cells, which secrete IL-4, IL-5, IL-6, IL-10 and IL-13, and activate B-lymphocytes result-ing in upregulation of antibody production. Thecharacteristic cytokines produced by Th1 and Th2 cellshave mutually inhibitory effects on the reciprocal phe-notype. Interferon g will inhibit the proliferation ofTh2 cells and IL-10 will downregulate Th1 cells.Although the factors involved in directing immuneresponses to these two subpopulations are not fullyelucidated, it appears that APCs and, in particular,dendritic cells (DCs) appear to be pivotal in drivingdifferentiation towards a Th1 or Th2 phenotype. Alsothe cytokine environment in which these cells developmay influence their ultimate phenotype. Therefore IL-12 produced by APCs will stimulate IFNg productionfrom natural killer (NK) cells, and both these cytokineswill drive differentiation of Th1 cells and inhibit Th2responses. On the other side of the coin, when Th areactivated by antigen in the presence of IL-4 theydevelop into Th2 cells (figure 8.4).

The different patterns of cytokine production bythese T-cell subtypes are of importance in protectionagainst different classes of microorganisms. Th1 cellsproducing cytokines such as IFNg would be especiallyeffective against viruses and intracellular organismsthat grow in macrophages. Th2 cells are very goodhelpers for B-cells and seem to be adapted for defenceagainst parasites that are vulnerable to IL-4-switchedIgE and IL-5-induced eosinophilia, and againstpathogens that are removed primarily through humoral mechanisms.

IL-5

IL-4/5/6

a Control of lymphocyte growth

T B Antibody

IL-2

IFNg

IL-10

b Activation of innate immune mechanisms

Activation Activation

InfectionMf

EOSIN

NK

Activatedkiller cell

LAKMONOPMN

LIVER

ActivationAcute phase proteins

IL-8TNF

IL-1 TNFIL-6

IFNa

IFNg

c Hematopoiesis in bone marrow

Formedelements

of the blood

FIBROBLAST OR ENDOTHELIAL CELL

STEM CELL

STROMAL CELL

IL-1/12

IL-1

M-CSFG-CSFGM-CSFIL-1IL-6

M-CSFG-CSFGM-CSFIL-6IL-7

T

IL-2

Th1 Th2

Figure 8.2 Cytokine action. A general but not entirely comprehen-sive guide to indicate the scope of cytokine interactions. EOSIN,eosinophil; G-CSF, granulocyte colony-stimulating factor; GM-CSF,granulocyte-macrophage colony-stimulating factor; IFN, interfe-ron; IL, interleukin; LAK, lymphokine activated killer; Mf,macrophage; M-CSF, macrophage colony-stimulating factor;MONO, monocyte; NK, natural killer cell; PMN, polymorphonu-clear neutrophil; TNF, tumor necrosis factor.


Infections with Mycobacterium tuberculosis, theetiologic agent of tuberculosis, demonstrate well theclinical significance of these Th subsets. Successfulcontrol of infection is dependent upon an adequateTh1 response. These cells, by promoting IFNg produc-tion, activate macrophages to reduce bacterial loadand to release TNF, which is essential for granulomaproduction.

Infections with Leishmania organisms are also influ-enced by the pattern of cytokines produced by Th1 andTh2 cells. In the localized cutaneous form of the dis-ease, IL-2 and IFNg predominate in the lesions, indicat-ing that Th1 cells are limiting the infection. In the moresevere chronic mucocutaneous disease, however, thelesions have an abundance of IL-4, suggesting a pre-dominantly Th2 response with inadequate cellularcontrol. In the most severe visceral form of the disease,circulating lymphocytes are unable to produce IFNg orIL-12 in response to the etiologic agent, Leishmaniadonovani, but addition of anti-IL-10 antibody results inan augmented IFNg response. These findings mayhave significant therapeutic possibilities as therapiesthat increase activity of Th1 cytokines or decrease Th2cytokines may enhance resolution of Leishmania le-sions. Interferon g in particular, since it enhancesmacrophage killing of parasites, has been shown tohave some beneficial effect in treating severely ill pa-tients and those with refractory disease.

Interactions with cells of the innate immunesystem biases the Th1/Th2 response

Antigen-presenting cells and, in particular, DCs ap-pear to be pivotal in driving differentiation towards aTh1 or Th2 phenotype. Whilst there is some evidenceindicating the existence of subpopulations of DCs spe-cialized for the stimulation of either Th1 or Th2 popu-lations, this is an area that is still under intensiveinvestigation. Interleukin-12 seems to be particularlyimportant for the production of Th1 cells and IL-4 forthe production of Th2 cells. Invasion of phagocyticcells by intracellular pathogens induces copious secre-tion of IL-12, which in turn stimulates IFNg productionby NK cells. These two cytokines selectively drive dif-ferentiation of Th1 development and inhibit Th2 re-sponses (figure 8.4). However, IL-4 effects appear to bedominant over IL-12 and, therefore, the amounts of IL-4 relative to the amounts of IL-12 and IFNg will be ofparamount importance in determining the differentia-tion of Th0 cells into Th1 or Th2.

ACTIVATED T-CELLS PROLIFERATE INRESPONSE TO CYTOKINES

Amplification of T-cells following activation is criti-cally dependent upon IL-2 (figure 8.5). This cytokineacts only on cells which express high-affinity IL-2 re-ceptors. These receptors are not present on restingcells, but are synthesized within a few hours after acti-vation (figure 8.1). The number of these receptors onthe cell increases under the action of antigen and IL-2,

+ +–

PANCREATIC b-CELL

cSynergism

TNF

IFNg

MHC class II

+ +

baCascade

Receptor transmodulation

Upregulation Downregulation

Mf

MACROPHAGE

TNF

RECEPTOR

TNF

IL-1

T-CELL

IL-4 IL-5a b

IL-2 RECEPTOR

TGFb

IL-2 RECEPTOR

T-CELL

+ + _

Figure 8.3 Network interactions of cytokines. (a) Cascade: in thisexample tumor necrosis factor (TNF) induces secretion of inter-leukin-1 (IL-1) and of itself (autocrine) in the macrophage. (Note alldiagrams in this figure are simplified in that the effects on the nucl-eus are due to messengers resulting from combination of cytokinewith its surface receptor.) (b) Receptor transmodulation showing

upregulation of each chain forming the high-affinity IL-2 receptor inan activated T-cell by individual cytokines and downregulation bytransforming growth factor-b (TGFb). (c) Synergy of TNF and inter-feron g (IFNg) in upregulation of surface major histocompatibilitycomplex (MHC) class II molecules on cultured pancreatic insulin-secreting cells. Mf, macrophage.


and as antigen is cleared, so the receptor numbers de-cline and, with that, the responsiveness to IL-2.

The T-cell blasts also produce an impressive array ofother cytokines and the proliferative effect of IL-2 is re-inforced by the action of IL-4 and IL-15 which reactwith corresponding receptors on the dividing T-cells.We must not lose sight of the importance of controlmechanisms such as TGFb, which blocks IL-2-inducedproliferation (figure 8.3b) and the cytokines IFNg, IL-4and IL-12, which mediate the mutual antagonism ofTh1 and Th2 subsets. Should the T-cells be repeatedlyactivated, IL-2 will be responsible for activation-induced cell death of the T-cells, thereby further con-trolling the extent of the immune response.

IL-4,5,6,10,13

IL-4IL-10

Humoralimmunity

Cell-mediatedimmunity

IL-3, GM-CSF, TNF

Intracellularinfection

ANTIGEN & ANTIGEN PRESENTING CELLSACCESSORY SIGNALS : IL-1, 2, 4, IFNg

? Helminth

NK

IL-2, IFNg ,IL-4,5

IL-3, GM-CSF

Mf

IL-2

IFNg

IL-2, IFNg, LT

IL-3, GM-CSF, TNF

IL-12

IL-4

Th1

Thp

Th2

Th0

Figure 8.4 The generation of Th1 and Th2 CD4 subsets. Followinginitial stimulation of T-cells, a range of cells producing a spectrum ofcytokine patterns emerges. Under different conditions, the resultingpopulation can be biased towards two extremes. Interleukin-12 (IL-12), possibly produced through an “innate” type effect of an in-tracellular infection on macrophages, encourages the developmentof Th1 cells which produce the cytokines characteristic of cell-mediated immunity. In contrast IL-4, possibly produced by interac-tion of infectious agents with natural killer (NK) cells, skews the de-velopment to production of Th2 cells whose cytokines assist theprogression of B-cells to antibody secretion and the provision of humoral immunity. Cytokines produced by polarized Th1 and Th2 subpopulations are mutually inhibitory. LT, lymphotoxin; Th0,early helper cell producing a spectrum of cytokines; Thp, T-helperprecursor; other abbreviations as in table 8.1.

CYTOKINES IL-2,3,4,5,6,9,10,13IFN�, TNF, LT, GM-CSF

Activation

Synthesis of IL-2and IL-2 receptor

IL-2 drivenproliferation

IL-2 RECEPTOR

IL-2

Th Th

Figure 8.5 Activated T-blasts expressing surface receptors for in-terleukin-2 (IL-2) proliferate in response to IL-2 produced by itselfor by another T-cell subset. The expanded population secretes awide variety of biologically active cytokines of which IL-4 also en-hances T-cell proliferation. GM-CSF, granulocyte-macrophagecolony-stimulating factor; IFNg, interferon g; LT, lymphotoxin; Th, T-helper cell; TNF, tumor necrosis factor.


T-CELL EFFECTORS IN CELL-MEDIATEDIMMUNITY

Cytokines mediate chronic inflammatory responses

In addition to their role in the adaptive response, the T-cell cytokines are responsible for generating antigen-specific chronic inflammatory reactionswhich deal with intracellular microorganisms (figure8.6).

Early events

The initiating event is probably a local inflammatoryresponse to tissue injury caused by the infectious agentthat would upregulate the synthesis of adhesion molecules such as VCAM-1 (vascular cell adhesionmolecule-1) and ICAM-1 (intercellular adhesion molecule-1) on adjacent vascular endothelial cells.

These would permit entry of memory T-cells to the infected site through their VLA-4 (very late antigen-4)and LFA-1 (lymphocyte function-associated antigen-1) homing receptors. Contact with processed antigenderived from the intracellular organism will activatethe specific T-cell and induce the release of secreted cytokines. Tumor necrosis factor will further enhancethe expression of endothelial accessory molecules andincrease the chances of other memory cells in the circulation homing in to meet the antigen provokingthe inflammation.

Chemotaxis

The recruitment of T-cells and macrophages to the in-flammatory site is greatly enhanced by the action ofchemotactic cytokines termed chemokines (chemoat-tractant cytokine). The main stimuli to the productionof chemokines are the proinflammatory cytokinessuch as IL-1 and TNF, but bacterial and viral products

T

IL-3 GM-CSFIL-4,5,6

Th2

CHEMOKINESIL-1,6G-CSF GM-CSF

M CYTOKINESfTNF IFN� IFN� TNF LT

IL-3 GM-CSF

Th1

T-CELL MEDIATED INFLAMMATION

Mf

B

ANTIGEN

ANTIBODY IgM, G, A & E

Plasma cell

Figure 8.6 Cytokines controlling the antibody and T-cell-mediated inflammatory responses.Abbreviations as in table 8.1.


may also do this. There are now more than 50chemokines described and they bind to at least 19 func-tional receptors, predominantly on leukocytes. Thereare four chemokine families termed the CC and CXCchemokines, which are the predominant ones, and theCX3C and the C chemokines, which are smaller fami-lies (table 8.2). Receptor activation leads to a cascade of

cellular events that ultimately results in activation ofthe cellular machinery necessary to direct the cell to aparticular location. Despite the fact that a singlechemokine can sometimes bind to more than one re-ceptor, and a single receptor can bind severalchemokines, many chemokines exhibit a strong tissueand receptor specificity. They play important roles in

FAMILY CHEMOKINE CHEMOTAXIS RECEPTORS

CXC

C

CX3C

CC

CXCL1CXCL2CXCL3CXCL4CXCL5CXCL6CXCL7CXCL8CXCL9CXCL10CXCL11CXCL12CXCL13CXCL14CXCL15

XCL1XCL2

CX3CL1

CCL1CCL2CCL3CCL4CCL5(CCL6)CCL7CCL8(CCL9/10)CCL11(CCL12)CCL13CCL14CCL15CCL16CCL17CCL18CCL19CCL20CCL21CCL22CCL23CCL24CCL25CCL26CCL27

GROa/MGSAaGROb/MGSAbGROg/MGSAgPF4ENA-78GCP-2/(CKa-3)NAP-2IL-8MigIP-10I-TACSDF-1a/bBLC/BCA-1BRAK/BolekineLungkine

Lymphotactin/SCM-1a/ATACSCM-1b

Fractalkine/Neurotactin

I-309/(TCA-3/P500)MCP-1/MCAFMIP-1a/LD78 aMIP-1bRANTES(C10/MRP-1)MCP-3MCP-2(MRP-2/CCF18/MIP-1g)Eotaxin-1(MCP-5)MCP-4HCC-1/HCC-3HCC-2/Leukotactin-1/MIP-1dHCC-4/LEC/(LCC-1)TARCDCCK1/PARC/AMAC-1MIP-3b/ELC/Exodus-3MIP-3a/LARC/Exodus-16Ckine/SLC/Exodus-2/(TCA-4)MDC/STCP-1/ABCD-1MPIF-1MPIF-2/Eotaxin-2TECKSCYA26/Eotaxin-3CTACK/ALP/ESkine

NeutroNeutroNeutroEosino,BasoNeutroNeutroNeutroNeutroT, NKT, NKT, NKT, B, DC, MonoB?Neutro

TT

T, NK, Mono

MonoT, NK, DC, Mono, BasoT, NK, DC, Mono, EosinoT, NK, DC, MonoT, NK, DC, Mono, Eosino, BasoMonoT, NK, DC, Mono, Eosino, BasoT, NK, DC, Mono, Baso?T, DC, Eosino, BasoT, NK, DC, Mono, BasoT, NK, DC, Mono, Eosino, BasoT, Mono, EosinoTTT, DC, MonoTT, B, DCDCT, DCT, DC, MonoTT, DC, Eosino, BasoT, DC, MonoTT

CXCR2>CXCR1CXCR2CXCR2CXCR3CXCR2CXCR1, CXCR2CXCR2CXCR1, CXCR2CXCR3CXCR3CXCR3CXCR4CXCR5??

XCR1XCR1

CX3CR1

CCR8CCR2CCR1, CCR5CCR5CCR1, CCR3, CCR5?CCR1,CCR2, CCR3CCR3?CCR3CCR2CCR2, CCR3CCR1CCR1, CCR3CCR1CCR4?CCR7CCR6CCR7CCR4CCR1CCR3CCR9CCR3CCR10

ALTERNATIVE NAMES

Table 8.2 Chemokines and their receptors. The chemokines are grouped according to the arrangement of their cysteines. The letter Ldesignatesligand (i.e. the individual chemokine), whereas the letter R designates receptors. Names in parentheses refer to the murine homologs of thehuman chemokine where the names of these differ, or the murine chemokine alone if no human equivalent has been described.

B, B-cell; Baso, basophil; DC, dendritic cell; Eosino, eosinophil; Mono, monocyte; Neutro, neutrophil; NK, natural killer; T, T-cell.


inflammation, lymphoid organ development, cell traf-ficking, cell compartmentalization within lymphoidtissues, Th1/Th2 development, angiogenesis andwound healing. Although the receptors are specific forchemokines, the CCR5 and CXCR4 receptors are nowknown to also act as co-receptors for the human im-munodeficiency virus (HIV), allowing the virus thathas attached to the CD4 molecule to gain entrance tothe T-cell or the monocyte.

Macrophage activation

Macrophages with intracellular organisms are acti-vated by agents such as IFNg, GM-CSF, IL-2 and TNFand become endowed with microbicidal powers. During this process, some macrophages may die (per-haps helped along by cytotoxic T-cells (Tc)) and releaseliving organisms, but these will be dealt with by freshmacrophages brought to the site by chemotaxis and activated by local cytokines.

Combating viral infection

Virally-infected cells require a different strategy, andone strand of that strategy exploits the innate IFNmechanism to deny the virus access to the cell’sreplicative machinery. Tumor necrosis factor has cyto-toxic potential against virally infected cells, which isparticularly useful since death of an infected cell be-fore viral replication has occurred is obviously benefi-cial to the host. The cytotoxic potential of TNF was firstrecognized using tumor cells as targets (hence thename), and IFNg and lymphotoxin can act synergisti-cally with TNF setting up the cell for destruction by in-ducing the formation of TNF receptors.

Killer T-cells

The generation of cytotoxic T-cells

Cytotoxic T-cells (Tc), also referred to as cytotoxic T-lymphocytes (CTLs), represent the other major arm ofthe cell-mediated immune response, and are of strate-gic importance in the killing of virally infected cellsand possibly in contributing to the postulated surveil-lance mechanisms against cancer cells.

The cytotoxic cell precursors (Tcp) recognize antigenon the surface of cells in association with class I majorhistocompatibility complex (MHC) molecules, andlike B-cells they usually, but not always, require helpfrom other T-cells. The mechanism by which help isproffered may, however, be quite different. Effective

T–B collaboration involves capture of the native anti-gen by the surface Ig receptors on the B-cells, whichprocess it internally and present it to the Th as a pep-tide in association with MHC class II. With Th and Tcpinteractions, it seems most likely that both cells bind tothe same APC which has processed viral antigen anddisplays processed viral peptides in association withboth MHC class II (for the Th cell) and class I (for theTcp) on its surface. It is possible that the APC could bethe virally infected cell itself. Cytokines from the trig-gered Th and from DCs will be released in close prox-imity to the Tcp, which is engaging the antigen–MHCsignal, and these cells will be stimulated to proliferateand differentiate into a CTLunder the influence of IL-2,IL-6, IL-12 and IL-15 (Figure 8.7).

The lethal process

Most Tc are of the CD8 subset and their binding to thetarget cell through TCR recognition of antigen plusclass I MHC is assisted by association between CD8and MHC class I and by other accessory moleculessuch as LFA-1 and CD2 (see figure 7.2). In some circum-stances CD4+ T-cells may also mediate cytotoxicitythrough the Fas–FasL(Fas ligand) pathway.

Cytotoxic T-cells are unusual secretory cells whichuse a modified lysosome to secrete their lytic proteins.Following delivery of the TCR/CD3 signal, the lyticgranules are driven along the microtubule system and

CD4+

ThCD8+

Tc

IL-2, IL-6, etc.

CD40L

CD40

TCR

MHC ΙΙ

TCR

MHC Ι

Dendritic cell

Figure 8.7 T-helper cell (Th) activation of cytotoxic T-cells (Tc).Ac-tivation of the CD4+ Th by the dendritic cell (DC) involves aCD40–CD40L (CD40 ligand) (CD154) costimulatory signal andrecognition of peptide presented to the T-cell receptor (TCR) bymajor histocompatibility complex (MHC) class II. The release of cy-tokines from the activated Th cells stimulates the differentiation ofthe CD8+ precursor into an activated, MHC class I-restricted Tc.


delivered to the point of contact between the Tc and itstarget (figure 8.8). This guarantees the specificity ofkilling dictated by TCR recognition of the target andlimits any damage to bystander cells. As with NK cellswhich have comparable granules, exocytosis of thegranule contents, including perforins, granzymes andTNF, produces lesions in the target cell membrane anddeath by inducing apoptosis. Cytotoxic T-cells are en-dowed with a second killing mechanism involving Fasand its ligand (cf. pp. 13 and 179).

Video microscopy shows that the Tc are serial killers.After the “kiss of death,” the T-cell can disengage andseek a further victim, there being rapid synthesis ofnew granules. At some stage the expansion of the CD8cells will need to be contracted and this occurs when

the newly activated CD8 cells kill the activating APCs.One should also not lose sight of the fact that CD8 cellssynthesize other cytokines such as IFNg which alsohave antiviral potential.

Inflammation must be regulated

Once the inflammatory process has cleared the incitingagent, the body needs to switch off the response to thatantigen. A variety of anti-inflammatory cytokines areproduced during humoral and cell-mediated immuneresponses. Interleukin-10 has profound anti-inflammatory and immunoregulatory effects, actingon macrophages and Th1 cells to inhibit release ofproinflammatory factors such as IL-1 and TNF. Fur-thermore IL-10 downregulates surface TNF receptorand induces the release of soluble TNF receptorswhich are endogenous inhibitors of TNF. Interleukin-1, a potent proinflammatory cytokine, can be regulatedby soluble IL-1 receptors released during inflamma-tion which act to decoy IL-1, and by the IL-1 receptorantagonist (IL-1Ra). This latter cytokine, which isstructurally similar to IL-1, is produced by monocytesand macrophages during inflammation and competeswith IL-1 for IL-1 receptors thereby antagonizing IL-1activity. Production of IL-1Ra is stimulated by IL-4,which also inhibits IL-1 production and, as we have indicated previously, also acts to constrain Th1 cells.The role of TGFb is more difficult to tease out because it has some pro and other anti-inflammatory effects, although it undoubtedly promotes tissue repair afterresolution of the inflammation.

These anti-inflammatory cytokines have consid-erable potential for the treatment of those human diseases where proinflammatory cytokines are re-sponsible for clinical manifestations. Such a situationexists in septic shock, where many of the clinical fea-tures are due to the massive production of IL-1, TNFand IL-6. Studies in rabbits showed that pre-treatmentwith IL-1Ra blocked endotoxin-induced septic shockand death. In studies in humans, however, treatmentwith IL-1Ra has not been shown to reduce mortality inpatients with sepsis, and further trials on this and otheranti-inflammatory cytokines are in progress.

PROLIFERATION AND MATURATION OF B-CELL RESPONSES ARE MEDIATED BYCYTOKINES

The activation of B-cells by Th through the TCR re-cognition of MHC-linked antigenic peptide plus thecostimulatory CD40L–CD40 interaction leads to up-regulation of the surface receptors for IL-4. Copious

Figure 8.8 Conjugation of a cytotoxic T-cell (on left) to its target,here a mouse mastocytoma, showing polarization of the granulestowards the target at the point of contact. The cytoskeletons of bothcells are revealed by immunofluorescent staining with an antibodyto tubulin (green) and the lytic granules with an antibody togranzyme A (red). Twenty minutes after conjugation the target cellcytoskeleton may still be intact (above), but this rapidly becomes disrupted (below). (Photographs kindly provided by Dr GillianGriffiths.)


local release of this cytokine from the Th then drivespowerful clonal proliferation and expansion of the ac-tivated B-cell population (figure 8.9). Interleukin-2and IL-13 also contribute to this process.

Immunoglobulin M plasma cells emerge under thetutelage of IL-4 plus IL-5, and IgG producers resultfrom the combined influence of IL-4, IL-5, IL-6, IL-13and IFNg. Under the influence of IL-4 and IL-13, the ex-panded clones can differentiate and mature into IgE-synthesizing cells. Transforming growth factor-band IL-5 encourage cells to switch their Ig class to IgA(figure 8.9).

Thymus-independent antigens can activate B-cellsdirectly but nonetheless still need cytokines for effi-cient proliferation and Ig production.

WHAT IS GOING ON IN THE GERMINAL CENTER?

Secondary challenge with antigen or immune com-

plexes induces enlargement of germinal centers, for-mation of new ones, appearance of B-memory cellsand development of Ig-producing cells of higher affin-ity. B-cells entering the germinal center become cen-troblasts, which divide with a very short cycle time of6 h, and then become nondividing centrocytes in thebasal light zone, many of which die from apoptosis(figure 8.10). As the surviving centrocytes mature, theydifferentiate either into immunoblast plasma cell pre-cursors, which secrete Ig in the absence of antigen, ormemory B-cells.

What then is the underlying scenario? Followingsecondary antigen challenge, primed B-cells may beactivated by paracortical Th cells in association withinterdigitating DCs or macrophages, and migrate tothe germinal center. There they divide in response to powerful stimuli from complexes on follicular dendritic cells (FDCs) and from cytokines released by T-cells in response to antigen-presenting B-cells.Somatic hypermutation of B-cell Ig genes occurs withhigh frequency during this stage of cell division. Mu-tated cells with surface antibody of higher affinity willbe positively selected, so leading to maturation of antibody affinity during the immune response. Thecells also undergo Ig class switching and further dif-ferentiation will now occur. The cells either migrate tothe sites of plasma cell activity (e.g. lymph nodemedulla) or go to expand the memory B-cell pool.

IMMUNOGLOBULIN CLASS-SWITCHINGOCCURS IN INDIVIDUAL B-CELLS

The synthesis of antibodies belonging to the various Ig classes proceeds at different rates. Usually there is an early IgM response which tends to fall off rapidly. Immunoglobulin G antibody synthesis builds up to its maximum over a longer time period. On secondarychallenge with antigen, the synthesis of IgG antibodiesrapidly accelerates to a much higher titer and there is a relatively slow fall-off in serum antibody levels(figure 8.11). The same probably holds for IgA, and in asense both these Ig classes provide the main im-mediate defense against future penetration by foreignantigens.

Antibody synthesis in most classes shows consider-able dependence upon T-cooperation in that the re-sponses in T-deprived animals are strikingly deficient.Similarly, the switch from IgM to IgG and other classesis largely under T-cell control critically mediated byCD40 and by cytokines as described earlier. In additionto being brisker, the secondary responses tend to be ofhigher affinity because once the primary response getsunder way and the antigen concentration declines to

IL-2,4,5,6,13, IFNg, TGFb

IL-4,5,6,13IFNg

G0

S

G1

IL-4,13 IL-4,5TGF ,bIL-5

IL-2, 4,13

Activation

Clonalproliferation

Maturation

IgE IgA IgM IgG

S

IICD40

TCR

CD40L Th

Figure 8.9 B-cell response to thymus-dependent (TD) antigen:clonal expansion and maturation of activated B-cells under the in-fluence of T-cell-derived soluble factors. Costimulation throughthe CD40 ligand (CD40L)–CD40 interaction is essential for primaryand secondary immune responses to TD antigens and for the for-mation of germinal centers and memory. IFNg, interferon g; IL, interleukin; Ig, immunoglobulin; TCR, T-cell receptor; TGFb, transforming growth factor-b; Th, T-helper cell.


low levels, only successively higher-affinity cells willbind sufficient antigen to maintain proliferation.

MEMORY CELLS

Memory of early infections such as measles is long-

lived and the question arises as to whether the memorycells are long-lived or are subject to repeated antigenstimulation from persisting antigen or subclinical rein-fection. Fanum in 1847 described a measles epidemicon the Faroe Islands in the previous year in which almost the entire population suffered from infection

Mf

GERMINAL CENTER

MATURATION OFSURVIVING CELLS

MEMORYB-CELL

PLASMA CELLPRECURSOR

B-BLAST

DARK ZONE

BASAL LIGHT ZONE

FOLLICULARDENDRITIC CELL

CENTROBLASTS

CENTROCYTES

FOLLICULARDENDRITIC CELL

T-HELPER

MACROPHAGEWITH PHAGOCYTOSEDLYMPHOCYTE

APICAL LIGHT ZONE

IMMUNE COMPLEX

CORONA MANTLEOF B-LYMPHOCYTES

(Ag Ig C3d)

PROLIFERATION

Ig SWITCH

SOMATIC MUTATION

APOPTOSIS

SELECTIVE SURVIVAL T T

T

Figure 8.10 The events occurring in lymphoid germinal centers.Germinal center B-cells show numerous mutations in antibodygenes. Expression of LFA-1 (lymphocyte function-associated anti-gen-1) and ICAM-1 (intercellular adhesion molecule-1) on B-cellsand follicular dendritic cells (FDCs) in the germinal center makesthem “sticky.” Through their surface receptors, FDCs bind immune

complexes containing antigen and C3 which, in turn, are very effec-tive B-cell stimulators since coligation of the surface receptors forantigen and C3 lowers their threshold for activation. The costimula-tory molecules CD40 and B7 play pivotal roles, and antibodies toCD40 or B7 prevent formation of germinal centers. Ag, antigen; Ig,immunoglobulin; Mf, macrophage.


except for a few old people who had been infected 65years earlier. While this evidence favors the long half-life hypothesis, it is envisaged that B-cell memory is adynamic state in which survival of the memory cells is maintained by recurrent signals from FDCs in thegerminal centers, the only long-term repository ofantigen.

T-cell memory exists for both CD4 and CD8 cells andmay be maintained even in the absence of the antigen.Usually, however, antigen persists as complexes onFDCs. There is, therefore, the potential for APCs with-

in the germinal center to capture and process this com-plexed antigen and then present it to memory T-cells.

The memory population is not simply anexpansion of corresponding naive cells

In general, memory cells are more readily stimulatedby a given dose of antigen because they have a higheraffinity. In the case of B-cells, we have been satisfied bythe evidence linking mutation and antigen selection tothe creation of high-affinity memory cells within thegerminal center of secondary lymph node follicles.Virgin B-cells lose their surface IgM and IgD andswitch receptor isotype on becoming memory cells.The costimulatory molecules B7.1 (CD80) and B7.2(CD86) are rapidly upregulated on memory B-cells,and their potent antigen-presenting capacity for T-cells could well account for the brisk and robust natureof secondary responses.

Memory T-cells augment their binding avidity forAPCs through increased expression of accessory adhe-sion molecules such as CD2, LFA-1, LFA-3 and ICAM-1. Since several of these molecules also function toenhance signal transduction, the memory T-cell ismore readily triggered than its naive counterpart. Indeed, memory cells enter cell division and secretecytokines more rapidly than naive cells, and there issome evidence that they may secrete a broader range ofcytokines than naive cells.

Antib

ody

titer

(lo

g sc

ale)

IgG

IgM0 1 2 3 4 5 6 7Weeks

1st injectionof antigen

2nd injectionof antigen

Figure 8.11 Synthesis of immunoglobulin M (IgM) and IgG anti-body classes in the primary and secondary responses to antigen.

REVISION

A succession of genes are upregulated by T-cell activation• Within 15–30 min genes for transcription factors con-cerned in the progression of G0 to G1 and control of IL-2are expressed.• Up to 14 h, cytokines and their receptors are expressed.• Later, a variety of genes related to cell division and ad-hesion are upregulated.

Cytokines act as intercellular messengers• Cytokines act transiently and usually at short range, al-though circulating interleukin-1 (IL-1) and IL-6 can medi-ate release of acute-phase proteins from the liver.• They act through high-affinity cell surface receptors.• Cytokines are pleiotropic, i.e. have multiple effects inthe general areas of (i) control of lymphocyte growth, (ii) activation of innate immune mechanisms (including

inflammation), and (iii) control of bone marrow hematopoiesis.• Cytokines may act sequentially, through one cytokineinducing production of another or by transmodulation ofthe receptor for another cytokine; they can also act syner-gistically or antagonistically.

Different CD4 T-cell subsets can make different cytokines• As immunization proceeds, T-helper cells (Th) tend todevelop into two subsets: Th1 cells concerned in inflam-matory processes, macrophage activation and delayedsensitivity make IL-2 and IL-3, interferon-g (IFNg), lym-photoxin and granulocyte-macrophage colony-stimulat-ing factor (GM-CSF); Th2 cells help B-cells to synthesizeantibody and secrete IL-3, 4, 5, 6 and 10, tumor necrosis factor-a (TNFa) and GM-CSF.


• Early interaction of antigen with macrophages produc-ing IL-12 or with a T-cell subset secreting IL-4 will skew theresponses to Th1 or Th2 respectively.• Different patterns of cytokine production influence theseverity and clinical manifestations of various infectiousdiseases.

Activated T-cells proliferate in response to cytokines• Interleukin-2 acts as an autocrine growth factor for Th1and paracrine for Th2 cells that have upregulated their IL-2 receptors.• Cytokines act on cells which express receptors.

T-cell effectors in cell-mediated immunity• Cytokines mediate chronic inflammatory responses.• a-Chemokines are cytokines which chemoattract neu-trophils, and b-chemokines attract T-cells, macrophagesand other inflammatory cells.• CCR5 and CXCR4 chemokine receptors act as co-receptors for the human immunodeficiency virus (HIV).• Tumor necrosis factor synergizes with IFNg in killingcells.

Killer T-cells• Cytotoxic T-cells (Tc) are generated against cells (e.g. vi-rally infected) that have intracellularly-derived peptideassociated with surface major histocompatibility complex(MHC) class I.• Cytotoxic T-cells proliferate under the influence of IL-2released by Th in close proximity.• Cytotoxic T-cells are CD8 cells that secrete lytic proteins to the point of contact between the Tc and its target.• The granules contain perforins and TNF, which pro-duce lesions in the target cell membrane, and granzymes,which cause death by apoptosis.

Control of inflammation• Various anti-inflammatory cytokines are produced dur-ing the immune response.• T-cell-mediated inflammation is strongly downregulat-ed by IL-10.

• Interleukin-1 is inhibited by the IL-1 receptor antagonist(IL-1Ra) and by IL-4.

Proliferation of B-cell responses is mediated by cytokines• Early proliferation is mediated by IL-4, which also aidsimmunoglobulin E (IgE) synthesis.• Immunoglobulin A producers are driven by transform-ing growth factor-b (TGFb) and IL-5.• Interleukin-4 plus IL-5 promotes IgM, and IL-4, IL-5, IL-6 and IL-13 plus IFNg stimulate IgG synthesis.

Events in the germinal center• There is clonal expansion, isotype switch and mutationin the centroblasts.• The B-cell centroblasts become nondividing centrocyteswhich differentiate into plasma cell precursors or intomemory cells.

Immunoglobulin class-switching occurs in individual B-cells• Immunoglobulin M produced early in the responseswitches to IgG, particularly with thymus-dependentantigens. The switch is under T-cell control.• Immunoglobulin G, but not IgM, responses improve onsecondary challenge.• Secondary responses show increased affinity for antigen.

Memory cells• It has been suggested that activated memory cells aresustained by recurrent stimulation with antigen.• This must occur largely in the germinal centers since the complexes on the surface of follicular dendritic cells (FDCs) are the only long-term source ofantigen.• Memory cells have higher affinity than naive cells.



FURTHER READING

Borish, L. & Steinke, J.W. (2003) Cytokines and chemokines. Journalof Allergy and Clinical Immunology, 111 (Suppl.), S460–75.

Camacho, S.A., Kosco-Vilbois, M.H. & Berek, C. (1998) The dynamicstructure of the germinal center. Immunology Today, 19, 511–14.

Kelsoe, G. (1999) VDJ hypermutation and receptor revision. CurrentOpinion in Immunology, 11, 70–5.

Sprent, J & Surh, C.D. (2001) Generation and maintenance of mem-ory T cells. Current Opinion in Immunology, 13, 248–54.

Weiss, A. & Cambier, J.C. (eds) (2004) Section on lymphocyte activa-tion. Current Opinion in Immunology, 16, 285–387.

97

ANTIGEN IS A MAJOR FACTOR IN CONTROL

The acquired immune response evolved so that itwould come into play when contact with an infectiousagent was first made. The appropriate antigen-specificcells expand, the effectors eliminate the antigen, andthen the response quietens down and leaves room forreaction to other infections. Feedback mechanismsmust operate to limit antibody production; otherwise,after antigenic stimulation, we would become over-whelmed by the responding clones of antibody-forming cells and their products. There is abundant evidence to support the view that antigen is a majorregulatory factor and that antibody production is dri-ven by the presence of antigen, falling off in intensity asthe antigen concentration drops (figure 9.1). Further-more, clearance of antigen by injection of excess anti-body during the course of an immune response leadsto a dramatic decrease in antibody synthesis and in thenumber of antibody-secreting cells.

ANTIBODY EXERTS FEEDBACK CONTROL

A useful control mechanism is to arrange for the prod-uct of a reaction to be an inhibitor, and this type of negative feedback is seen with antibody. Thus, re-moval of circulating antibody by plasmapheresis during an ongoing response leads to an increase insynthesis, whereas injection of preformed im-munoglobulin G (IgG) antibody markedly hastens thefall in the number of antibody-forming cells consistentwith feedback control on overall synthesis probablymediated through the B-cell Fcg receptor.

IDIOTYPE NETWORK

Jerne’s network hypothesis

The hypervariable loops on the Ig molecule whichform the antigen-combining site have individual char-acteristic shapes that can be recognized by the appro-priate antibodies as idiotypic determinants. There arehundreds of thousands of different idiotypes in one individual.

Jerne reasoned that the great diversity of idiotypeswould, to a considerable extent, mirror the diversity ofantigenic shapes in the external world. Thus, Jernestated that if lymphocytes can recognize a whole rangeof foreign antigenic determinants, then they should beable to recognize the idiotypes on other lymphocytes.They would therefore form a large network or series of networks depending upon idiotype–anti-idiotyperecognition between lymphocytes of the various T-and B-subsets (figure 9.2). The response to an externalantigen would be proliferation of the specific clone oflymphocytes with expansion of the particular idiotypeand then subsequent triggering of anti-idiotype re-sponses to downregulate the specific lymphocyte population. There is no doubt that the elements whichcan form an idiotypic network are present in the body.Individuals can be immunized against idiotypes ontheir own antibodies, and such autoanti-idiotypeshave been identified during the course of responses in-duced by antigens. It seems likely that similar interac-tions will be established for newly expanding T-cellclones, possibly linking with the B-cell network. Cer-tainly, anti-idiotypic reactivity can be demonstrated

CHAPTER 9

Control mechanisms

Antigen is a major factor in control, 97Antibody exerts feedback control, 97Idiotype network, 97

Jerne’s network hypothesis, 97T-cell regulation, 98

Activation-induced cell death, 98Activated T-cells exchange CD28 for cytotoxic T-lymphocyte

antigen-4 (CTLA-4), 99Regulatory T-cells, 100

The influence of genetic and other factors, 100Some genes affect general responsiveness, 100Psychoimmunology, 101Sex hormones come into the picture, 102Decreased immune response in the elderly

(immunosenescence), 102Malnutrition diminishes the effectiveness of the

immune response, 103


in T-cell populations, and it is likely that relativelyclosed idiotype–anti-idiotype circuits contribute to aregulatory system.

T-CELL REGULATION

Activation-induced cell death

Termination of T-cell responses may result from ultra-violet or gamma-irradiation, hypoxia, high-dose corti-costeroids, or various cytotoxic cytokines. It may alsofollow a decline in activating stimuli or growth factors,or be due to programmed cell death (apoptosis). Acti-vation of T-cells by antigen does not continually pro-duce proliferation but eventually sets off a train ofevents that culminate in activation-induced cell deathby apoptosis. There are two main pathways of apopto-sis, the CD95–CD95L (CD95 ligand) (Fas) death sys-tem and the mitochondrial pathway. Both pathwaysdepend on the activation of a group of proteases be-longing to the caspase family and result in T-cell num-bers declining to pre-immunization levels.

ANTIGENCONCN.

ANTIBODYRESPONSE

TIME AFTER INFECTION

EARLY LATEAntigen catabolism & removal

by immune response

Figure 9.1 Antigen drives the immune response. As the antigenconcentration falls due to catabolism and elimination by antibody,the intensity of the immune response declines but is maintained forsome time at a lower level by antigen trapped on the follicular den-dritic cells of the germinal centers.

ANTIGEN

ANTIGEN-REACTIVE

LYMPHOCYTE

Nonspecificparallel

set

Branch

Ab2αAb1Ab2β

ANTI-ANTI-IDIOTYPEANTI-IDIOTYPEIDIOTYPEANTI-IDIOTYPE (internal image)

Ab3

Internal image

αld1

ld1

αld1

α(αld1)

α(αld1)

Figure 9.2 Elements in an idiotypic network in which the antigenreceptors on one lymphocyte reciprocally recognize an idiotype onthe receptors of another. T–T interactions could occur through directrecognition of one T-cell receptor (TCR) by the other, or more usually

by recognition of a processed TCR peptide associated with majorhistocompatibility complex (MHC). One of the anti-idiotype sets,Ab2b, may bear an idiotype of similar shape to (i.e. provides an inter-nal image of ) the antigen.

CHAPTER 9—Control mechanisms 99

In the CD95 system, Fas (CD95) expressed on thesurface of all lymphoid cells interacts with its ligand,which is expressed on T-cells after they are activated.The FasL (Fas ligand) may combine with numerousFas molecules on the same or on neighboring cells andeven on B-cells resulting in their death. FasL binds anintracellular protein called FADD (Fas-associateddeath domain) which recruits the inactive form of caspase 8 and cleaves this into an active form. The activated caspase 8 now cleaves downstream caspasesultimately leading to endonuclease digestion of DNA, resulting in nuclear fragmentation and celldeath. Details are shown in figure 9.3.

In the mitochondrial pathway, cytochrome c is re-leased from the mitochondria into the cytoplasmwhere, together with a protein called Apaf-1 (apopto-sis activating factor-1), it activates caspase 9 resultingin apoptosis. Like many other biologic systems, apop-

tosis needs to be regulated. Several gene products inhibit apoptosis and these belong mainly to the bcl-2 family. Those bcl-2 family members with anti-apoptotic activity probably do so by controlling the re-lease of cytochrome c.

The cytokine interleukin-2 (IL-2) plays an importantrole in activation-induced cell death. Early in the im-mune response IL-2 acts as an important T-cell growthpromoter. However, as the response develops and levels of this cytokine increase, it develops growth inhibitory effects by rendering T-cells more sensitive to the induction of apoptosis.

Activated T-cells exchange CD28 for cytotoxic T-lymphocyte antigen-4 (CTLA-4)

Early in the response, B7 molecules bind to CD28,which is important for T-cell activation. After activa-

Death receptor induced apoptosis Stress induced apoptosis

CELLMEMBRANE

CELLMEMBRANE

CELLMEMBRANE

FASL (CD178)

FAS (CD95)

FADD

DISC

DD DD DD DD

DED DED

DED

8

6 3 7

Caspase

Effectorcaspases

+

UV Hypoxia Cytotoxins g-irradiation

APOPTOSOME

9 Cyt c

Apaf-1 Pro-apoptotic

Anti-apoptotic

bcl-2 bcl-XL

Smacbax

+

ENDONUCLEASE TRANSLOCATION TO NUCLEUS

M+

Figure 9.3 Activation-induced cell death. Receptor-based induc-tion of apoptosis involves the trimerization of Fas by FasL (Fas-ligand). This brings together cytoplasmic death domains (DD)which can recruit a number of death effector domain (DED)-contain-ing adapter molecules, such as FADD (Fas-associated protein withdeath domain) to form the death-inducing signaling complex(DISC). The DISC induces the cleavage of inactive procaspase 8 intoactive caspase 8, with subsequent activation of downstream effectorcaspases. A second pathway of apoptosis induction, often triggeredby cellular stress, involves a number of mitochondria-associatedproteins including cytochrome c, Smac/DIABLO and the bcl-2 family member bax. Caspase 9 activation is the key event in this

pathway and requires association of the caspase with a number ofother proteins including the cofactor Apaf-1 (apoptosis activatingfactor-1); the complex formed incorporates cytochrome c and is re-ferred to as the apoptosome. The activated caspase 9 then cleavesprocaspase 3. Although the death receptor and mitochondrial path-ways are shown as initially separate in the figure, there is some cross-talk between them. Thus, caspase 8 can cleave the bcl-2 familymember bid (not shown), a process which promotes cytochrome c re-lease from mitochondria. Other members of the bcl-2 family, such asbcl-2 itself and bcl-XL, inhibit apoptosis, perhaps by preventing therelease of pro-apoptotic molecules from the mitochondria. M, mito-chondrion; UV, ultraviolet.


tion, however, CTLA-4 is induced on T-cells and thiscompetes with CD28 for B7 on the antigen-presentingcell (APC). Cytotoxic T-lymphocyte antigen-4 inhibitsthe transcription of IL-2 and subsequent T-cell prolif-eration, which will cause a decline of the immune response.

Regulatory T-cells

For many years immunologists have demonstratedthe presence of suppressor or regulatory cells thatcould modulate a variety of humoral and cellular re-sponses. We now recognize that when T-cells are acti-vated they develop high levels of IL-2 receptor (CD25)on their surface, and that a population of these acti-vated CD4+ CD25+ cells have regulatory activity on T-cell responses. It is thought that regulation is mediatedby immunosuppressive cytokines which inhibit pro-liferation of other T-cells. These cytokines include IL-10 and transforming growth factor-b (TGFb) whichcontrol the expansion of specific T-cells, and, by in-hibiting interferon g (IFNg) production by the T-cells,will bring activated APCs back to their resting state.Other populations of CD4+CD25+ regulatory T-cellsappear to act via a poorly defined cytokine-indepen-dent, but cell contact-dependent, mechanism.

THE INFLUENCE OF GENETIC AND OTHER FACTORS

Some genes affect general responsiveness

Mice can be selectively bred for high or low antibodyresponses through several generations to yield twolines, one of which consistently produces high-titer antibodies to a variety of antigens (high responders),and the other antibodies of relatively low titer (low responders). Out of the several different genetic loci involved, some give rise to a higher rate of B-cell proliferation and differentiation, whilst others affectmacrophage behavior.

There was much excitement when it was first dis-covered that the antibody responses to a number ofthymus-dependent antigenically simple substancesare determined by genes mapping to the major histo-compatibility complex (MHC) and three mechanismshave been proposed to account for MHC-linked highand low responsiveness.1 Defective processing and presentation. In a high re-sponder, processing of antigen and its recognition by acorresponding T-cell lead to lymphocyte triggeringand clonal expansion (figure 9.4a). Sometimes, the

a bHIGH RESPONDER LOW RESPONDER

stimulation

T-CELL

T-CELL RECEPTOR

Antigen binds MHCT-cell sees antigen

MHC II

ANTIGENIC PEPTIDE

Antigen does not bind MHCNo antigen for T-cell to see

MHC II

c

Self peptide + MHCtolerizes immature T-cell

X-reacting foreign peptide + MHC.No T-cell to see antigen

LOW RESPONDER

TCR

THYMUS

MHC II

IMMATURET-CELL tolerance

NO T-CELL

a ab b

d LOW RESPONDER

T-cell sees antigen but triggering suppressed by TS cell

SUPPRESSORT-CELL

T-CELL

TCR

suppression

a b

MHC II

MHC II

a ab b

Figure 9.4 Different mechanisms can account for low T-cell re-sponse to antigen in association with major histocompatibilitycomplex (MHC) class II. TCR, T-cell receptor; Ts, T-suppressor.


natural processing of an antigen in a given individualdoes not produce a peptide that fits well into theirMHC molecules. Furthermore, it is known that varia-tion in certain key residues of the MHC influence thebinding to individual peptides. If a given MHC is un-able to bind the peptide then obviously it cannot pre-sent antigen to the reactive T-cell (figure 9.4b).2 Defective T-cell repertoire. T-cells with moderate tohigh affinity for self MHC molecules and their com-plexes with processed self antigens will be renderedunresponsive (cf. tolerance induction), so creating a“hole” in the T-cell repertoire. If there is a cross-reaction, i.e. similarity in shape at the T-cell recognitionlevel between a foreign antigen and a self moleculewhich has already induced unresponsiveness, the hostwill lack T-cells specific for the foreign antigen andtherefore be a low responder (figure 9.4c).3 T-suppression. Major histocompatibility complex-restricted low responsiveness has been observed to relatively complex antigens containing several differ-ent epitopes. Such observations support the notionthat low responder status can arise as an expression ofregulatory cell activity (figure 9.4d).

Psychoimmunology

Attention has been drawn increasingly to interactionsbetween immunologic and neuroendocrine systemsgenerating the subject of “psychoimmunology.” Thereis now extensive evidence that psychosocial stress canresult in immune modulation resulting in detrimentalchanges in health. Many of the studies have examinedimmune function in individuals facing psychosocialstress such as students taking examinations, peopleproviding long-term care to a spouse with Alzheimer’sdisease, or recently bereaved individuals. In all thesegroups depressed immune function was documented.Depressive symptoms in human immunodeficiencyvirus (HIV) infected men have been associated withdecreased CD4+ counts and a more rapid immune de-cline. Individuals with generalized anxiety disordersshowed reduced natural killer (NK) cell activity anddecreased IL-2 receptor expression on activated T-cells, but an increase in production of proinflam-matory cytokines such as IL-6 and tumor necrosisfactor-a (TNFa). Stress influenced the response ofmedical students to hepatitis B vaccinations in thatthose students who were less stressed mounted an antibody response earlier than those who were moreanxious. It is likely therefore that the immune responseto infectious agents will be reduced in this latter group.There are also studies documenting that wound heal-

Hypothalamicparaventricular nucleus Anterior pituitary

CRH

ACTH

Adrenal

–

+

–

GLUCOCORTICOIDS

CYTOKINES

IL-1, IL-6TNF

Th1

Mf

++

Figure 9.5 Glucocorticoid negative feedback on cytokine produc-tion. Cells of the immune system in both primary and secondarylymphoid organs can produce hormones and neuropeptides, while classical endocrine glands as well as neurones and glial cellscan synthesize cytokines and appropriate receptors. ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hor-mone; IL, interleukin; Mf, macrophage; TNF, tumor necrosis factor.

ing is impeded by stress and this will enhance the riskfor wound infection after surgery or trauma.

It is likely that many of the immunologic effects ofstress are mediated via the endocrine system. Socialstress has been shown to elevate hormones such as catecholamines and cortisol which have multiple immunomodulatory effects.

The secretion of glucocorticoids is a major responseto stress induced by a wide range of stimuli such as ex-treme changes of temperature, fear, hunger and physi-cal injury. Steroids are also released as a consequenceof immune responses and limit those responses in aneuroendocrine feedback loop. Thus, IL-1, IL-6 andTNFa are capable of stimulating glucocorticoid syn-thesis and do so through the hypothalamic–pituitary–adrenal axis. This in turn leads to downregulation ofTh1 and macrophage activity, so completing the negative feedback circuit (figure 9.5).


Sex hormones come into the picture

Estrogen is said to be the major factor influencing themore active immune responses in females relative tomales. Women have higher serum Igs and secretoryIgA levels, a higher antibody response to T-independent antigens, relative resistance to T-cell tol-erance and greater resistance to infections. Females arealso far more susceptible to autoimmune disease, anissue that will be discussed in greater depth in Chapter17, but here let us note that oral contraceptives can induce flares of the autoimmune disorder systemiclupus erythematosus (SLE).

Decreased immune response in the elderly(immunosenescence)

Aging is associated with complex changes in manyparts of the normal immune response (table 9.1), whichaccounts for the relative susceptibility of the elderly toviral and bacterial infections. Looking at early eventsin the response, the phagocytic and microbicidal activ-ity of neutrophils is significantly diminished in laterlife. The reduced ability to phagocytose Staphylococcusaureus is of particular importance because of the in-creased susceptibility to this pathogen in the elderly.Although NK cell numbers are increased in this popu-lation they have decreased cytotoxic activity.

T-cell function and number are significantly alteredwith increasing age. T-cells, particularly the CD8 com-ponent, decrease significantly, probably due to an in-crease in apoptotic activity. This is associated withimpairment of many T-cell functions including expres-sion of CD28, proliferation, cytotoxicity, IL-2 secretionand delayed hypersensitivity reactions. It is primarilythe Th1 subset that diminishes in the aged, and there isan increase in cytokine production by the Th2 subset.Therefore, levels of IL-6 are raised in the elderly popu-lation whilst IL-2 levels are decreased (figure 9.6).

Humoral immunity is less affected by age, althoughthere is an increase in autoantibody levels. CirculatingIgG and IgA levels are generally increased. However,the primary antibody response is diminished in the elderly, resulting in lower antibody levels that decrease far sooner after reaching their peak than inyounger people.

Healthy elderly individuals show evidence of low-grade inflammatory activity in their blood with highcirculating levels of TNFa, IL-6 and the IL-1 receptorantagonist (IL-1Ra). Although the cause of this is un-clear, these proinflammatory cytokines may play a rolein the pathogenesis of age-related diseases such as

Alzheimer’s disease, Parkinson’s disease and cer-tainly atherosclerosis. The high levels of cytokines alsocontribute to muscle breakdown, which is found in theelderly, and increased osteoclastic activity resulting inosteoporosis. The low-grade inflammation may alsoplay a role in the depressed cell-mediated immune re-sponses by inhibiting T-cell activity through long-termactivation. Aging therefore is associated with impairedcellular immunity combined with low-grade chronicinflammation.

NK activity

T-cells

B-cellsArbi

trary

uni

ts

Age in years0 60 120

Arbi

trary

uni

ts

Age in years0 60 120

IL-1/6TNF

IL-2

Figure 9.6 Age trends in some immunological parameters. IL, in-terleukin; NK, natural killer; TNF, tumor necrosis factor. (Based onC., Franceschi, D., Monti, P., Sansoni & A., Cossarizza. (1995) Im-munology Today 16, 12.)

NEUTROPHILS

Decreased phagocytic activity

Decreased microbicidal activity

CELL-MEDIATED IMMUNITY

Decreased CD3+ cells

Increased Th2 subset and decreased Th1 subset

Decreased lymphocyte proliferation

Decreased CD28 expression

Decreased delayed type hypersensitivity

Increased production of pro-inflammatory cytokines

HUMORAL IMMUNITY

Increased autoantibodies

Decreased ability to generate primary immune responses

NATURAL KILLER CELLS

Increased percentage

Decreased cytotoxic activity

Table 9.1 Immune deficiencies observed in normal elderly individuals.


REVISION

Control by antigen• Immune responses are largely antigen driven and as thelevel of exogenous antigen falls, so does the intensity of theresponse.

Feedback control by antibody• Immunoglobulin G (IgG) antibodies inhibit responsesvia the Fcg receptor on B-cells.

T-cell regulation• Activated T-cells express Fas and FasL (Fas ligand)which can restrain unlimited clonal expansion.• The costimulatory molecule CD28 is replaced by the in-hibitory molecule CTLA-4 (cytotoxic T-lymphocyte anti-gen-4) following T-cell activation.• During the immune response regulatory T-cells emergewhich suppress T-helpers, presumably as feedback con-trol of excessive T-helper cell (Th) expansion.

Idiotype networks• Antigen-specific receptors on lymphocytes can interactwith the idiotypes on the receptors of other lymphocytesto form a network (Jerne).

Genetic factors influence the immune response• A number of genes control the overall antibody re-sponse to complex antigens: some affect macrophage anti-gen processing and microbicidal activity and some the rateof proliferation of differentiating B-cells.

Immunoneuroendocrine networks• Immunologic, neurologic and endocrinologic systemscan all interact.• The field of psychoimmunology examines the state ofthe immune system in individuals living through acute orchronic stressful episodes.• Stress may result in diminished natural killer (NK) cellactivity, antibody production and cellular immunity, re-sulting in increased susceptibility to infection and de-pressed would healing.• Estrogens may be largely responsible for the more ac-tive immune responses in females relative to males.

Effects of aging and diet on immunity• Protein–calorie malnutrition grossly impairs cell-mediated immunity and phagocyte microbicidal potency.• The elderly exhibit impaired immune responses to in-fectious agents.• The pattern of cytokines produced by peripheral blood cells changes with age, interleukin-2 (IL-2) decreasing and tumor necrosis factor-a (TNFa), IL-1 andIL-6 increasing.

Malnutrition diminishes the effectiveness of theimmune response

The greatly increased susceptibility of undernour-ished individuals to infection can be attributed tomany factors including a stressful lifestyle, poor sani-tation and personal hygiene, overcrowding and inade-quate health education. But, in addition, there aregross effects of protein calorie malnutrition on im-munocompetence. The widespread atrophy of lym-phoid tissues and the 50% reduction in circulating CD4 T-cells underlies serious impairment of cell-

mediated immunity. Antibody responses may be in-tact but they are of lower affinity; phagocytosis of bac-teria is relatively normal but the subsequentintracellular destruction is defective.

It should be remembered that undernutrition is verycommon amongst the elderly and further depressescell-mediated immunity including lymphocyte prolif-eration, cytokine synthesis and decreased antibody responses to vaccine. The immunosuppression of theelderly is further enhanced by zinc deficiency, which isextremely frequent in aged individuals.



FURTHER READING

Bruunsgaard, H., Pederson, M. & Pederson, B.K. (2001) Aging andproinflammatory cytokines. Current Opinion in Hematology, 8,131–6.

Chandra, R.K. (1998) Nutrition and the immune system. In: Encyclo-pedia of Immunology (eds P.J. Delves & I.M. Roitt ), 2nd edn, pp.1869–71. Academic Press, London. [See also other relevant articlesin the Encyclopedia: “Cinader, B. Aging and the immune system,”pp. 4559–61; “Cohen, N., Moynihan, J.A., Ader, R. Behavioural

regulation of immunity,” pp. 336–40; “Yamamoto, N. Vitamin Dand the immune response,” pp. 2494–9.]

Lesourd, B.M., Mazari, L. & Ferry, M. (1998) The role of nutrition inimmunity in the aged. Nutrition Reviews, 56, S113–25.

Padgett, D.A. & Glaser, R. (2003) How stress influences the immuneresponse. Trends in Immunology, 24, 444–8.

Selin, L.K. (ed.) (2004) Section on lymphocyte effector function. Cur-rent Opinion in Immunology, 16, 257–83.

Swain, S.L. & Cambier, J.C. (eds) (1996) Lymphocyte activation andeffector functions. Current Opinion in Immunology, 8, 309–418.

105

THE MULTIPOTENTIAL HEMATOPOIETICSTEM CELL GIVES RISE TO THE FORMEDELEMENTS OF THE BLOOD

Hematopoiesis originates in the early yolk sac and, asembryogenesis proceeds, this function is taken over bythe fetal liver and finally by the bone marrow where itcontinues throughout life. The hematopoietic stem cellwhich gives rise to the formed elements of the blood(figure 10.1) can be shown to be multipotent, to seedother organs and to have a relatively unlimited capa-city to renew itself through the creation of further stemcells. Thus, an animal can be completely protectedagainst the lethal effects of high doses of radiation byinjection of stem cells which will repopulate its lym-phoid and myeloid systems. This forms the basis ofhuman bone marrow transplantation.

We have come a long way towards the goal of isolat-ing highly purified populations of hematopoietic stemcells, although not all agree that we have yet achievedit. CD34 is a marker of an extremely early cell but thereis some debate as to whether this identifies the holypluripotent stem cell itself. The stem cells differentiatewithin the microenvironment of stromal cells whichproduce various growth factors such as interleukin-3(IL-3), IL-4, IL-6, IL-7, granulocyte-macrophagecolony-stimulating factor (GM-CSF) and others (fig-ure 10.1).

THE THYMUS PROVIDES THE ENVIRONMENTFOR T-CELL DIFFERENTIATION

The thymus is organized into a series of lobules basedupon meshworks of epithelial cells which form well-defined cortical and medullary zones (figure 10.2).This framework of epithelial cells provides the mi-croenvironment for T-cell differentiation. There aresubtle interactions between the extracellular matrixproteins and a variety of integrins on different lym-phocyte subpopulations which, together withchemokines and chemokine receptor expression, playa role in the homing of progenitors to the thymus andtheir subsequent migration within the gland. In addi-tion, the epithelial cells produce a series of peptide hor-mones including thymulin, thymosin a1, thymichumoral factor (THF) and thymopoietin, which are capable of promoting the appearance of T-cell diffe-rentiation markers.

The specialized large epithelial cells in the outer cor-tex are known as “nurse” cells because they can each beassociated with large numbers of lymphocytes whichappear to be lying within their cytoplasm. The epithe-lial cells of the deep cortex have branched dendriticprocesses rich in class II major histocompatibility com-plex (MHC). They connect through desmosomes toform a network through which cortical lymphocytesmust pass on their way to the medulla (figure 10.2). Thecortical lymphocytes are densely packed compared

CHAPTER 10

Ontogeny

The multipotential hematopoietic stem cell givesrise to the formed elements of the blood, 105

The thymus provides the environment for T-celldif ferentiation, 105

T-cell ontogeny, 106Differentiation is accompanied by changes in surface

markers, 106Receptor rearrangement, 107Cells are positively selected for self MHC restriction in the

thymus, 107Self-reactive cells are removed in the thymus by negative

selection, 108

T-cell tolerance, 109The induction of immunologic tolerance is necessary to avoid

self-reactivity, 109Dendritic cells are important in the induction of T-cell

tolerance, 109Development of B-cell specificity, 109

The sequence of immunoglobulin gene rearrangements, 109The importance of allelic exclusion, 111

Natural kil ler (NK) cell ontogeny, 111The overall response in the neonate, 111


with those in the medulla, many are in division andlarge numbers are undergoing apoptosis as a result ofpositive and negative selection (see later). Anumber ofbone marrow-derived interdigitating dendritic cells(DCs) are present in the medulla and the epithelial cellshave broader processes than their cortical counter-parts and express high levels of both class I and class IIMHC.

In the human, thymic involution commences withinthe first 12 months of life, reducing by around 3% ayear to middle age and by 1% thereafter. The size of theorgan gives no clue to these changes because there isreplacement by adipose tissue. In a sense, the thymusis progressively disposable because, as we shall see, itestablishes a long-lasting peripheral T-cell pool whichenables the host to withstand loss of the gland without

catastrophic failure of immunologic function, witnessthe minimal effects of thymectomy in the adult com-pared with the dramatic influence in the neonate.

T-CELL ONTOGENY

Differentiation is accompanied by changes insurface markers

T-lymphocytes originate from hematopoietic stemcells which express a number of chemokine receptorsand are attracted to the thymus by chemokines secret-ed by the thymic stromal cells. The T-cell precursorsstain positively for CD34 and for the enzyme terminaldeoxynucleotidyl transferase (TdT) (figure 10.3),which inserts nontemplated (“N-region”) nucleotides

MULTIPOTENTIALSTEM CELL

COMMONPROGENITORS

SPECIALIZEDPROGENITORS

DIFFERENTIATEDPROGENY

M-CSF GM-CSFPROMONOCYTE

GRANULOCYTE

B-CELL

T-CELL

LYMPHOID

MYELOID

IL-2IL-4IL-5IL-6

IL-3IL-3GM-CSF

STEMCELL

LIF

MONOCYTE/Mf

NEUTROPHIL

EOSINOPHIL

BASOPHIL

MAST CELL

B1

B2

ab CD4 Th

ab CD8 Tc

gd T

NK

IL-3IL-6

EPO

MEGAKARYOCYTE

ERYTHROCYTE

PLATELET

RETICULOCYTE

DENDRITIC CELL

SCFTGFb?

IL-1IL-3IL-6

IL-7

EPO

IL-11 TPO

GM-CSF

TPO

GM-CSF

IL-2IL-3IL-7IL-11

IL-1IL-7TNF

IL-2IL-4IL-7

IL-2 IL-5 IL-21

GM-CSF G-CSF

IL-5 GM-CSF

IL-4 IL-9 GM-CSF

IL-9?

IL-9? IL-10?

SELFRENEWAL

Figure 10.1 The multipotential hematopoietic stem cell and itsprogeny which differentiate under the influence of a series ofgrowth factors within the microenvironment of the bone marrow.EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor;GM-CSF, granulocyte-macrophage colony-stimulating factor, socalled because it promotes the formation of mixed colonies of thesetwo cell types from bone marrow progenitors either in tissue culture

or on transfer to an irradiated recipient where they appear in thespleen; IL-3, interleukin-3, often termed the multi-CSF because itstimulates progenitors of platelets, mast cells and all the other typesof myeloid and erythroid cells; LIF, leukemia inhibitory factor; Mf,macrophage; M-CSF, macrophage colony-stimulating factor; NK,natural killer; SCF, stem cell factor; TGFb, transforming growth factor-b; TNF, tumor necrosis factor; TPO, thrombopoietin.

CHAPTER 10—Ontogeny 107

at the junctions between the V, D and J variable regionsegments to increase diversity of the T-cell receptors(TCRs). Under the influence of IL-1 and tumor necrosisfactor (TNF) the T-cell precursors differentiate intoprothymocytes, committed to the T-lineage. At thisstage the cells begin to express various TCR chains andare then expanded, ultimately synthesizing CD3, theinvariant signal-transducing complex of the TCR, andbecoming double-positive for CD4+ and CD8+. Finally, under the guiding hand of chemokines, thecells traverse the cortico-medullary junction to themedulla, where they appear as separate immunocom-petent populations of single-positive CD4+ T-helpersand CD8+ cytotoxic T-cell precursors. The gd cells re-main double-negative, i.e. CD4–8–, except for a smallsubset which express CD8.

Receptor rearrangement

The development of TCRs

The earliest T-cell precursors have TCR genes in thegerm-line configuration, and the first rearrangementsto occur involve the g and d loci. The ab receptors areonly detected a few days later. The Vb is first

rearranged in the double-negative CD4–8– cells and associates with an invariant pre-a-chain and the CD3molecules to form a single “pre-TCR.” Expression ofthis complex leads the pre-T-cells to proliferate and become double-positive CD4+8+ cells. Further devel-opment now requires rearrangement of the Va genesegments so allowing formation of the mature ab TCR,and the cells are now ready for subsequent bouts ofpositive and negative receptor editing, as will be dis-cussed shortly. Rearrangement of the Vb genes on thesister chromatid is suppressed by a process called allelic exclusion so that each cell only expresses a sin-gle TCR b chain.

Cells are positively selected for self MHCrestriction in the thymus

The ability of T-cells to recognize antigenic peptides inassociation with self MHC is developed in the thymusgland. A small proportion of the double-positive(CD4+8+) T-cells bearing their TCR will bind with lowavidity to MHC molecules expressed on thymic corti-cal epithelial cells and will be positively selected tocomplete their progress to mature T-cells. The rest ofthe cells that do not recognize their own MHC are

CAPSULEAPOPTOTIC

CELLDIVIDING

LYMPHOCYTE NURSE CELL

CORTEX

CORTICAL DENDRITICEPITHELIAL CELL

MEDULLA

SEPTUMINTERDIGITATINGCELL

HASSALL'SCORPUSCLE

LYMPHOBLAST

DENSEAGGREGATES OFCORTICAL SMALLLYMPHOCYTES

MACROPHAGE

SPARSERMEDULLARYSMALLLYMPHOCYTES

MEDULLARYEPITHELIAL CELLFigure 10.2 Cellular features of a thymus

lobule. See text for description. (Adaptedfrom L.E. Hood, I.L. Weissman, W.B. Wood& J.H. Wilson (1984) Immunology, 2nd edn,p. 261. Benjamin Cummings, California.)


eliminated and will die in 3 or 4 days. Another featureof this selection phase of T-cell development is thatCD4+8+ cells bearing TCR which recognize self MHCon the epithelial cells are positively selected for differ-entiation to CD4+8– or CD4–8+ single-positive cells. Acell coming into contact with self MHC class I mole-cules will mature as a CD8+ cell, whereas if contact ismade with MHC class II molecules the cells will dev-elop as CD4+ T-cells. In the rare immunodeficiency dis-order called the bare lymphocyte syndrome, MHC

molecules do not appear on the surface of cells. As a result CD8+ cells will not develop in those cases whichlack class I MHC, while CD4+ cells fail to appear inthose cases without class II MHC molecules.

Self-reactive cells are removed in the thymus bynegative selection

Many of the cells that survive positive selection havereceptors for self antigen and if allowed to mature

4–8– 4+ 8+

abTCR

4+

gdTCR

4–8–4–8– 4+ 8+ 8+

Class Irestrictedcytotoxic

4–8– 4+ 8+

abTCR

4–8–4–8–

pre TCR(preTa+b)gdTCR

anti-self anti-self anti-self

34+44+117+TdT+

EXTRA THYMICTISSUE

THYMICCORTEX

THYMICMEDULLA

PERIPHERY ImmunocompetentT-cells

MHC restriction

or CD8+

Active proliferationand early differentiationof PROTHYMOCYTESReceptorrearrangementin PRE-T-CELLS

Chemotactic attractionof stem cell to thymus

Class IIrestricted

helper8+4+4–8–

abgd

abTCR

Proliferation

Expanded doublepositive CD4+8+

population

Mature abTCRreceptorrearrangement

Self tolerance throughNEGATIVE SELECTIONi.e. elimination orinactivation of anti-self-cells reacting withintrathymic Ag+MHC

3+5+

ab

throughPOSITIVE SELECTIONi.e. expansion of self-MHC reacting cellsDifferentiation to singlepositive CD4+

gdTCR

*Mainlynon-MHCrestricted

Figure 10.3 Differentiation of T-cells within the thymus. Num-bers refer to CD designation. TdT, terminal deoxynucleotidyl trans-ferase. Negatively selected cells in gray. The diagram is partlysimplified for the sake of clarity. *gd cells mainly appear to recognize

antigen directly, in a manner analogous to the antibody molecule onB-cells, although some may be restricted by major histocompatibili-ty complex (MHC) class I or II.


would produce immune responses to autoantigens.These thymocytes therefore undergo negative selec-tion, which is crucial for maintaining tolerance to selfantigens. In this process self antigens are presented tothe maturing thymocytes by DCs or macrophages, andany cell responding with high avidity is eliminated bythe induction of apoptosis. The result of positive andnegative selection is that all mature T-cells in thethymic medulla will recognize foreign peptide only inthe context of self MHC, and will not have the potentialto mount immune responses against self antigens andin the case of ab TCR cells, will either be CD4 or CD8positive.

T-CELL TOLERANCE

The induction of immunologic tolerance isnecessary to avoid self-reactivity

In essence, lymphocytes recognize foreign antigensthrough complementarity of shape mediated by the intermolecular forces we have described previously.To a large extent the building blocks used to form microbial and host molecules are the same, so it is theassembled shapes of self and nonself molecules whichmust be discriminated by the immune system if potentially disastrous autoreactivity is to be avoided.Many self-reacting T-cells are eliminated by apoptosisduring negative selection in the thymus and others aretolerized in the periphery; for example, by being madeanergic (functionally inactivated) following contactwith self antigens in a “nonstimulatory” context. Therestriction of each lymphocyte to a single specificitymakes the job of establishing self-tolerance that mucheasier, simply because it just requires a mechanismwhich functionally deletes self-reacting cells andleaves the remainder of the repertoire unscathed. Themost radical difference between self and nonself mole-cules lies in the fact that, in early life, the developinglymphocytes are surrounded by self and normallyonly meet nonself antigens at a later stage and thenwithin the context of the adjuventicity and cytokine release characteristic of infection. With its customaryefficiency, the blind force of evolution has exploitedthese differences to establish the mechanisms of immunologic tolerance to host constituents.

Dendritic cells are important in the induction of T-cell tolerance

We have previously described how DCs, when encountering foreign antigens or cytokines from an inflamed site, become activated and develop costim-

ulatory molecules important in the activation of T-cells. In the absence of microorganisms or inflamma-tion, DCs will capture the remains of cells that diephysiologically and present these to autoreactive T-cells. However, because they are not activated theywill present tissue antigens without costimulatorymolecules resulting in T-cell anergy rather than T-cellactivation.

DEVELOPMENT OF B-CELL SPECIFICITY

The B-lymphocyte precursors, pro-B-cells, are presentin the fetal liver by 8–9 weeks gestation. Production ofB-cells by liver gradually wanes and is mostly takenover by bone marrow for the remainder of life.

The sequence of immunoglobulin gene rearrangements

Stage 1 Initially, the D–J segments on both heavychain coding regions (one from each parent) rearrange(figure 10.4).

Stage 2 A V–DJ recombinational event now occurs onone heavy chain. If this proves to be a nonproductiverearrangement (i.e. adjacent segments are joined in anincorrect reading frame or in such a way as to generatea termination codon downstream from the splicepoint), then a second V–DJ rearrangement will occuron the sister heavy chain region. If a productive re-arrangement is not achieved, we can wave the pre-B-cell a fond farewell.

Stage 3 Assuming that a productive rearrangement ismade, the pre-B-cell can now synthesize m heavychains. At around the same time, two genes, VpreB andl5, with homology for the VL and CL segments of l-light chains respectively, are temporarily transcribedto form a “pseudo light chain,” which associates with the m chains to generate a surface surrogate “immunoglobulin M (IgM)” receptor together withthe Ig-a and Ig-b chains conventionally required toform a functional B-cell receptor. This surrogate receptor closely parallels the pre-Ta/b receptor on pre-T-cell precursors of ab TCR-bearing cells.

Stage 4 The surface receptor is signaled, perhaps by astromal cell, to suppress any further rearrangement ofheavy chain genes on a sister chromatid. This is termedallelic exclusion.

Stage 5 It is presumed that the surface receptor nowinitiates the next set of gene rearrangements which


PROGENITOR

STAGE 1

GERM LINEIg GENES

DJHREARRANGEMENT

VDJHREARRANGEMENT

V

V

J

J

D

D

CH

CH

(MATERNAL)

(PATERNAL)

V

V

CH

CH

DJ

DJ

V

CH

CHDJ

VDJOR

CHV~DJ

CHVDJ

SYNTHESIS OF SURROGATESURFACE ‘IgM’ RECEPTOR

CHVDJ

s ‘IgM’

�5

ALLELIC EXCLUSIONOF SISTER H GENES

CHVDJ

DJV CH

s ‘IgM’ EXTERNAL SIGNAL

–

VJL REARRANGEMENTsIgM SYNTHESIS CkVJ

JV

+

Ck

CkV~J

VJ Ck

k s IgM

m+

ALLELIC EXCLUSIONOF REMAINING LIGHTCHAIN GENES

CkVJ

JV Ck

JV

JV Cl

Cl

s IgM–

–

STAGE 2

STAGE 3

STAGE 4

STAGE 5

STAGE 6

m

‘L’

s ‘IgM’

OR

s ‘IgM’

+

VDJ = productive rearrangement; V~DJ = non-productive rearrangement

VpreB

Figure 10.4 Postulated sequence of B-cell gene rearrangements and mechanism of allelic exclusion (see text).


occur on the k light chain gene loci. These involve V–Jrecombinations on first one and then the other k alleleuntil a productive Vk–J rearrangement is accom-plished. Were that to fail, an attempt would be made toachieve productive rearrangement of the l alleles. Synthesis of conventional surface IgM (sIgM) now proceeds.

Stage 6 The sIgM molecule now prohibits any furthergene shuffling by allelic exclusion of any unrearrangedlight chain genes. The further addition of surface IgDnow marks the readiness of the virgin (naive) B-cell forpriming by antigen. B-cells bearing self-reactive recep-tors of moderately high affinity are eliminated by anegative selection process akin to that operating on autoreactive T-cells in the thymus.

Following encounter with specific antigen, the naiveB-cell can develop into an IgM-secreting plasma cell, orcan undergo class switching in which case the surfaceIgM and IgD will be replaced with, usually, a singleclass of immunoglobulin (IgG, IgA or IgE). At the ter-minal stages in the life of a fully mature plasma cellonly the secreted version of the Ig molecule is madewith virtually no sIg present.

The importance of allelic exclusion

Since each cell has chromosome complements derivedfrom each parent, the differentiating B-cell has fourlight- and two heavy-chain gene clusters to choosefrom. We have described how once a productive VDJDNA rearrangement has occurred within one heavy-chain cluster, and a productive VJ rearrangement inone light-chain cluster, the V genes on the other fourchromosomes are held in the germ line configurationby an allelic exclusion mechanism. Thus, the cell is ableto express only one light and one heavy chain. This isessential for clonal selection to work since the cell isthen programmed only to make the one antibody ituses as a cell surface receptor to recognize antigen.

NATURAL KILLER (NK) CELL ONTOGENY

The precise lineage of NK cells is still to be established.They share a common early progenitor with T-cellsand express the CD2 molecule which is also present onT-cells. Furthermore, they have IL-2 receptors, are dri-ven to proliferate by IL-2 and produce interferon g.However, they do not develop in the thymus and theirTCR V genes are not rearranged. The current view istherefore that they separate from the T-cell lineagevery early on in their differentiation.

THE OVERALL RESPONSE IN THE NEONATE

Lymph node and spleen remain relatively under-developed in the fetus except where there has been intrauterine exposure to antigens, as in congenital in-fections with rubella or other organisms. The ability toreject grafts and to mount an antibody response is rea-sonably well developed by birth, but the immunoglob-ulin levels, with one exception, are low, particularly inthe absence of intrauterine infection. The exception isIgG, which is acquired by placental transfer from themother, a process dependent upon Fc structures spe-cific to this Ig class. Maternal IgG is catabolized with ahalf-life of approximately 30 days so that serum levelsfall over the first 3 months, accentuated by the increasein blood volume of the growing infant. Thereafter therate of synthesis overtakes the rate of breakdown ofmaternal IgG and the overall concentration increasessteadily. The other immunoglobulins do not cross theplacenta and the low but significant levels of IgM incord blood are synthesized by the baby (figure 10.5).Immunoglobulin M reaches adult levels by 9 monthsof age. Only trace levels of IgA, IgD and IgE are presentin the circulation of the newborn.

-3 0 6 12 180

50

100IgM

IgG

IgA,IgD,IgE

BIRTH

INFANT MOTHERAge (months)

% A

dult

leve

l

Figure 10.5 Development of serum immunoglobulin levels in thehuman. (After J.R. Hobbs (1969) In: Immunology and Development (ed.M. Adinolfi), p. 118. Heinemann, London.)


REVISION

Multipotential stem cells from the bone marrow give rise to allthe formed elements of the blood• Expansion and differentiation are driven by solublegrowth (colony-stimulating) factors and contact withreticular stromal cells.

The differentiation of T-cells occurs within themicroenvironment of the thymus• Precursor T-cells arising from stem cells in the marrowneed to travel to the thymus under the influence ofchemokines. There they become immunocompetent T-cells.• Numerous lymphocytes undergo apoptosis either due to not being positively selected or due to negative selection.• Thymic involution commences within the first 12months of life.

T-cell ontogeny• Differentiation to immunocompetent T-cell subsets isaccompanied by changes in the surface phenotype whichcan be recognized with monoclonal antibodies.• CD34 positive stem cells differentiate into prothymo-cytes and begin expressing various T-cell receptor (TCR)chains and CD3.• Double-negative CD4–8– pre-T-cells are expanded to be-come double-positive CD4+8+.• As the cells traverse the cortico-medullary junctionunder the influence of chemokines, they become eitherCD4+ or CD8+.

Receptor rearrangement• The first TCR rearrangement involves the g and d loci.• Rearrangements of first the Vb locus and later the Valocus form the mature ab TCR.

T-cells are positively and negatively selected in the thymus• The thymus epithelial cells positively select CD4+8+ T-cells with avidity for their major histocompatibility com-plex (MHC) haplotype so that single-positive CD4+ orCD8+ T-cells develop that are restricted to the recognitionof antigen in the context of the epithelial cell haplotype.

• The rest of the cells that do not recognize their ownMHC are eliminated.• Cells that have receptors for self antigen undergo negative selection and are also eliminated.

T-cell tolerance• The induction of immunological tolerance is necessaryto avoid self-reactivity.• Many T-cells reacting with self antigens are eliminatedby negative selection in the thymus. Others are tolerized inthe periphery when they come into contact with self anti-gens in the absence of costimulatory molecules, as whenthey are presented by non-activated dendritic cells (DCs).

Development of B-cell specificity• The sequence of immunoglobulin (Ig) heavy chain vari-able gene rearrangements is D to J and then V to DJ.• VDJ transcription produces m chains which associatewith VpreB.l5 chains to form a surrogate surface IgM-likereceptor.• This receptor signals allelic exclusion of unrearrangedheavy chains.• If the rearrangement at any stage is unproductive, i.e.does not lead to an acceptable gene reading frame, the allele on the sister chromosome is rearranged.• The next set of gene rearrangements is V to J on the klight gene, or, if this fails, on the l light chain gene.• The cell now develops a commitment to producing aparticular class of antibody.• The mechanisms of allelic exclusion ensure that eachlymphocyte is programmed for only one antibody (figure10.4).

The overall response in the neonate• Maternal IgG crosses the placenta and provides a highlevel of passive immunity at birth.



FURTHER READING

Banchereau, J., Paezesny, S., Blanco, P. et al. (2003) Dendritic cells:Controllers of the immune system and a new promise for im-munotherapy. Annals of the New York Academy of Sciences, 987,180–7.

von Boehmer, H. (2000) T-cell lineage fate: Instructed by receptor sig-nals? Current Biology, 10, R642–5.

Hardy, R.R. & Hayakawa, K. (2001) B cell development pathways.Annual Review of Immunology, 19, 595–621.

Herzenberg, L.A. (2000) B-1 cells, the lineage question revisited. Immunological Reviews, 175, 9–22.

Kamradt, T. & Mitchison, N.A. (2001) Tolerance and autoimmunity.New England Journal of Medicine, 344, 655–64.

Phillips, R.L., Ernst, R.E., Brunk, B. et al. (2000) The genetic programof hematopoietic stem cells. Science, 288, 1635–40.

leukin-1 (IL-1) and tumor necrosis factor (TNF), whichupregulate the expression of adherence molecules onboth endothelial and white cells, and chemokines,which lead the leukocytes to the inflamed site.

Initiation of the acute inflammatory response

Avery early event in inflammation is the upregulationof the adhesion molecules E-selectin on endotheliumand L-selectin on leukocytes. Engagement of P-selectin with ligands on polymorphonuclear neu-trophils (PMNs) causes a leukocyte adhesion cascade.The cells are tethered to the endothelium and then rollalong it, followed by firm adhesion and migrationthrough the endothelium. Inflammatory mediatorsalso increase surface expression of the integrins LFA-1(named lymphocyte function-associated antigen-1,but in fact found on all leukocytes) and Mac-1. Underthe influence of cytokines, ICAM-1 (intercellular adhe-sion molecule-1) is induced on endothelial cells andbinds the various integrins on the PMN surface caus-ing PMN arrest (figure 11.1). Exposure of the PMN toIL-8 from endothelial cells and to various cytokinesproduced at the site of infection will also cause activa-tion of the PMN. This makes them more responsive tochemotactic agents, and they will exit from the circula-tion under the influence of C5a, leukotriene-B4 andchemokines (chemotactic cytokines; table 11.1). Theymove purposefully through the gap between endothe-lial cells, across the basement membrane (diapedesis)

114

INFLAMMATION REVISITED

The acute inflammatory process involves a protectiveinflux of white cells, complement, antibody and otherplasma proteins into a site of infection or injury andwas discussed in broad outline in the introductorychapters.

Mediators of inflammation

A complex variety of mediators are involved in acuteinflammatory responses. Some act directly on thesmooth muscle wall surrounding the arterioles to alterblood flow. Others act on the venules to cause contrac-tion of the endothelial cells with transient opening ofthe inter-endothelial junctions and consequent transu-dation of plasma. The migration of leukocytes from thebloodstream is facilitated by cytokines, such as inter-

CHAPTER 11

Adversarial strategies during infection

Inflammation revisited, 114Mediators of inflammation, 114Initiation of the acute inflammatory response, 114The ongoing inflammatory process, 115Regulation and resolution of inflammation, 116Chronic inflammation, 117

Extracellular bacteria susceptible to kil l ing byphagocytosis and complement, 117Bacterial survival strategies, 117The host counterattack, 117

Bacteria that grow in an intracellular habitat, 119

Cell-mediated immunity is crucial for the control of intracellularorganisms, 119

Activated macrophages kill intracellular parasites, 120Immunity to viral infection, 120

Innate immune mechanisms, 120Protection by serum antibody, 121Cell-mediated immunity gets to the intracellular virus, 121

Immunity to fungi, 123Immunity to parasit ic infections, 123

The host responses, 123

We are engaged in constant warfare with the microbes that surround us, and the processes ofmutation and evolution have tried to select microorganisms with the means of evading our defense mechanisms. In this chapter, we look at thevaried, often ingenious, adversarial strategieswhich our enemies and we have developed oververy long periods of time.

CHAPTER 11—Adversarial strategies during infection 115

and up the chemotactic gradient to the inflammationsite.

The ongoing inflammatory process

During the inflammatory process, not only are leuko-cytes recruited from the blood but also cells such asmacrophages and mast cells, which are pre-stationedin the tissue, will become activated. Mast cells respondearly to anaphylatoxins (C3a and C5a) produced bythe complement cascade and to bacteria and theirproducts. They will release histamine, platelet activat-ing factor (PAF), TNF, newly synthesized cytokinesand various chemokines. Tissue macrophages under

the stimulus of local infection or injury secrete an im-posing array of mediators. These include the cytokinesIL-1 and TNFa that stimulate the endothelial cells toupregulate the adhesion molecule E-selectin, and agroup of chemokines such as IL-8, which are highly ef-fective PMN chemotaxins. In general, chemokines ofthe C-X-C subfamily (defined in table 11.1) such as IL-8are specific for neutrophils and, to varying extents,lymphocytes, whereas chemokines with the C-C motifare chemotactic for monocytes and natural killer (NK)cells, basophils and eosinophils. Eotaxin (CCL11) ishighly specific for eosinophils and the presence of sig-nificant concentrations of this mediator together withRANTES (regulated upon activation, normal T-cell

1 2 3

4 5 6

PMN

ROLLING ACTIVATION STABLE ADHESION

DIAPEDESIS LATE CYTOKINE ACTIVATION LATE DIAPEDESIS

CHEMOTAXIS IL-1, TNF, LPS CHEMOTAXIS

E-Selectin

ESL-1

IL-8

HISTAMINETHROMBIN

BASEMENT MEMBRANE

HISTAMINETHROMBIN

C5aLTB4

ICAM-1

LFA-1

PAF

PAF-R

P-Selectin

PSGL-1

P-Selectin

PSGL-1

PSG

L-1

LFA-

1

ENDOTHELIALCELLS

Blood

Figure 11.1 Early events in inflammation affecting neutrophilmargination and diapedesis. Induced upregulation of P-selectin on the vessel walls plays the major role in the initial leukocyte–endothelial interaction (rolling) by interaction with ligands on theneutrophil such as the mucin-like P-selectin glycoprotein ligand-1(PSGL-1, CD162). Recognition of extracellular gradients of thechemotactic mediators by receptors on the polymorphonuclear neu-trophil (PMN) surface triggers intracellular signals which generate

motion. The cytokine-induced expression of E-selectin, which is recognized by the glycoprotein E-selectin ligand-1 (ESL-1) on theneutrophil, occurs as a later event. Chemotactic factors such as inter-leukin-8 (IL-8), which is secreted by a number of cell types includingthe endothelium itself, are important mediators of the inflammatoryprocess. ICAM-1, intercellular adhesion molecule-1; LFA-1, lym-phocyte function-associated antigen-1; LPS, lipopolysaccharide;PAF, platelet activating factor; TNF, tumor necrosis factor.

PART 4—Immunity to infection116

expressed and secreted) in mucosal surfaces, could ac-count for the enhanced population of eosinophils inthose tissues.

Clearly this whole operation serves to focus the im-mune defenses around the invading microorganisms.These become coated with antibody, C3b and certainacute phase proteins and are ripe for phagocytosis bythe activated PMN and macrophages.

Of course it is beneficial to recruit lymphocytes tosites of infection and we should remember that en-dothelial cells in these areas express VCAM-1 (vascu-lar cell adhesion molecule-1), which acts as a homingreceptor for VLA-4 (very late antigen-4) -positive acti-vated memory T-cells. In addition, the endothelial cellsthemselves, when activated, release the chemokinesIL-8 and lymphotactin, which will attract lymphocytesto the site of the inflammatory response.

Regulation and resolution of inflammation

With its customary prudence, evolution has estab-lished regulatory mechanisms to prevent inflamma-tion from getting out of hand. The complement systemis controlled by a series of complement regulatory pro-

teins such as C1 inhibitor, the C3 control proteins factors H and I, complement receptor CR1, and decayaccelerating factor (DAF). Regulation of inflammatorycells is mediated by prostaglandin E2 (PGE2), trans-forming growth factor-b (TGFb), IL-10 and other cytokines, which were discussed more fully in Chapter8. Prostaglandin E2 is a potent inhibitor of lymphocyteproliferation and cytokine production by T-cells and macrophages. Transforming growth factor-b andIL-10 deactivate macrophages by inhibiting the pro-duction of reactive oxygen intermediates and down-regulating major histocompatibility complex (MHC)class II expression. Most importantly these cytokinesinhibit the release of TNF and other proinflammatorycytokines.

Endogenous glucocorticoids produced via the hypothalamic–pituitary–adrenal axis exert their anti-inflammatory effects both through the repression of anumber of genes, including those for proinflammatorycytokines and adhesion molecules, and the inductionof inhibitors of inflammation such as lipocortin-1, se-cretory leukocyte proteinase inhibitor and IL-1 recep-tor antagonist (IL-1Ra). Once the inflammatory agenthas been cleared, these regulatory processes will

Chemokine

Chemoattractant specificity

Neutrophils Basophils Eosinophils NK cells Monocytes Lymphocytes

ENA-78 (CXCL5)

IL-8 (CXCL8)

IP-10 (CXCL10)

NAP-2 (CXCL7)

Eotaxin

MCP-1 (CCL2)

MCP-2 (CCL8)

MCP-3 (CCL7)

MIP-1a (CCL3)

MIP-1b (CCL4)

RANTES (CCL5)

Lymphotactin (XCL1)

Fractalkine (CX3CL1)

+

+ +

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

C, lacks first and third cysteines of the motif; except for IP-10, the CXC motif is preceded by the amino acids E-L-R; CC, no intervening residue; CXC, chemokine with any amino acid X intervening between the first and second conserved cysteines of the four which characterize the chemokine structural motif; ENA-78, epithelial derived neutrophil attractant-78; IP-10, interferon-inducible protein-10; MCP, monocyte chemotactic proteins; MIP, macrophage inflammatory protein; NAP-2, neutrophil activating protein-2; RANTES, regulated upon activation normal T-cell expressed and secreted.

(Data summarized from Schall T.J. & Bacon K.B. (1994) Current Opinion in Immunology 6, 865.)

+

-1 (CCL11)

Table 11.1 Chemokines: leukocyte chemo-attractant specificities.


normalize the site. When the inflammation trauma-tizes tissues through its intensity and extent, TGFbplays a major role in the subsequent wound healing by stimulating fibroblast division and the laying downof new extracellular matrix elements forming scar tissue.

Chronic inflammation

If an inflammatory agent persists, either because of itsresistance to metabolic breakdown or through the in-ability of a deficient immune system to clear an infec-tious microbe, the character of the cellular responsechanges. The site becomes dominated by macrophageswith varying morphology: many have an activated appearance, some form arrays of what are termed “epithelioid” cells and others fuse to form giant cells.Collectively these macrophages produce the charac-teristic granuloma which walls off the persisting agentfrom the remainder of the body (see section on type IVhypersensitivity in Chapter 14, and figure 14.13). If anadaptive immune response is involved, lymphocytesin various guises will also be present.

EXTRACELLULAR BACTERIA SUSCEPTIBLETO KILLING BY PHAGOCYTOSIS AND COMPLEMENT

Bacterial survival strategies

The variety and ingenuity of escape mechanismsdemonstrated by bacteria are most intriguing and, aswith virtually all infectious agents, if you can think of apossible avoidance strategy, some microbe will al-ready have used it.

A common mechanism by which virulent forms es-cape phagocytosis is by synthesis of an outer capsule.This does not adhere readily to phagocytic cells andcovers carbohydrate molecules on the bacterial surfacethat could otherwise be recognized by phagocyte re-ceptors. Other organisms have actively antiphago-cytic cell surface molecules, and some go as far as tosecrete exotoxins, which actually poison the leuko-cytes. Many organisms have developed mechanismsto resist complement activation and lysis. For example,Gram-positive organisms have evolved thick peptido-glycan layers which prevent the insertion of the lyticC5b–9 membrane attack complex into the bacterial cellmembrane, and certain strains of group B streptococciactually produce a C5a-ase which cleaves and in-activates C5a.

The host counterattack

The defense mechanisms exploit the specificity andvariability of the antibody molecule. Antibodies candefeat these devious attempts to avoid engulfment byneutralizing the antiphagocytic molecules and bybinding to the surface of the organisms to focus the sitefor fixation of complement, so “opsonizing” the organ-isms for ingestion by polymorphs and macrophages orpreparing them for the terminal membrane attackcomplex.

Most antigen-presenting cells (APCs) have recep-tors on their surface, which detect bacterial compo-nents such as lipopolysaccharide. These receptorsinclude CD14 and Toll-like receptor 4 (TLR4) whichwhen activated lead to the expression of a broad rangeof proinflammatory genes. These include IL-1, IL-6, IL-12 and TNF and the B7.1 (CD80) and B7.2 (CD86)costimulatory molecules. A related receptor TLR2 recognizes Gram-positive bacterial cell wall components.

Toxin neutralization

Circulating antibodies neutralize the soluble anti-phagocytic molecules and other exotoxins released bybacteria. These antibodies, which in this context arealso called antitoxins, inhibit the binding of the toxinto specific receptors on target cells and thereby preventthe toxin from damaging tissue.

Opsonization of bacteria

Mannose-binding lectin (MBL) is a molecule with asimilar ultrastructure to C1q which can bind to termi-nal mannose on the bacterial surface and can lead to complement activation. Mannose-binding lectin does this by interacting with two MBL-associated serine proteases called MASP-1 and MASP-2 whichare homologous in structure to C1r and C1s. This leadsto antibody-independent activation of the classicalpathway. Encapsulated bacteria which resist phagocy-tosis become extremely attractive to polymorphs andmacrophages when coated with antibody and C3b,and their rate of clearance from the bloodstream isstrikingly enhanced (figures 11.2 & 11.3). Furthermore,complexes containing C3b may show immune adher-ence to the CR1 complement receptors on red cells toprovide aggregates which are transported to the liverfor phagocytosis.

Some elaboration on complement receptors may bepertinent at this stage. The CR1 receptors for C3b arealso present on neutrophils, macrophages, B-cells and


follicular dendritic cells in lymph nodes. Togetherwith the CR3 receptor, they have the main responsibili-ty for clearance of complexes containing C3.

CR2 receptors, which bind to various breakdownproducts of C3 such as C3dg and iC3b, are present onB-cells and follicular dendritic cells and transduce ac-cessory signals for B-cell activation, especially in thegerminal centers. Their affinity for the Epstein–Barrvirus (EBV) provides the means for entry of the virusinto the B-cell.

CR3 receptors on polymorphs, macrophages andNK cells all bind the inactivated form of C3b calledC3bi.

The secretory immune system protects the externalmucosal surfaces

We have earlier emphasized the critical nature of themucosal barriers, particularly in the gut where there isa potentially hostile interface with the gut flora. With ahuge surface area of around 400m2, the epithelium ofthe adult mucosae represents the most frequent portalof entry for common infectious agents, allergens andcarcinogens. The need for well-marshalled, highly effective mucosal immunity is glaringly obvious.

The mucosal surfaces are defended by both antigen-specific and nonantigen-specific mechanisms. The latter include a group of antibacterial peptides calleddefensins which are produced by neutrophils,

INNATE

ACQUIRED

100

10

1

0.1

0.010 1 2

C3badherence

+ C9 killing

Fc adherence

MICE

IN NORMAL MICE

Hours

% S

urvi

val o

f bac

teria

UNCOATED BACTERIA

Ab-COATEDBACTERIA INC'-DEFICIENT

Ab-COATED BACTERIAIN NORMAL MICE

MICE

Figure 11.2 Effect of opsonizing antibody and complement on rateof clearance of virulent bacteria from the blood. The uncoated bac-teria are phagocytosed rather slowly (innate immunity) but, on coat-ing with antibody, adherence to phagocytes is increased many-fold(acquired immunity). The adherence is less effective in animals tem-porarily depleted of complement. This is a hypothetical but realisticsituation; the natural proliferation of the bacteria has been ignored.Ab, antibody.

IgM + C3bIgG

IgG + C3b

IgA

C3bRECEPTORS

IgGRECEPTORS IgA

RECEPTORSNONSPECIFIC

BINDINGLECTIN

BINDING SITES

CH0

BACTERIUM

UNCOATED

IgM

Phagocyte

Figure 11.3 Coating with immunoglobulin (Ig) and complementgreatly increase the adherence of bacteria (and other antigens) tomacrophages and polymorphs. Uncoated bacteria adhere to lectin-like sites, including the mannose receptor. There are no specific bind-ing sites for IgM ( ) but there are high-affinity receptors for IgG (Fc) ( ) and iC3b ( : CR1 and CR3 types) on the macrophage surface which considerably enhance the strength of binding. The

augmenting effect of complement is due to the fact that two adjacentIgG molecules can fix many C3b molecules, thereby increasing thenumber of links to the macrophage. Although IgM does not bindspecifically to the macrophage, it promotes adherence through com-plement fixation. Specific receptors for the Fca domains of IgAhavealso been defined.


macrophages and mucosal epithelium. The produc-tion of these antibacterial substances is upregulated bycytokines such as IL-1 and TNF. Specific immunity isprovided by secretory immunoglobulin A (IgA) andIgM. The size of the task is highlighted by the fact that80% of the Ig-producing B-cells in the body are presentin the secretory mucosae and exocrine glands. Im-munoglobulin A antibodies afford protection in the external body fluids, tears, saliva, nasal secretions andthose bathing the surfaces of the intestine and lung, bycoating bacteria and viruses and preventing their ad-herence to the epithelial cells of the mucous mem-branes. In addition high-affinity Fc receptors for this Igclass have been identified on macrophages and poly-morphs and can mediate phagocytosis (figure 11.4).

If an infectious agent succeeds in penetrating theIgAbarrier, it comes up against the next line of defenseof the secretory system which is manned by IgE anti-bodies. It is worth noting that most serum IgE arisesfrom plasma cells in mucosal tissues and in the lymphnodes that drain them. Although present in low con-centration, IgE is bound very firmly to the Fc receptorsof the mast cell. Contact with antigen leads to the re-lease of mediators which effectively generate a localacute inflammatory reaction. Thus, histamine, by in-creasing vascular permeability, causes the transuda-tion of IgG and complement into the area, whilechemotactic factors for neutrophils and eosinophils at-tract the effector cells needed to dispose of the infec-tious organism coated with specific IgG and with C3b.Engagement of the Fcg and C3b receptors on localmacrophages by such complexes will lead to secretionof cytokines and chemokines which further reinforcethese vascular permeability and chemotactic events.

Where the opsonized organism is too large forphagocytosis, it can be killed by antibody-dependentcellular cytotoxicity (ADCC), discussed earlier (p. 23).This is particularly important in controlling parasiticinfections.

BACTERIA THAT GROW IN ANINTRACELLULAR HABITAT

Cell-mediated immunity is crucial for the controlof intracellular organisms

Some strains of bacteria, such as the tubercle and lep-rosy bacilli and Listeria and Brucella organisms, escapethe immune system by taking up residence insidemacrophages (figure 11.5). Entry of opsonized bacteriais facilitated by phagocytic uptake after attachment to

INFECTIOUS AGENT

IgA

PLASMACELL

LUMEN

MUCOSALSURFACE

sIgA

FcaR

Blockadherence

Opsonize

Figure 11.4 Defense of the mucosal surfaces. Immunoglobulin A(IgA) opsonizes organisms and prevents adherence to the mucosa.sIgA, secretory IgA.

1 2 3Inhibit lysosome fusion

Intracellular bacterium

Mf

LYSOSOME

Resist oxidative &lysosomal attack Escape from phagosome

PHAGOSOME

Figure 11.5 Evasion of phagocytic deathby intracellular bacteria. Mf, macrophage.


Fcg and C3b receptors but, once inside, many of themdefy the mighty macrophage by subverting the innatekilling mechanisms.

In an elegant series of experiments, Mackanessdemonstrated the importance of cell-mediated immu-nity (CMI) reactions for the killing of these intracellu-lar parasites and the establishment of an immune state.Animals infected with moderate doses of Mycobac-terium tuberculosis overcome the infection and are im-mune to subsequent challenge with the bacillus. Theimmunity can be transferred to a normal recipient bymeans of T-lymphocytes but not macrophages orserum from an immune animal. Supporting this view,that specific immunity is mediated by T-cells, is thegreater susceptibility of patients with T-cell defectssuch as human immunodeficiency virus (HIV) infec-tion to infection with mycobacteria or other intracellu-lar organisms.

Activated macrophages kill intracellular parasites

Resting macrophages can be activated in severalstages, but the ability to kill obligate intracellularmicrobes only comes after stimulation by macrophageactivating factor(s) such as interferon g (IFNg) releasedfrom stimulated cytokine-producing T-cells or NKcells. Primed T-cells react with processed antigenderived from the intracellular bacteria present on thesurface of the infected macrophage in association withMHC class II. The activated T-cells express CD40L(CD40 ligand) which reacts with CD40 on themacrophage, and, in the presence of IFNg, themacrophage is activated and endowed with the abilityto kill the organisms it has phagocytosed (figure 11.6).Foremost amongst the killing mechanisms which areupregulated, are those mediated by reactive oxygenintermediates and nitric oxide (NO). The activatedmacrophage is undeniably a remarkable and formi-dable cell, capable of secreting a vast array of cytokinesand microbicidal substances which are concerned inchronic inflammatory reactions (figure 11.7).

Where the host has difficulty in effectively eliminat-ing these organisms, the chronic CMI response to localantigen leads to the accumulation of densely packedmacrophages, which release angiogenic and fibro-genic factors and stimulate the formation of granula-tion tissue and ultimately fibrosis. The activatedmacrophages, perhaps under the stimulus of IL-4,transform to epithelioid cells and fuse to become giantcells. As suggested earlier, the resulting granulomarepresents an attempt by the body to isolate a site ofpersistent infection.

IMMUNITY TO VIRAL INFECTION

Innate immune mechanisms

Early in the infection, before the primary antibody re-sponse has developed, the rapid production of type 1interferons (IFNa and IFNb) by infected cells is themost significant mechanism used to counter the viral

a

MfCD4

IFNg

DEAD TB

ACTIVATEDNEW

IMMIGRANT

MfCD4

IL-2

NK

CD8

RELEASED TB

CYTOTOXICITY

INTRACELLULARKILLING

MfCD4

LIVE TB

IFNg

MfCD4

KILLEDTB

T-CELLMEDIATED

MfACTIVATION

INTRACELLULARKILLING

d

b

c

NO

ACTIVATED

NO

SENILE

Figure 11.6 The “cytokine connection”: nonspecific murinemacrophage killing of intracellular bacteria triggered by a specificT-cell-mediated immune reaction. (a) Specific CD4 Th1 cell recognizes mycobacterial peptide associated with major histo-compatibility complex (MHC) class II and releases macrophage(Mf)-activating interferon g (IFNg). (b) The activated Mf kills the intracellular tubercle bacilli (TB), mainly through generation of toxicnitric oxide (NO). (c) A“senile” Mf, unable to destroy the intracellu-lar bacteria, is killed by CD8 and CD4 cytotoxic cells and possibly byinterleukin-2 (IL-2) -activated natural killer (NK) cells. The Mf thenreleases live TB, which are taken up and killed by newly recruitedMf susceptible to IFNg activation.


infection. The production of IFNa during viral infec-tion not only protects surrounding cells but also acti-vates NK cells and upregulates MHC expression onthe adjacent cells. The surface of a virally infected cellundergoes modification, probably in its surface carbo-hydrate structures, making it an attractive target forNK cells. As described previously, NK cells possesstwo families of surface receptors. One binds to thenovel structures expressed by infected cells and theother recognizes specificities common to several MHCclass I alleles. The first activates killing but the recogni-tion of class I delivers an inhibitory signal to the NKcell and protects normal cells from NK attack. Thus,the sensitivity of the target cell is closely related to selfMHC class I expression. Virally infected cells, by in-hibiting protein synthesis, may block production ofclass 1 MHC molecules, thereby turning off the in-hibitory signal and allowing NK cells to attack. There-fore, whereas T-cells search for the presence of foreignshapes, NK cells survey tissues for the absence of selfas indicated by aberrant or absent expression of MHCclass I, which might occur in tumorigenesis or viral infections.

Protection by serum antibody

The antibody molecule can neutralize viruses by a variety of means. It may stereochemically inhibitcombination with the receptor site on cells, therebypreventing penetration and subsequent intracellularmultiplication . The protective effect of antibodies to

influenza virus is an example of this. Antibody may de-stroy a free virus particle directly through activation ofthe classical complement pathway or it may produceaggregation, enhanced phagocytosis and intracellulardeath by mechanisms already discussed. With someviral diseases, such as influenza and the common cold,there is a short incubation time as the final target organfor the virus is the same as the portal of entry. Antibody,as assessed by the serum titer, seems to arrive on thescene much too late to be of value in aiding recovery.However, antibody levels may be elevated in the localfluids bathing the infected surfaces, for example nasalmucosa and lung, despite low serum titers, and it is theproduction of antiviral antibody (most prominentlyIgA) by locally deployed immunologically primedcells which is of major importance for the prevention ofsubsequent infection. Unfortunately, in so far as thecommon cold is concerned, a subsequent infection islikely to involve an antigenically unrelated virus sothat general immunity to colds is difficult to achieve.

Cell-mediated immunity gets to the intracellularvirus

In Chapter 2 we emphasized the general point that antibody dealt with extracellular infective agents andCMI with intracellular ones. The same holds true forviruses which have established themselves in an intra-cellular habitat. Local or systemic antibodies can blockthe spread of cytolytic viruses released from the hostcell that they have just killed. Antibodies alone, how-

INFLAMMATIONAND FEVER

IL-1, TNFa, IL-6IFNb

LeukotrienesProstaglandins

Complement factorsClotting factors

LYMPHOCYTEACTIVATION

Antigen processingAntigen presentation

IL-1 production

TISSUEREORGANIZATION

Angiogenesis factorFibrogenesis factor

Elastase, CollagenaseHyaluronidase

TISSUE DAMAGE

H202Acid hydrolases

C3a

O2 dependent

H2O2, .O2–

.OH, hypohalite

O2 independent

NO.Lysozyme

Acid hydrolasesCationic proteins

Lactoferrin

TUMORICIDALACTIVITY

Direct cytotoxicityH2O2, C3aArginase

Cytolytic proteaseTNFa, NO.

MICROBICIDALACTIVITY

Figure 11.7 The role of the activatedmacrophage in the initiation and media-tion of chronic inflammation with con-comitant tissue repair, and in the killing ofmicrobes and tumor cells. It is possible thatmacrophages differentiate along distinctpathways to subserve these different func-tions. The electron micrograph shows ahighly activated macrophage with manylysosomal structures, which have beenhighlighted by the uptake of thorotrast; one(arrowed) is seen fusing with a phagosomecontaining the protozoan Toxoplasma gondii.(Photograph kindly supplied by ProfessorC. Jones.) IFNg, interferon g; IL-1, inter-leukin-1; NO, nitric oxide; TNF, tumornecrosis factor.


ever, are usually inadequate in controlling those viruses which bud off from the surface as infectiousparticles because they may spread to adjacent cellswithout becoming exposed to antibody (figure 11.8).The importance of CMI for recovery from infectionwith these agents is underlined by the inability of chil-dren with primary T-cell immunodeficiency to copewith such viruses, whereas patients with Ig deficiencybut intact CMI are not troubled in this way.

Cytotoxic T-cells (Tc) are crucial elements inimmunity to infection by budding viruses

T-lymphocytes from a sensitized host are directly cytotoxic to cells infected with viruses, the new MHC-associated peptide antigens on the target cell surfacebeing recognized by specific ab receptors on the ag-gressor CD8+ cytotoxic T-cells. Although gd T-cells,which recognize native viral coat protein (e.g. herpessimplex virus glycoprotein) on the cell surface mayplay a role in controlling viral infection, most cytotoxicT-cells are CD8+ ab T-cells (figure 11.8). Specific Tc cells

undergo significant proliferation during a viral infec-tion. They can usually be detected in the peripheralblood in 3–4 days and then decline as the infection iscontrolled. Studies on volunteers, showing that highlevels of cytotoxic activity before challenge with liveinfluenza correlated with low or absent shedding ofvirus, speak in favor of the importance of Tc in humanviral infection.

Cytokines recruit effectors and provide a “cordon sanitaire”

CD4+ T-cells also play an important role in antiviral defence by producing cytokines such as IFNg that hasantiviral activity, and IL-2 which recruits and activatesCD8+ cells. Cytokines can also be produced by CD8cells themselves, an activity that may well be crucialwhen viruses escape the cytotoxic mechanism andmanage to sidle laterally into an adjacent cell. T-cellsstimulated by viral antigen release cytokines such asIFNg and macrophage or monocyte chemokines. Themononuclear phagocytes attracted to the site will be

‘CYTOKINE-PRODUCING’ T-CELL

ChemotaxisMf

INFECTED

Budding

FREE VIRUSNeutralize& destroy

Attack

Attack

ANTIBODYCOMPLEMENT

Host membrane

Viral coat protein

CYTOTOXICab T-CELL

NKCELL

IFNgTNF

NON-PERMISSIVE

Class IMHC

CYTOTOXICgd T-CELL

Figure 11.8 Control of infection by “bud-ding” viruses. Free virus released by bud-ding from the cell surface is neutralized by antibody. Specific cytotoxic T-cells killvirally infected targets directly. Interac-tion with a (separate?) subpopulation of T-cells releases cytokines, which attractmacrophages, prime contiguous cells withinterferon g (IFNg) and tumor necrosis fac-tor (TNF) to make them resistant to viral in-fection, and activate cytotoxic natural killer(NK) cells. Natural killer cells are powerfulproducers of IFNg. They recognize a lack ofmajor histocompatibility complex (MHC)class 1 on the infected cell membrane. Theycan also lyse cells by antibody-dependentcellular cytotoxicity (ADCC) if antibody toviral coat protein is bound to the infectedcell. Mf, macrophage; TCR, T-cell receptor.


activated to secrete TNF, which will synergize with theIFNg to render the contiguous cells nonpermissive forthe replication of any virus acquired by intercellulartransfer (figure 11.8). In this way, a cordon of resistantcells can surround the site of infection. Like IFNa, IFNgmay also increase the nonspecific cytotoxicity of NKcells for infected cells. This generation of “immune in-terferon” (IFNg) and TNF in response to nonnucleicacid viral components provides a valuable back-upmechanism when dealing with viruses which are in-trinsically poor stimulators of interferon synthesis.

After a natural infection, both antibody and Tc cellsare generated; subsequent protection is long livedwithout reinfection. By contrast, injection of killed in-fluenza produces antibodies but no Tc and protectionis only short term.

IMMUNITY TO FUNGI

Many fungal infections become established in im-munocompromised hosts or when the normal com-mensal flora is upset by prolonged administration ofbroad-spectrum antibiotics. Patients with neutrophildeficiency or neutrophil dysfunction such as in chronicgranulomatous disease are particularly susceptible toCandida albicans and other fungi. Cell-mediated immu-nity is crucial in the defence against fungi as evidencedby the high opportunistic fungal infection rate in pa-tients with HIV infection. T-cells function primarily byactivating macrophages containing ingested organ-isms, but some T-cells and NK cells can exert a directcytotoxic effect on a number of organisms such asCryptococcus neoformans and Candida albicans.

IMMUNITY TO PARASITIC INFECTIONS

The consequences of infection with the major parasiticorganisms could be at one extreme a lack of immuneresponse leading to overwhelming superinfection,and at the other an exaggerated life-threatening im-munopathologic response. Such a response may occurwhen parasites persist chronically in the face of an immune reaction which frequently produces tissue-damaging reactions. One example is the immune complex-induced nephrotic syndrome of Nigerianchildren associated with quartan malaria. Another example is the liver damage resulting from IL-4-mediated granuloma formation around schistosomeeggs. Cross-reaction between parasite and self maygive rise to autoimmunity, and this has been proposedas the basis for the cardiomyopathy in Chagas’ disease.It is also pertinent that the nonspecific immunosup-

pression that is so widespread in parasitic diseasestends to increase susceptibility to superimposed bacte-rial and viral infections.

The host responses

Awide variety of defensive mechanisms are deployedby the host but the rough generalization may be madethat a humoral response develops when the organismsinvade the bloodstream (e.g. malaria, trypanosomia-sis), whereas parasites which grow within the tissues(e.g. cutaneous leishmaniasis) usually elicit CMI.

Humoral immunity

Antibodies of the right specificity present in adequateconcentrations and affinity are reasonably effective inproviding protection against blood-borne parasitessuch as Trypanosoma brucei and the sporozoite andmerozoite stages of malaria. Thus, individuals receiv-ing IgG from solidly immune adults in malaria endem-ic areas are themselves temporarily protected againstinfection, the effector mechanisms being phagocytosisof opsonized organisms, and complement-dependentlysis.

A marked feature of the immune reaction tohelminthic infections such as Trichinella spiralis is theeosinophilia and the high level of IgE antibody pro-duced. Serum levels of IgE can rise from normal valuesof around 100ng/ml to as high as 10000ng/ml. Thesechanges have all the hallmarks of a response to Th2-type cytokines, and it is notable that in animals in-fected with helminths, injection of anti-IL-4 greatlyreduces IgE production and anti-IL-5 suppresses theeosinophilia. It is relevant to note that schistosomules,the early immature form of the schistosome, can bekilled in cultures containing both specific IgG andeosinophils by the ADCC mechanism.

Cell-mediated immunity

Just like mycobacteria, many parasites have adaptedto life within the macrophage despite the possessionby that cell of potent microbicidal mechanisms includ-ing NO·. Intracellular organisms such as Toxoplasmagondii, Trypanosoma cruzi and Leishmania spp. use a variety of ploys to subvert the macrophage killing systems but again, as with mycobacterial infections,cytokine-producing T-cells are crucially important forthe stimulation of macrophages to release their killingpower and dispose of the unwanted intruders.

In vivo, the balance of cytokines produced may be of


the utmost importance. Infection of mice with Leishma-nia major is instructive in this respect: the organismproduces fatal disease in susceptible mice but other strains are resistant. In susceptible mice there isexcessive stimulation of Th2 cells producing IL-4,whereas resistant strains are characterized by the ex-pansion of Th1 cells which secrete IFNg in response toantigen presented by macrophages harboring livingprotozoa.

Organisms such as malarial plasmodia, rickettsiaeand chlamydiae that live in cells, which are not pro-fessional phagocytes, may be eliminated through activation of intracellular defense mechanisms byIFNg released from CD8+ T-cells or even by direct cytotoxicity.

When it comes to worm infection, a vigorous re-sponse by Th2 cells will release a variety of cytokines

which are responsible for the high IgE levels and theeosinophilia observed in these patients. This Th2 re-sponse is necessary for protection against these wormsand to suppress Th1 cytokine induced immunopathol-ogy. In numerous mouse experiments it has beenshown that IL-4 and IL-13 are crucial for expulsion ofgastrointestinal worms, and that IL-5 and theeosinophils that it promotes are important in destroy-ing larval forms of some worms as they migratethrough the body. Human worm infections are also associated with Th2 cytokine responses includingeosinophilia, mastocytosis and high levels of serumIgE (figure 11.9). Th2 cytokines also appear to protectagainst ectoparasites such as ticks, probably by de-granulation of mast cells, thereby initiating inflamma-tion and skin edema which prevent the tick fromlocating a host blood vessel.

INTESTINE

GUT LUMEN

B

T

CYTOKINESANTIGEN

NEMATODE

IgG

INFLAMMATORYRESPONSE

IgE

Metabolic damage Expulsion

Stimulategoblet cells

Figure 11.9 The expulsion of nematodeworms from the gut. The parasite is firstdamaged by immunoglobulin G (IgG) anti-body passing into the gut lumen, perhaps asa consequence of IgE-mediated inflamma-tion and possibly aided by accessory antibody-dependent cellular cytotoxicity(ADCC) cells. Cytokines released by antigen-specific triggering of T-cells stimu-late proliferation of goblet cells and secre-tion of mucous materials, which coat thedamaged worm and facilitate its expulsionfrom the body by increased gut motility in-duced by mast cell mediators, such asleukotriene-D4, and diarrhea resultingfrom inhibition of glucose-dependent sodium absorption by mast cell-derivedhistamine and prostaglandin E2 (PGE2). B, B-cell; T, T-cell.


REVISION

Immunity to infection involves a constant battle betweenthe host defenses and the mutant microbes trying to evolveevasive strategies. Specific acquired responses amplifyand enhance innate immune mechanisms.

Inflammation revisited• Inflammation is a major defensive reaction initiated byinfection or tissue injury.

The acute inflammatory response• The mediators released upregulate adhesion moleculessuch as P-selectin on endothelial cells. These pair with ligands on leukocytes, initially causing rolling of leuko-cytes along the vessel wall and then passage across theblood vessel up the chemotactic gradient to the site of inflammation.• Under the influence of interleukin-1 (IL-1) and tumornecrosis factor (TNF), ICAM-1 (intercellular adhesionmolecule-1) is induced on endothelial cells and binds to in-tegrins on polymorphonuclear neutrophil (PMN) surfacescausing their arrest.• Macrophages and mast cells present in the inflamed tissue are activated and release a variety of mediators, cytokines and chemokines. Endothelial cells themselvesmay release cytokines and chemokines.• Activated granulocytes and macrophages easily phago-cytose organisms coated with antibody and complementproteins.• Cytokines such as transforming growth factor-b (TGFb)and IL-10 are powerful regulators of inflammation and endogenous glucocorticoids.• When tissue is severely traumatized, TGFb may stimu-late fibroblasts to lay down collagen forming scar tissue,which is the end result of many inflammatory events.• Inability to eliminate the initiating agent leads to achronic inflammatory response dominated by macro-phages and often forming granulomas.

Extracellular bacteria susceptible to killing by phagocytosisand complement• Bacteria try to evade the immune response by sur-rounding themselves with capsules to avoid phagocyto-sis, secreting exotoxins which kill phagocytes or impedeinflammatory reactions, resisting insertion of the comple-

ment membrane attack complex, or by secreting enzymeswhich destroy C5.• Antibody combats these tricks by neutralizing the tox-ins, and by overcoming the antiphagocytic nature of thecapsules by opsonizing them with immunoglobulin G(IgG) and C3b.• Antigen-presenting cells (APCs) have receptors for microorganisms, such as the Toll-like receptors (TLRs),which when activated lead to the production of proinflam-matory cytokines.• Mannose binding lectin (MBL) binds to mannose onbacterial surfaces. In association with MASP-1 and MASP-2 it leads to a further pathway of complement activation.• Complement activation is controlled by a number of in-hibitory proteins and by various complement receptors.

Protection of mucosal surfaces• Defensins are antimicrobial proteins produced bymacrophages and mucosal cells. Their production is up-regulated by proinflammatory cytokines.• The secretory immune system protects the external mucosal surfaces. Immunoglobulin A inhibits adherenceof bacteria and can opsonize them.• Immunoglobulin E bound to mast cells can be found in mucosal tissue. In contact with antigen it will cause degranulation of the mast cells. This initiates release of mediators which generate a local inflammatory reaction.

Bacteria which grow in an intracellular habitat• Intracellular bacteria such as tubercle and leprosy bacilli grow within macrophages.• They are killed by cell-mediated immunity (CMI):specifically sensitized T-cells become activated and re-lease interferon g (IFNg) which activates the macrophageto kill the organisms.• When intracellular organisms are not destroyed, achronic inflammatory reaction will lead to the formationof a macrophage rich granuloma.

Immunity to viral infection• Infected cells release type 1 interferons which have antiviral activity.• Natural killer (NK) cells are activated by IFNg and IL-2.They can now attack virally infected cells which have

(continued)


FURTHER READING

Bloom, B. & Zinkernagel, R. (1996) Immunity to infection. CurrentOpinion in Immunology, 8, 465–6.

Brandtzaeg, P. (1995) Basic mechanisms of mucosal immunity: Amajor adaptive defense system. The Immunologist, 3, 89–96.

Finkelman, F.D. & Urban, J.F. (2001) The other side of the coin: Theprotective role of the Th2 cytokines. Journal of Allergy and ClinicalImmunology, 107, 772–80.

Ley, K. (2002) Integration of inflammatory signals by rolling neutrophils. Immunological Reviews, 186, 8–18.

Nathan, C. (2002) Points of control in inflammation. Nature, 420,846–52.

Price, D.A., Klenerman, P., Booth, B.L., Phillips, R.E. & Sewell, A.K.(1999) Cytotoxic T lymphocytes, chemokines and antiviral immu-nity. Immunology Today, 20, 212–16.

Rappuoli, R., Pizza, M., Douce, G. & Dougan, G. (1999) Structure andmucosal adjuvanticity of cholera and Escherichia coli heat-labileenterotoxins. Immunology Today, 20, 493–500.

downregulated major histocompatibility complex (MHC)class 1 expression.• Antibody neutralizes free virus and is particularly ef-fective when the virus has to travel through the blood-stream before reaching its final target.• Antibody is important in preventing reinfection.• “Budding” viruses that can invade lateral cells withoutbecoming exposed to antibody are combated by CMI. In-fected cells express a processed viral antigen peptide ontheir surface in association with MHC class I.• Rapid killing of the cell by cytotoxic ab T-cells preventsviral multiplication.• Cytokines produced by CD4+ and CD8+ cells activateAPCs and control the replication of virus particles.• Natural infection generates specific antibody and T-cytotoxic (Tc) cells with subsequent long-term protectionagainst reinfection.

Immunity to fungi• Fungal infections are common in individuals with neu-trophil dysfunction and in patients with defective CMI.

Immunity to parasitic infections• Chronic parasitic infection can cause exaggerated im-mune responses leading to severe tissue injury.

• Antibodies are usually effective against the blood-borne parasitic diseases.• Most parasitic infections stimulate a Th2 response, withIgE production and eosinophilia. These are important indestruction of parasites such as schistosomes, which whencoated with IgG or IgE are killed by adherent eosinophilsthrough the mechanism of antibody-dependent cellularcytotoxicity (ADCC).• Organisms such as Leishmania spp., Trypanosoma cruziand Toxoplasma gondii hide from antibodies insidemacrophages and use the same strategies as intracellularparasitic bacteria to survive. Like them, they are killedwhen the macrophages are activated by cytokines pro-duced during CMI responses.• Cytokines produced by Th2 cells are important in theexpulsion of gastrointestinal worms and in destroying larval forms. They may also protect against ectoparasitessuch as ticks by degranulating mast cells.


127

PASSIVELY ACQUIRED IMMUNITY

Temporary protection against infection can be estab-lished by giving preformed antibody from another in-dividual of the same or a different species (table 12.1).The advantage of this form of therapy is that humoralimmunity is acquired immediately. The disadvantageis that the administered antibodies are rapidly utilizedby combination with antigen or catabolized in the nor-mal way. Therefore protection is quite quickly lost andmemory is not conferred. An additional problem withpassive immunotherapy is the potential transmissionof infectious agents present in donor serum.

In the past, horse globulins containing antitetanusand antidiphtheria toxins were extensively employedprophylactically, but with the advent of antibiotics therequirement for such antisera has diminished. Onecomplication of this form of therapy was serum sick-ness developing in response to the foreign protein. Al-though horse serum is now seldom used in modernmedical practice, monoclonal antibodies produced inmice and humanized monoclonal antibodies are beingincreasingly utilized.

Maternally acquired antibody

In the first few months of life, protection is afforded bymaternally derived antibodies acquired by placentaltransfer and by intestinal absorption of colostral im-munoglobulins (Igs). The major Ig in both colostrumand milk is secretory IgA(sIgA), which is not absorbedby the baby but remains in the intestine to protect themucosal surfaces. The surface IgA antibodies are directed against bacterial and viral antigens often pre-sent in the intestine, and it is presumed that in themother, IgA-producing cells, responding to gut anti-gens, migrate and colonize breast tissue where the antibodies they produce appear in the milk.

Pooled human gammaglobulin

Regular injection of pooled human adult gamma-globulin collected from the plasma of normal donors isan essential treatment for patients with long-standing humoral immunodeficiency. Preparations containing

CHAPTER 12

Prophylaxis

Passively acquired immunity, 127Maternally acquired antibody, 127Pooled human gammaglobulin, 127

Vaccination, 128Herd immunity, 128Strategic considerations, 128

Killed organisms as vaccines, 129Live attenuated organisms have many advantages

as vaccines, 129Classical methods of attenuation, 129Attenuation by recombinant DNA technology, 130

Microbial vectors for other genes, 130Subunit vaccines containing individual

protective antigens, 131Use of transgenic plants, 131Antigens can be synthesized through gene cloning, 131The naked gene itself acts as a vaccine, 131

Adjuvants, 132Current vaccines, 132

Immunization in transplant patients, 133Immunization in adults, 133

Immunization against infectious disease repre-sents one of science’s greatest triumphs. Many ofthe epidemic diseases of the past are now con-trolled and all but eliminated in developed coun-tries, and new vaccines promise protection againsta variety of more recently recognized infectiousdiseases including the various forms of hepatitis,Lyme disease and hopefully human immunodefi-ciency virus (HIV) infection. Modern vaccine biol-ogy is now targeting not only infectious diseasesbut also autoimmune and malignant disease. Thischapter will review various procedures for stimu-lating antibody production or activating T-cell responses against defined prophylactically admin-istered antigens.


high titers of antibodies to specific antigens may beused to modify the effects of respiratory syncytial virus(RSV) infection, chickenpox or measles especially inpatients with defective immune responses due to pre-maturity, protein malnutrition, steroid treatment orleukemia. Contacts of infectious hepatitis patientsmay also be afforded protection by gammaglobulin,especially when the material is derived from the serumof individuals vaccinated some weeks previously. Cytomegalovirus (CMV) human immune globulin isnow administered to recipients of organ transplantstaken from CMV-positive donors, and rabies immuneglobulin may be given together with active immuniza-tion to patients bitten by a potentially rabid animal. It isof interest that pooled gammaglobulin is being in-creasingly used as immune-modulating therapy in thetreatment of autoimmune diseases such as idiopathicthrombocytopenic purpura although the mechanismbehind the beneficial effect is currently unclear. Thebioengineering of custom-designed antibodies is of in-creasing importance (see Chapter 3).

VACCINATION

Herd immunity

In the case of tetanus, active immunization is of benefitto the individual but not to the community since it willnot eliminate the organism, which persists in the soil ashighly resistant spores. Where a disease depends on

human transmission, immunity in just a proportion ofthe population can help the whole community if itleads to a fall in the reproduction rate (i.e. the numberof further cases produced by each infected individual)to less than one. Under these circumstances the diseasewill die out. An example of this is the disappearance ofdiphtheria from communities in which around 75% ofthe children have been immunized (figure 12.1) In con-trast, focal outbreaks of poliomyelitis have occurred incommunities that object to immunization on religiousgrounds, raising an important point for parents in general.

Strategic considerations

The objective of vaccination is to provide effective im-munity by establishing adequate levels of appropriateimmune effector mechanisms, together with a primedpopulation of memory cells which can rapidly expandon renewed contact with antigen and so provide pro-tection against infection. Sometimes, as with polio in-fection, a high blood titer of antibody is required; inmycobacterial diseases such as tuberculosis (TB), a macrophage-activating cell-mediated immunity(CMI) is most effective, whereas with influenza virusinfection, antibodies and cytotoxic T-cells (Tc) play asignificant role. The site of the immune responseevoked by vaccination may also be most important.For example in cholera, antibodies need to be in the gutlumen to inhibit adherence to and colonization of theintestinal wall.

In addition to an ability to engender effective immu-nity, a number of mundane but nonetheless crucial

INFECTIONSOURCE OF ANTIBODY

TetanusDiphtheria

BotulismGas gangrene

Snake orscorpion bite

Varicella zoster

Hepatitis B

Hepatitis A

Measles

USE

Rabies

HORSE HUMAN

–

–

–

–

–

ProphylaxisTreatment

Treatment

Treatment immunodeficiency

Post-exposure to vaccine

Treatment

Treatment

Prophylaxis (Travel)

Cytomegalovirus

RSV*

Prophylaxisin patients receivingimmunosuppression

Treatment

–

–

Table 12.1 Passive immunotherapy with antibody.

Year

Not

ifica

tions

per

100

000

pop

ulat

ion

19210

1 000

5 000

15 000

10 000

'25 '29 '33 '37 '41 '45 '49 '53 '57 '61 '65 '69 '73

Immunization

Figure 12.1 Notification of diphtheria in England and Wales per100 000 population showing a dramatic fall after immunization.(Reproduced from G. Dick (1980) Immunisation. Leiden UniversityPress, Leiden, The Netherlands, with kind permission of the authorand publishers.)

*Ahumanized monoclonal antibody is also available.RSV, respiratory syncytial virus.

CHAPTER 12—Prophylaxis 129

conditions must be satisfied for a vaccine to be consid-ered successful (table 12.2). The antigens must be read-ily available, the preparation should be stable onstorage, and it should be cheap, easy to administerand, certainly, safe.

KILLED ORGANISMS AS VACCINES

The simplest way to destroy the ability of microbes tocause disease yet maintain their antigenicity is to pre-vent their replication by killing in an appropriate man-ner, such as with formaldehyde treatment. Parasiticworms and, to a lesser extent, protozoa are extremelydifficult to grow up in bulk to manufacture killed vac-cines. This problem does not arise for many bacteriaand viruses and, in these cases, the inactivated microorganisms have generally provided safe anti-gens for immunization. Examples are typhoid, choleraand killed poliomyelitis (Salk) vaccines. Care has to betaken to ensure that important protective antigens arenot destroyed in the inactivation process.

LIVE ATTENUATED ORGANISMS HAVE MANYADVANTAGES AS VACCINES

The objective of attenuation is to produce a modifiedorganism that mimics the natural behavior of the origi-nal microbe without causing significant disease. Inmany instances the immunity conferred by killed vaccines, even when given with adjuvant (see below),is often inferior to that resulting from infection withlive organisms. This must be partly because the repli-cation of the living microbes confronts the host with alarger and more sustained dose of antigen and that,with budding viruses, infected cells are required for

the establishment of good Tc memory. Another signifi-cant advantage of using live organisms is that the im-mune response takes place largely at the site of thenatural infection. This is well illustrated by the nasopharyngeal IgA response to immunization withpolio vaccine. In contrast with the ineffectiveness ofparenteral injection of killed vaccine, intra-nasal ad-ministration evoked a good local antibody responsethat declined over a period of 2 months. Oral immu-nization with live attenuated virus produced an evenbetter response with a persistently high IgA antibodylevel (figure 12.2).

Classical methods of attenuation

The objective of attenuation, that of producing an or-ganism that causes only a very mild form of the naturaldisease, can be equally well attained by employingheterologous strains which are virulent for anotherspecies, but avirulent in humans. The best example ofthis was Jenner’s seminal demonstration that cowpoxinfection would protect against smallpox. Since then, atruly remarkable global effort by the World Health Organization, combining extensive vaccination andselective epidemiological control methods, has com-pletely eradicated the human disease — a wonderfulachievement.

Attenuation itself can be achieved by modifying theconditions under which an organism grows. Pasteur

FACTOR REQUIREMENTS

Effectiveness

Availability

Stability

Cheapness

Safety

Must evoke protective levels of immunity: at the appropriate site of relevant nature (Ab, Tc, Th1, Th2) of adequate duration

Readily cultured in bulk or accessiblesource of subunit

Stable under extreme climatic conditions,preferably not requiring refrigeration

What is cheap in the West may be expensive indeveloping countries but WHO tries to help

Eliminate any pathogenicity

Table 12.2 Factors required for a successful vaccine.

Ab, antibody; WHO, World Health Organization.

intra-muscularkilled

per oral live attenuated

Months after immunization with polio vaccine

Nas

opha

ryng

eal I

gA a

ntib

ody

titer

0

<2

2

8

32

128

intra-nasal killed

1 2 3 4 12 24

Figure 12.2 Local immunoglobulin A (IgA) response to polio vac-cine. Local secretory antibody synthesis is confined to the specificanatomical sites, which have been directly stimulated by contactwith antigen. (Data from P.L. Ogra et al. (1975) In: Viral Immunologyand Immunopathology (ed. A.L. Notkins), p. 67. Academic Press, NewYork.)


first achieved the production of live but nonvirulentforms of chicken cholera bacillus and anthrax by cul-turing at higher temperatures and under anaerobicconditions. A virulent strain of Mycobacterium tubercu-losis became attenuated by chance in 1908 when Cal-mette and Guérin at the Institut Pasteur, Lille, addedbile to the culture medium in an attempt to achieve dis-persed growth. After 13 years of culture in bile-containing medium, the strain remained attenuatedand was used successfully to vaccinate childrenagainst tuberculosis. The same organism, BCG (bacillebilié de Calmette–Guérin), is widely used today forimmunization of tuberculin-negative individuals inmany countries. It should be stressed that an individ-ual receiving BCG immunization will convert frombeing tuberculin skin test-negative to the positivestate. Although the immunization often confers pro-tection, the feeling in the USA is that a negative skintest is important in excluding a diagnosis of TB, andBCG negates the use of the skin test. For this reason,therefore, BCG is not employed in the USA.

Attenuation of viruses usually requires cultivationin nonhuman cells where after many rounds of culturethe viruses develop multiple random genetic muta-tions, some of which lead to a loss of ability to infecthuman cells. Unfortunately on very rare occasionspathogenicity may return by further mutations of theorganism, as was seen with the polio virus vaccinewhere reversions to the wild type resulted in clinicalcases of the disease occurring in vaccinated children.Another problem with live attenuated vaccines is therisk to immunocompromised patients in whom theymay behave as opportunistic pathogens.

Attenuation by recombinant DNA technology

Instead of the random mutations achieved by classicattenuation procedures, genetic modification tech-niques are now being used to develop various atten-uated strains of viruses.

Microbial vectors for other genes

An ingenious trick is to use a virus as a “piggyback” forgenes from another virus, particularly one that cannotbe grown successfully, or which is inherently danger-ous. Large DNA viruses, such as vaccinia, can act ascarriers for one or many foreign genes while retaininginfectivity for animals and cultured cells. The proteinsencoded by these genes are appropriately expressed invivo, and are processed for major histocompatibilitycomplex (MHC) presentation by the infected cells,

thus effectively endowing the host with both humoraland CMI.

Awide variety of genes have been expressed by vac-cinia virus vectors including hepatitis B surface anti-gen (HBsAg), which has protected chimpanzeesagainst the clinical effects of hepatitis B virus (figure12.3). Spectacular neutralizing antibody titers wereproduced by a construct with the gene encoding rabiesvirus glycoprotein, which protected animals againstintra-cerebral challenge. Since approximately 10% ofthe poxvirus genome can be replaced by foreign DNA,the potential exists for producing multivalent vaccinesin these vectors.

Another approach is to employ attenuated bacteriasuch as BCG or Salmonella as vehicles for antigens re-quired to evoke CD4-mediated T-cell immunity. BCGis avirulent, has a low frequency of serious complica-tions, can be administered any time after birth, hasstrong adjuvant properties, gives long-lasting CMIafter a single injection and is inexpensive. The use ofSalmonella is particularly interesting because thesebacteria elicit mucosal responses by oral immuniza-tion. It is possible therefore that the oral route of immu-nization may be applicable to develop gut mucosalimmunity and perhaps systemic protection.

CMI AbT B

DNA

Vacciniapromoter

‘Foreign’HBsAggene

Vaccinia Infected cell

Protein synthesis

Foldingglycosylation

Antigenprocessing

HBsAgparticle

HBsAgpeptideMHC

Figure 12.3 Hepatitis B surface antigen (HBsAg) vaccine using anattenuated vaccinia virus carrier. The HBsAg protein is synthesizedby the machinery of the host cell: some is secreted to form the HBsAg22 nm particle which stimulates antibody (Ab) production, andsome follows the antigen processing pathway to stimulate cell-mediated immunity (CMI) and T-helper activity. B, B-cell; T, T-cell.


SUBUNIT VACCINES CONTAININGINDIVIDUAL PROTECTIVE ANTIGENS

A whole parasite or bacterium usually contains manyantigens that are not concerned in the protective re-sponse of the host but may provoke hypersensitivity.Vaccination with the isolated protective antigens mayavoid these complications, and identification of theseantigens then opens up the possibility of producingthem synthetically.

Bacterial exotoxins such as those produced by diph-theria and tetanus bacilli have long been used as im-munogens. First, they must of course be detoxified andthis is achieved by formaldehyde treatment, which for-tunately does not destroy the major immunogenic determinants (figure 12.4). Immunization with the re-sulting toxoid will therefore provoke the formation ofprotective antibodies, which neutralize the toxin bystereochemically blocking the active site. The toxoid isgenerally given after adsorption to aluminum hydrox-ide, which acts as an adjuvant and produces higher an-tibody titers.

Other isolated protective antigens include purifiedextracts of some component of the organism such asthe subunit acellular pertussis vaccine or the subunitinfluenza vaccine. Soluble capsular material, such as isfound in the pneumococcal or hemophilus influenzavaccines, stimulate significant protection but are suboptimal in young children and therefore are conjugated to carrier proteins such as tetanus or diphtheria toxoids.

Use of transgenic plants

Viral and bacterial antigens such as HBsAg, rabies gly-coprotein and Escherichia coli enterotoxin may be pro-duced in transgenic plants. The advantage of such anapproach is the low cost and the possibility that theperson may only have to eat the transgenic plant to beimmunized. In addition to antigens, specific antibod-

ies called plantibodies are being produced in plants.Studies with antibodies produced in this way againstStreptococcus mutans (the cause of dental caries) showpromising results, and numerous other plantibodiesare now available.

Antigens can be synthesized through gene cloning

The emphasis now is to move towards gene cloning ofindividual proteins once they have been identified im-munologically and biochemically. Recombinant DNAtechnology enables us to make genes encoding part orthe whole of a protein peptide chain almost at will, andto express them in an appropriate vector. This ap-proach has been used to produce a commercial hepati-tis vaccine employing the product secreted by yeastcells expressing the HBsAg gene. A similar approachhas been used to produce a vaccine against Borreliaburgdorferi, the cause of Lyme disease. The potential ofgene cloning is clearly vast and, in principle, economi-cal, but there are sometimes difficulties in identifying agood expression vector and in obtaining correct fold-ing of the peptide chain to produce an active protein.

One restriction to gene cloning has been that carbohydrate antigens cannot be synthesized directlyby recombinant DNA technology. However, the glycosylation pathway in the yeast Pichia pastoris hasrecently been humanized by replacing the endoge-nous yeast glycosylation pathways with their eukary-otic equivalents, thereby permitting the synthesis ofcomplex carbohydrates.

The naked gene itself acts as a vaccine

It has recently been appreciated that injected DNAfunctions as a source of immunogen and can inducestrong immune responses, both humoral and cell-mediated. The gene is stitched in place in a DNA plas-mid with appropriate promoters and enhancers andinjected into muscle where it can give prolonged ex-pression of protein. The plasmids may even be intro-duced into the body using a biolistics gun whichdelivers gold microspheres coated with plasmids ofthe gene encoding the vaccine antigen. As mentionedabove, DNAhas also been placed into microorganismssuch as vaccinia, adenovirus or Salmonella spp. Broadimmune responses are observed including inductionof Tc cells, presumably reflecting the cytosolic expres-sion of the protein and its processing with MHC class I.

DNA for this procedure can be obtained directlyfrom current clinical material without having to selectspecific mutant strains. Altogether, the speed and sim-

EPITOPES

PATHOGENIC EXOTOXIN HARMLESS PREPARATION (TOXOID)

CHEMICALMODIFICATION

SITE RELATED TOPATHOGENICITY

Figure 12.4 Modification of toxin to harmless toxoid without los-ing many of the antigenic determinants. Thus, antibodies to thetoxoid will react well with the original toxin.


plicity mean that the 2 years previously needed tomake a recombinant vaccine can be reduced tomonths. DNA vaccines do not need the cumbersomeand costly protein synthesis and purification proce-dures that subunit formulations require; they can beprepared in a highly stable powder and, above all, theyare incredibly cheap. Before there is widespread use inhumans, however, a number of safety considerations,such as the possibility of permanent incorporation of aplasmid into the host genome, need to be addressed.

ADJUVANTS

For practical and economic reasons, prophylactic im-munization should involve the minimum number ofinjections and the least amount of antigen. We have re-ferred to the undoubted advantages of replicating at-tenuated organisms in this respect, but nonlivingorganisms, and especially purified products, frequent-ly require an adjuvant. This is a substance that by defi-nition is incorporated into or injected simultaneouslywith antigen and potentiates the immune response(Latin adjuvare, to help). The mode of action of adju-vants involves a number of mechanisms, one of whichis to counteract the dispersion of free antigen and local-ize it, either at an extracellular location or withinmacrophages. The most common adjuvants of the typeused in humans are aluminum compounds (phos-phate and hydroxide). Virtually all adjuvants stimu-late antigen-presenting cells (APCs) and improve theirimmunogenicity by the provision of accessory costim-ulatory signals to direct lymphocytes towards an im-mune response rather than tolerance, and by thesecretion of soluble stimulatory factors (e.g. inter-leukin-1 (IL-1)), which influence the proliferation oflymphocytes.

CURRENT VACCINES

The established vaccines in current use and the sched-ules for their administration are set out in table 12.3.

Because of the pyrogenic reactions and worriesabout possible hypersensitivity responses to thewhole-cell component of the conventional pertussisvaccine, a new generation of vaccines containing oneor more purified components of Bordetella pertussis,and therefore termed “acellular” vaccines, has been li-censed in the USA. The combination of diphtheria andtetanus toxoids with acellular pertussis (DTP) is rec-ommended for the later fourth and fifth “shots” andfor children at increased risk for seizures. Childrenunder 2 years of age make inadequate responses to theT-independent Haemophilus influenzae capsular poly-

saccharide, so they are now routinely immunized withthe antigen conjugated with tetanus or diphtheria toxoids.

A resurgence of measles outbreaks in recent yearshas prompted recommendations for the reimmuniza-tion of children, at the age of school entry or at thechange to middle (secondary) school, with measlesvaccine. The considerable morbidity and mortality as-sociated with hepatitis B infection, its complex epi-demiology and the difficulty in identifying high-riskindividuals have led to recommendations for vaccina-tion in the 6–18-month age group.

Relevant information about protocols for immu-nization and immunization-related problems areavailable at the web site of the Centers for Disease Control and Prevention (http://www.cdc.gov/nip)

VACCINEADMINISTRATION

Triple (DTP) vaccine:diphtheria, tetanus,pertussis

CHILDREN

Polio: live

killed

MMR vaccine:measles, mumpsrubella

BCG (TB, leprosy)

Haemophilus

Varicella

ELDERLY

Pneumococcalpolysaccharide serotypes

SPECIAL GROUPS

Hepatitis B

Hepatitis AMeningitis (A+C)

Yellow feverTyphoid

Rabies

Primary 2–6 mo (3x/4 weekly)

15 mo, 4 yrhigh-risk adult

Immunocompromised

UK USA

12–15 mo

BoostDT

15 mo/4 yrDT every 10 yr

10–14 yr high risk only

Neonates at risk,immunocompromised

Aged & high risk

Travelers, high risk groups

Travelers to endemic areas

Travelers to endemic areas

Prophylactically in high riskgroupsPost-exposure tocontacts in endemic areas

Japan: 2 yr

OTHERCOUNTRIES

Africa: 6 mo

Tropics: at birth

Tropics: infantsYellow fever:boost residentsand frequentvisitors every10 yr

3–5yr

Concomitant with DTP

4/6 yr

Primary

Boost

Primary

Boost 4–5 yr10–14 yr seronegative girls

selectively with rubella

Aged & high riskInfluenza

Concomitant with DTP

Table 12.3 Current vaccination practice.


and the UK Health Protection Agency(http://www.hpa.org.uk).

Immunization in transplant patients

Many common infections produce a more severe clinical picture in immunosuppressed patients than innormal individuals. It is important therefore to immu-nize these patients wherever possible. However, theimmunosuppression associated with transplantationpresents a number of special immunization problemsfor patients, especially children. Immunization withlive viral vaccines may lead to vaccine-associated dis-ease, and are not usually administered after transplan-tation. Children, therefore, who have not beenimmunized, should receive immunizations before the transplant takes place. Inactivated vaccines do notreplicate and therefore do not cause vaccine-

associated disease. However, due to the immunosup-pression, immune responses are suboptimal and multiple booster immunizations are required.

Immunization in adults

Many childhood immunizations do not last a lifetimeand adults need to be reimmunized against tetanus,diptheria and other illnesses. Adults need to be pro-tected against chickenpox if they have not had the dis-ease or the vaccine, and a measles, mumps and rubella(MMR) vaccine may be indicated if they have an unre-liable history of being previously immunized. Adultsover the age of 50 years should be immunized annuallyagainst current influenza strains, and individuals aged65 years and older should receive the pneumococcalvaccine.

REVISION

Passively acquired immunity• Horse antisera have been extensively used in the pastbut their use is now more restricted because of the dangerof serum sickness.• Passive immunity can be acquired by maternal antibod-ies or from homologous pooled gammaglobulin.• Sera containing high-titer antibodies against a specificpathogen are useful in treating or preventing various viraldiseases such as severe chickenpox, cytomegalovirus(CMV) infection or rabies.

Vaccination• Active immunization provides a protective statethrough contact with a harmless form of the organism.• Agood vaccine should be based on antigens that are eas-ily available, cheap, stable under extreme climatic condi-tions and nonpathogenic.

Killed organisms as vaccines• Killed bacteria and viruses have been widely used.

Live attenuated organisms• The advantages are that replication of organisms pro-duces a bigger dose, and the immune response is pro-duced at the site of the natural infection.• Attenuated vaccines are produced by altering the con-ditions under which organisms are cultivated.

• Recombinant DNAtechnology is now used to manufac-ture attenuated strains.• Attenuated vaccinia can provide a “piggyback” carrierfor genes from other organisms that are difficult to attenuate.• BCG is a good vehicle for antigens requiring CD4 T-cellimmunity, and Salmonella constructs may give oral andsystemic immunity. Intra-nasal immunization is fast gain-ing popularity.• Risks are reversion to the virulent form and danger toimmunocompromised individuals.

Subunit vaccines• Whole organisms have a multiplicity of antigens, someof which are not protective, may induce hypersensitivityor might even be frankly immunosuppressive.• It makes sense in these cases to use purified components.• Toxoids are exotoxins treated with formaldehyde thatdestroys the pathogenicity of the organism but leaves anti-genicity intact.• There is greatly increased use of recombinant DNAtechnology to produce these antigens.• Microbial antigens can be produced in transgenic plantsand plantibodies are specific antibodies also produced inplants.• Naked DNAencoding the vaccine subunit can be inject-

(continued)


FURTHER READING

Ada, G. (2001) Advances in immunology: Vaccines and vaccination.New England Journal of Medicine, 345, 1042–53.

Dietrich, G., Gentschev, I., Hess, J. et al. (1999) Delivery of DNA vac-cines by attenuated intracellular bacteria. Immunology Today, 20,251–3.

Faix, R. (2002) Immunization during pregnancy. Clinical Obstetricsand Gynecology, 45, 42–58.

Kumar, V. & Sercarz, E. (1996) Genetic vaccination: The advantagesof going naked. Nature Medicine, 2, 857–9.

Moylett, E.H. & Hanson, I.C. (2003) Immunization. Journal of Allergyand Clinical Immunology, 111, S754–65.

Sherwood, J.K., Zeitlin, L., Whaley, K.J. et al. (1996) Controlled re-lease of antibodies for long-term topical passive immunoprotec-tion of female mice against genital herpes. Nature Biotechnology,14, 468–71.

ed directly into muscle, where it expresses the protein andproduces immune responses. The advantages are stability,ease of production and cheapness.

Adjuvants• Adjuvants work by producing depots of antigen, andby activating antigen-presenting cells (APCs); they some-times have direct effects on lymphocytes.

Current vaccines• Children in the USA and UK are routinely immunizedwith diphtheria and tetanus toxoids and pertussis (DTPtriple vaccine) and attenuated strains of measles, mumpsand rubella (MMR) and polio. BCG is given at 10–14 yearsin the UK.• Subunit forms of pertussis lacking side effects are beingintroduced.• The capsular polysaccharide of Haemophilus influenzaehas to be linked to a carrier.• Vaccines for hepatitis Aand B, meningitis, yellow fever,typhoid, cholera and rabies are available for travelers andhigh-risk groups.

Immunization in transplant patients• Transplant patients are highly susceptible to commoninfections and should be protected wherever possible.• Live attenuated vaccines are generally not adminis-tered to these patients because of the concern of vaccine-associated disease.• Killed vaccines are safe in transplanted individuals butmultiple booster immunizations may be required

Immunization in adults and the elderly• Childhood vaccine effects do not last a lifetime andadults should receive booster vaccines at regular intervals.• Influenza vaccine for people over the age of 50 years andpneumococcal vaccine for those over 65 years is recommended.


135

PRIMARY IMMUNODEFICIENCY STATES IN THE HUMAN

A multiplicity of immunodeficiency states in humansthat are not secondary to environmental factors havebeen recognized. We have earlier stressed the mannerin which the interplay of complement, antibody andphagocytic cells constitutes the basis of a tripartite de-fense mechanism against pyogenic (pus-forming) in-fections. Hence it is not surprising that deficiency inany one of these factors may predispose the individualto repeated infections of this type. Patients with T-celldeficiency of course present a markedly different pat-tern of infection, being susceptible to those viruses, intracellular bacteria, fungi and parasites that are normally eradicated by cell-mediated immunity(CMI).

The following sections examine various forms ofthese primary immunodeficiencies.

DEFICIENCIES OF INNATE IMMUNEMECHANISMS

Phagocytic cell defects

Primary defects in neutrophil number are rare as mostcases of neutropenia are due to treatment with cytoto-xic drugs usually associated with malignant disease.As would be expected, neutropenic patients are gener-ally prone to pyogenic bacterial infections but not to

parasitic or viral infections. A number of primary im-munodeficiency disorders involve qualitative defectsof phagocytic cells:

Chronic granulomatous disease (CGD) is a rare dis-ease in which the monocytes and neutrophils fail toproduce reactive oxygen intermediates (figure 13.1) re-quired for the intracellular killing of phagocytosed or-ganisms. This results in severe recurrent bacterial andfungal infections, which when unresolved may lead togranulomas that can obstruct the gastrointestinal andurogenital systems. Curiously, the range of infectiouspathogens that trouble these patients is relatively re-stricted. The most common pathogen is Staphylococcusaureus but certain Gram-negative bacilli and fungisuch as Aspergillus fumigatus and Candida albicans arefrequently involved.

The disease is a heterogeneous disorder due to defects in any one of four subunits of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase which is crucial for the formation of hydrogenperoxide.

Prophylactic daily treatment with antibiotics con-siderably reduces the frequency of infections. Interfe-ron g (IFNg) has been shown to stimulate the productionof superoxide by CGD neutrophils resulting in consid-erably fewer infectious episodes. Because CGD resultsfrom a single gene defect in phagocytic cells, the idealtreatment of the disease would be the transfer of thecorrect gene into the bone marrow stem cells. Initialwork in this area is encouraging. Some patients have

CHAPTER 13

Immunodeficiency

Primary immunodeficiency states in the human, 135

Deficiencies of innate immune mechanisms, 135Phagocytic cell defects, 135Complement system deficiencies, 136

Immunoglobulin deficiencies, 138Primary T-cell deficiency, 139Primary T-cell dysfunction, 139Severe combined immunodeficiency (SCID), 140

Mutation in the common cytokine receptor gc chain is thecommonest cause of SCID, 140

Severe combined immunodeficiency can be due to mutations inpurine salvage pathway enzymes, 140

Other SCID variants, 140Other combined immunodeficiency disorders, 141Recognition of immunodeficiencies, 141Secondary immunodeficiency, 141Acquired immunodeficiency syndrome (AIDS), 142

Characteristics of HIV, 143The infection of cells by HIV, 143The AIDS infection depletes helper T-cells, 144Laboratory diagnosis of AIDS, 145Treatment of HIV disease, 145

been effectively treated by bone marrow transplanta-tion from a normal major histocompatibility complex(MHC) -matched sibling, but the significant mortalityof this procedure needs to be weighed against themuch-improved quality of life that patients can nowexpect from conservative treatments with antibioticsand IFNg.

Chediak–Higashi disease is a rare autosomal reces-sive disease due to various mutations in a gene calledthe CHS gene. In this condition, neutrophils, mono-cytes and lymphocytes contain giant lysosomal gran-ules which result from increased granule fusion. Thisgives rise to phagocytic cells which are defective inchemotaxis, phagocytosis and microbicidal activity,and to natural killer (NK) cells which lack cytotoxic activity. In addition to increasing susceptibility to pyogenic infections, these lysosomal defects affectmelanocytes causing albinism and platelets giving riseto bleeding disorders.

Leukocyte adhesion deficiency results from lack ofthe CD18 b-subunit of the b2-integrins, which includeLFA-1 (lymphocyte function-associated antigen-1).Children with this defect suffer from repeated pyo-genic infections because phagocytic cells do not bind

to intercellular molecules on endothelial cells andtherefore cannot emigrate through the vessel walls tothe infected tissue. Patients also have problems withwound healing, and delayed separation of the umbili-cal cord is often the earliest manifestation of this defect.Fortunately, bone marrow transplantation has result-ed in restoration of neutrophil function and is now thetreatment of choice.

Defects in the IFNg–interleukin-12 (IL-12) axis.Normally initiators of the Th1 cytokine pathway, my-cobacteria induce macrophages and dendritic cells(DCs) to secrete IL-12, which then activates both NKcells and Th1-cells; these then produce IFNg, whichswitch on macrophage killing mechanisms providingprotection against pathogenic mycobacteria.

An interesting group of patients who are selectivelysusceptible to poorly pathogenic mycobacterialspecies and other intracellular bacteria have defects inthis Th1 cytokine pathway. These patients have muta-tions in the IFNg receptor, the IL-12 receptor or IL-12 itself, resulting in defective activation of macrophagesand inability to control intracellular infections.

Complement system deficiencies

As we have seen previously, activation of the comple-ment system plays an important role in host defenseagainst bacterial infections and is also crucial for theclearance of immune complexes from the circulation.Genetic deficiencies in almost all the complement pro-teins have now been described and, as would be ex-pected, these result in either recurrent infections orfailure to clear immune complexes.

Deficiency in the mannose-binding lectin (MBL)pathway results in recurrent infection

The MBL pathway is an important system for activat-ing complement and for depositing complement frag-ments on microorganisms where they facilitatephagocytosis and initiate inflammatory reactions. It istherefore not surprising that a deficiency of MBL is associated with susceptibility to infections and withthe development of immunological diseases particu-larly in children. This suggests that it is important foropsonin production during the interval between theloss of passively acquired maternal immunity and thetime of development for a mature immune system. Re-cently a patient with MBL-associated serine protease-2(MASP-2) deficiency has been described who alsoshows significant recurrent bacterial infections.

PART 5—Clinical immunology136

Time (min)

Supe

roxi

de (

nmol

s)CHRONIC GRANULOMATOUS DISEASE

PHORBOL ESTER

NORMAL

CARRIER

PATIENT 2

PATIENT 1

20

40

0 2 4

Figure 13.1 Defective respiratory burst in neutrophils of patientswith chronic granulomatous disease (CGD). The activation of the NADP (nicotinamide adenine dinucleotide phosphate)/cytochrome oxidase is measured by superoxide anion (·O-

2; cf. figure1.6) production following stimulation with phorbol myristate ac-etate. Patient 2 has a p92phox mutation which prevents expression ofthe protein, whilst patient 1 has the variant p92phox mutation produc-ing very low but measurable levels. Many carriers of the X-linkeddisease express intermediate levels, as in the individual shown whois the mother of patient 2. (Data from R.M. Smith & J.T. Curnutte(1991) Blood, 77, 673–86.)

Deficiency of early classical pathway proteinsresults in increased susceptibility to immune-complex disease

This has been observed in patients with deficiency ofthe C1 proteins, C2 or C4 who have some increase insusceptibility to infection but also have an unusuallyhigh incidence of a systemic lupus erythematosus(SLE) -like disease. Although the reason for this is un-clear, it could be due to a decreased ability to mount anadequate host response to infection with a putative etiologic agent or, more probably, to eliminate anti-gen–antibody complexes effectively.

Deficiency of C3 or the control proteins factor H orfactor I, results in severe recurrent infections

Although deficiency of C3 is rare, the clinical pictureseen in these patients is instructive in emphasizing thecrucial role of C3 in both the classical and alternativepathways. Complete absence of this component caus-es a block in both pathways resulting in defective op-sonizing and chemotactic activities. Factor H or factor Ideficiency produce a similar clinical picture, becausethere is inability to control C3b. This results in continu-ous activation of the alternative pathway through thefeedback loop, leading to consumption of C3, the levels of which may fall to zero.

Permanent deficiencies in C5, C6, C7 and C8 result insusceptibility to Neisseria infection

Many of the individuals with these rare disorders arequite healthy and not particularly prone to infectionapart from an increased susceptibility to disseminatedNeisseria gonorrhoeae and N. meningitidis infection. Pre-sumably the host requires a complete and intact com-plement pathway with its resulting cytolytic activity todestroy these particular organisms.

Deficiency of the C1 inhibitor causes hereditary angioedema

C1INH acts on the classical complement system by in-hibiting the binding of C1r and C1s to C1q, and it alsoinhibits factor XII of the clotting system (figure 13.2).Absence of C1INH leads to recurring episodes of acutecircumscribed noninflammatory edema mediated bythe unrestricted production of the vasoactive C2 frag-ment. Lack of C1INH also leads to an increase in gener-ation of bradykinin, which adds to the increased

permeability of small vessels causing the edema asso-ciated with this disease. These patients therefore pre-sent with recurrent episodes of swelling of the skin,acute abdominal pain due to swellings of the intestine,and obstruction of the larynx which may rapidly causecomplete laryngeal obstruction. The majority of cases(type 1) are due to a mutant gene leading to markedlysuppressed amounts of C1INH whereas a less com-mon form of the disease (type 2) is due to a dysfunc-tional C1INH protein.

Attacks are precipitated by trauma or excessive ex-ercise and can be controlled by anabolic steroids suchas danazol, which for unknown reasons suppress thesymptoms of hereditary angioedema.

GPI (glycosylphosphatidylinositol) -anchorprotein deficiencies

A mutation in the PIG-A glycosyltransferase geneleads to an inability to synthesize the GPI-anchors forthe complement regulatory proteins decay accelerat-ing factor (DAF, CD55), homologous restriction factor(HRF, CD59), and membrane cofactor protein (MCP,CD46). The absence of these proteins from the erythro-cyte surface results in a condition known as paroxys-mal nocturnal hemoglobinuria due to the inability toinactivate bound C3b generated from spontaneous hydrolysis of fluid phase C3.

CHAPTER 13—Immunodeficiency 137

EDEMA

C2 KININ

PLASMIN

DANAZOLe-AMINO

CAPROIC ACID

C1INHC1

C2bC2

Figure 13.2 C1 inhibitor deficiency and angioedema. C1 inhibitorstoichiometrically inhibits C1, plasmin, kallikrein and activatedHageman factor and deficiency leads to formation of the vasoactiveC2 kinin by the mechanism shown. The synthesis of C1 inhibitor canbe boosted by methyltestosterone or preferably the less masculiniz-ing synthetic steroid, danazol; alternatively, attacks can be con-trolled by giving e-aminocaproic acid to inhibit the plasmin.

IMMUNOGLOBULIN DEFICIENCIES

X-linked agammaglobulinemia (XLA) is one of seve-ral immunodeficiency syndromes that have beenmapped to the X-chromosome (figure 13.3). The defectis due to mutations in the Bruton’s tyrosine kinase(Btk) gene, which is expressed early in B-cell develop-ment and which is crucial for maturation of the B-cellseries from pre-B cells. Over 100 different Btk muta-tions are now recorded in the XLA mutation registry.Early B-cells are generated in the bone marrow but failto mature, and the production of immunoglobulin inaffected males is grossly depressed. B-cells are reducedor absent in the peripheral blood and there are few lymphoid follicles or plasma cells in the lymphnodes.

Affected boys are usually well for the first fewmonths of life as they are protected by transplacentallyacquired maternal antibody. They are then subject to repeated infection by pyogenic bacteria —Staphylococcus aureus, Streptococcus pyogenes and pneu-moniae, Neisseria meningitidis, Haemophilus influenzae —and by Giardia lamblia which chronically infects thegastrointestinal tract. Resistance to viruses and fungi,which depends on T-lymphocytes, is normal, with theexception of hepatitis and enteroviruses. Therapy involves the repeated administration of human gammaglobulin to maintain adequate concentrationsof circulating immunoglobulin.

Immunoglobulin A (IgA) deficiency due to a fail-ure of IgA-bearing lymphocytes to differentiate intoplasma cells is encountered with relative frequency.Many of these individuals are clinically asymptomaticbut those with symptoms have sinopulmonary andgastrointestinal infections. An increased frequency ofautoimmune and allergic disorders is noted in thesepatients who are at risk for developing anti-IgA anti-bodies on receipt of blood products.

Common variable immunodeficiency (CVID) isthe most common form of primary immunodeficiencybut the least well characterized. It is a heterogeneousdisease usually appearing in adult life and is asso-ciated with a high incidence of autoimmune disordersand malignant disease, particularly of the lymphoidsystem. It probably results from inadequate T-cell sig-nal transduction, which causes imperfect interactionsbetween T- and B-cells. In the absence of suitable T-cellinteractions, there is defective terminal differentiationof B-lymphocytes into plasma cells in some cases or de-fective ability to secrete antibody in others. Althoughsome patients with CVID have a homozygous partialdeletion of the ICOS gene (the “inducible costimula-tor” on activated T cells), the genetic basis of this dis-ease in most patients is still unknown.

X-linked hyper-IgM syndrome is a rare disordercharacterized by recurrent bacterial infections, verylow levels or absence of IgG, IgA and IgE, and normalto raised concentrations of serum IgM and IgD. Thedisease is due to point mutations, usually single aminoacid substitutions in the CD40L (CD40 ligand) ex-pressed by CD4+ T-cells. Such defects will interferewith the normal interaction between CD40L and itscounter-receptor CD40 on B-cells, a reaction which iscrucial for the generation of memory B-cells and for Igclass-switching. Furthermore, it has been shown thatinteraction between CD40L on activated T-cells andCD40 on macrophages will stimulate IL-12 secretion,which is important in eliciting an adequate immune re-sponse against intracellular pathogens. This may ex-


CHRONICGRANULOMATOUS DISEASE

p

q

X

28

27

26

25

24

23

22.

21.

13

12

11.

11.

22.

21.

3

2

1

3

2

1

4

3

23

2221

1

1

2

1

2

3

123

WISKOTT–ALDRICH SYNDROME

SEVERE COMBINEDIMMUNODEFICIENCY

XL-AGAMMAGLOBULINEMIA

XL-HYPER IgMSYNDROME

XL-LYMPHOPROL-IFERATIVE SYNDROME

Figure 13.3 Loci of the X-linked (XL) immunodeficiency syndromes.

plain why these patients are also unduly susceptible tothe intracellular organism Pneumocystis carinii.

Transient hypogammaglobulinemia of infancy isassociated with an abnormal delay in the onset of Igsynthesis, which can extend into the second or thirdyear of life. It is characterized by recurrent respiratoryinfections, and low IgG levels which return to normalby 4 years of age.

PRIMARY T-CELL DEFICIENCY

DiGeorge syndrome is characterized by a failure of thethymus to develop normally from the third and fourthpharyngeal pouches during embryogenesis. Thesechildren also lack parathyroids and have severe car-diovascular abnormalities, and most show microdele-tion of specific DNA sequences from chromosome22q11.2. Consequently, stem cells cannot differentiateto become T-lymphocytes and the “thymus-depen-dent” areas in lymphoid tissue are sparsely populated;in contrast lymphoid follicles are seen but even theseare poorly developed (figure 13.4). Cell-mediated im-mune responses are undetectable, and, although theinfants can deal with common bacterial infections,they may be overwhelmed by vaccinia (figure 13.5) ormeasles, or by BCG (bacille bilié de Calmette–Guérin)

if given by mistake. Humoral antibodies can be elicitedbut the response is subnormal, presumably reflectingthe need for the cooperative involvement of T-cells.Transplantation of cultured, mature thymic epithelialcells has successfully reconstituted immune functionin these patients and is considered as the treatment ofchoice. Partial hypoplasia of the thymus is more fre-quent than total aplasia, and immunologic treatment isusually not indicated.

PRIMARY T-CELL DYSFUNCTION

A number of syndromes due to abnormal T-cell func-tion have been described. These differ from severecombined immunodeficiency (SCID; see below) byhaving T-cells in the peripheral blood. They includepatients with mutations in the genes encoding the g or echain of the CD3 molecule resulting in T-cells with de-fective function. Another group have a defective formof ZAP-70, a tyrosine kinase crucial for T-cell signaltransduction, and a further subset of patients have ab-normal IL-2 production resulting from an IL-2 genetranscription failure. The clinical picture produced bythese defects is similar to that seen in patients withSCID, and bone marrow transplantation offers theonly hope of long-term cure.


Figure 13.4 Lymph node cortex. (a) From patient with DiGeorgesyndrome showing depleted thymus-dependent area (TDA) andsmall primary follicles (PF). (b) From normal subject: the populatedT-cell area and the well-developed secondary follicle with its mantle

of small lymphocytes (M) and pale-staining germinal center (GC)provide a marked contrast. (DiGeorge material kindly supplied byDr D. Webster; photograph by Mr C.J. Sym.)

SEVERE COMBINED IMMUNODEFICIENCY(SCID)

Severe combined immunodeficiency is a heteroge-neous group of diseases involving T-cell immuno-deficiency with or without B-cell or NK-cellimmunodeficiency, resulting from any one of severalgenetic defects. The cases are characterized by severelymphopenia with deficient cellular and humoral im-munity. These children suffer recurrent infectionsearly in life. Prolonged diarrhea resulting from gas-trointestinal infections and pneumonia due to Pneumo-cystis carinii are common; Candida albicans growsvigorously in the mouth or on the skin. If vaccinatedwith attenuated organisms (figure 13.5) these childrenusually die of progressive infection, and most will not

survive the first year of life unless their immune sys-tems are reconstituted by hematopoietic stem celltransplantation.

Mutation in the common cytokine receptor gc chain is the commonest cause of SCID

Severe combined immunodeficiency occurs in both anX-linked recessive and an autosomal form, but 50–60%of the cases are X-linked (X-SCID) and derive from mutations in the common g chain of the IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors. A number of muta-tions of this gene have been described and each resultsin a complex association of defects in the six affected cytokine/cytokine receptor systems.

Severe combined immunodeficiency can be due to mutations in purine salvage pathway enzymes

Many SCID patients with the autosomal recessiveform of the disease have a genetic deficiency of thepurine degradation enzymes, especially adenosinedeaminase (ADA) and less commonly purine nucleo-side phosphorylase (PNP). This results in the accumu-lation of metabolites that are toxic to lymphoid stemcells. Enzyme replacement therapy with injections ofpolyethylene glycol-modified adenosine deaminaseresults in clinical and immunologic improvement, andgene therapy using transfer of the ADA gene into ei-ther peripheral blood lymphocytes or hematopoieticstem cells has also met with some success. Bone mar-row transplantation remains the treatment of choice.

Other SCID variants

A variety of other rare defects have been identified inindividual SCID patients. These include mutations inthe RAG genes, which initiate VDJ recombinationevents, and in another gene called the Artemis gene,which is crucial for repairing DNA after double-stranded cuts have been made by the RAG genes. Severe combined immunodeficiency will also resultfrom mutations of the gene encoding JAK3 kinase,which is crucial for transducing the signal when IL-2and other cytokines bind their receptors. Other pa-tients with SCID have mutations of the CD45 commonleukocyte antigen surface protein, which is normallyrequired to regulate the Src kinase required for T- andB-cell antigen receptor signal transduction. There arealso patients with a defect in the CD3d gene who showa complete block in the development of T-cells. In thebare lymphocyte syndrome there is an MHC class II


Figure 13.5 A child with severe combined immunodeficiency(SCID) showing skin lesions due to infection with vaccinia gan-grenosum resulting from smallpox immunization. Lesions werewidespread over the whole body. (Reproduced by kind permissionof Professor R.J. Levinsky and the Medical Illustration Departmentof the Hospital for Sick Children, Great Ormond Street, London.)

deficiency; few CD4+ cells developing in the thymus,and those that do are inadequately stimulated by antigen-presenting cells lacking class II molecules. Be-cause MHC class I molecules are normal, CD8+ cellsare present. The condition is due to mutations in one ofa number of genes that regulate class II MHC expres-sion rather than in the class II MHC genes themselves.Rare patients with mutations in TAP1 or TAP2 haveMHC class I deficiency and are also prone to recurrentinfections. Interaction between MHC class I moleculeson thymic cells is essential for CD8+ cell maturation,and therefore these patients have very low or absentCD8+ cells but normal numbers of functioning CD4+

cells.

OTHER COMBINED IMMUNODEFICIENCYDISORDERS

Wiskott–Aldrich syndrome (WAS) is an X-linked dis-ease characterized by thrombocytopenia, type I hyper-sensitivity and immunodeficiency. This results inbleeding, eczema and recurrent infections. The condi-tion is due to mutations of the gene encoding the so-called Wiskott–Aldrich syndrome protein (WASP)that is important in the organization of the cytoskele-ton of both lymphocytes and platelets. This accountsfor the disorganization of the cytoskeleton and loss ofmicrovilli seen in T-cells from these patients, a featurethat can be used to diagnose these cases prenatally. Innumerous patients the platelet and immunologic ab-normalities have been corrected by allogeneic stem celltransplantation. Gene therapy is likely to be the treat-ment of the future, and in in vitro studies retroviral in-fection of WASP-negative cells with WASP can restorenormal function.

Ataxia telangiectasia is an autosomal recessive dis-order of childhood characterized by progressive cere-bellar ataxia due to degeneration of Purkinje cells,associated with vascular malformations (telangiecta-sia), increased incidence of malignancy and defects ofboth T- and B-cells. These patients also display a hy-persensitivity to X-rays, which, together with the un-duly high incidence of cancer, has been laid at the doorof a defect in DNArepair mechanisms.

X-linked lymphoproliferative disease, also calledDuncan’s syndrome, is an immunodeficiency syn-drome associated with infection by the Epstein–Barrvirus (EBV). The condition is due to a mutation of thegene encoding SAP (SLAM-associated protein), whichregulates signal transduction from the “signaling lym-phocyte activation molecule” (SLAM) present on thesurfaces of T and B-cells. Since activation of SLAM en-hances IFNg production by T-cells and proliferation of

B-cells, significant immune deficiency results from thismutation.

A summary of the various primary immunodefi-ciency states is shown in Table 13.1.

RECOGNITION OF IMMUNODEFICIENCIES

Defects in humoral immunity can be assessed by quan-titative Ig estimations and by measuring the antibodyresponses that follow active immunization with diph-theria, tetanus, pertussis and killed poliomyelitis. B-cells can be enumerated by flow cytometry usingantibodies against CD19, CD20 and CD22.

Enumeration of T-cells is most readily achieved byflow cytometry using CD3 monoclonal antibody orother antibodies directed against T-cells such as CD2,CD5, CD7 or CD4 and CD8. Patients with T-cell defi-ciency will be hyporeactive or unreactive in skin teststo such antigens as tuberculin, Candida, tricophytin,streptokinase/streptodornase and mumps, and theirlymphocytes demonstrate reduced cytokine produc-tion when stimulated by phytohemagglutinin (PHA)or other nonspecific mitogens.

In vitro tests for complement and for the bactericidaland other functions of polymorphs are available, whilethe reduction of nitroblue tetrazolium (NBT) or thestimulation of superoxide production provides a measure of the oxidative enzymes associated with active phagocytosis and bactericidal activity.

SECONDARY IMMUNODEFICIENCY

Immune responsiveness can be depressed nonspecifi-cally by many factors. Cell-mediated immunity in par-ticular may be impaired in states of malnutrition, evenof the degree which may be encountered in urban areasof the more affluent regions of the world.

Viral infections are not infrequently immunosup-pressive, and the depressed CMI, which accompaniesmeasles infection, has been attributed to suppressionof IL-12 production by the virus. The most notoriousimmunosuppressive virus, human immunodeficiencyvirus (HIV), is elaborated upon below.

Many therapeutic agents such as X-rays, cytotoxicdrugs and corticosteroids may have dire effects on theimmune system. B-lymphoproliferative disorderslike chronic lymphocytic leukemia, myeloma andWaldenström’s macroglobulinemia are associatedwith varying degrees of hypogammaglobulinemiaand impaired antibody responses. These patients aresusceptible to infections with common pyogenic bacteria, in contrast to the situation in Hodgkin’s disease where the patients display all the hallmarks of


defective CMI —susceptibility to tubercle bacillus,Brucella, Cryptococcus and herpes zoster virus.

ACQUIRED IMMUNODEFICIENCYSYNDROME (AIDS)

Acquired immunodeficiency syndrome is due to thehuman immunodeficiency virus (HIV) of which there

are two main variants called HIV-1 and HIV-2. Thevirus is transmitted inside infected CD4 T cells andmacrophages, and the disease is therefore spread sexu-ally or through blood or blood products such as en-countered by intravenous drug users. The virus mayalso be transmitted from an infected mother to her in-fant, in which case babies born with a high viral loadprogress more rapidly than those with lower viral


DEFECTIVE GENE PRODUCT(S) DISORDER

COMPLEMENT DEFICIENCES

PHAGOCYTIC DEFECTS

PRIMARY B-CELL DEFICIENCY

PRIMARY T-CELL DEFICIENCY

COMBINED IMMUNODEFICIENCY

MBL, MASP-2

C1, C2, C4

C1 inhibitor

PIG-A glycosyltransferase

C3, Factor H, Factor I

C5, C6, C7, C8

Recurrent bacterial infections

Immune complex disease (SLE)

Angioedema

Paroxysmal nocturnal hemoglobinuria

Recurrent pyogenic infections

Recurrent infectionsNeisseria

NADPH oxidase

CD18 (b2-integrin b chain)

CHS

IFNgR1/2, IL-12p40, IL-12Rb1

Chronic granulomatous disease

Leukocyte adhesion deficiency

Chediak–Higashi disease

Mendelian susceptibility to mycobacterial infection

Bruton’s tyrosine kinase

?

ICOS

?

X-linked agammaglobulinemia

IgA deficiency

Common variable immunodeficiency

Transient infant hypogammaglobulinemia

?

RAG-1/2

MHC class II promoters

Atm

CD154 (CD40L)

CD3 e and g chains, ZAP-70

DiGeorge and Nezelof syndromes; failure of thymic development

Omenn’s syndrome; partial recombination

Bare lymphocyte syndrome

Ataxia telangiectasia; defective DNA repair

Hyper-IgM syndrome

Severe T-cell deficiency

VDJ

?

IL-7Ra

gc, JAK3

RAG-1/2

ADA

PNP

SAP

WASP

Reticular dysgenesis; defective production of myeloid and lymphoid

SCID due to defective IL-7 signaling

SCID due to defective cytokine signaling

Complete failure of recombination

Adenosine deaminase deficiency; toxic to early lymphoid stem cells

Purine nucleoside phosphorylase deficiency toxic to T-cells

X-linked lymphoproliferative disease; defective cell signaling

Wiskott–Aldrich syndrome; defective cytoskeletal regulation

precursors

VDJ

Table 13.1 Summary of primary immuno-deficiency states.

Ig, immunoglobulin; IL-7, interleukin-7; SCID, severe combined immunodeficiency; SLE, sys-temic lupus erythematosus.

loads. The disease causes widespread immune dys-function, with a protracted latent period but progres-sive decrease in CD4 cells leading eventually toseverely depressed CMI. Once the absolute CD4 countfalls below 200 cells/cu mm an individual becomessusceptible to opportunistic infections. These involvemost commonly, Pneumocystis carinii, cytome-galovirus, EBV, herpes simplex virus, fungi such asCandida, Aspergillus and Cryptococcus, and the proto-zoan Toxoplasma; additionally, there is exceptional susceptibility for Kaposi’s sarcoma induced by humanherpesvirus 8 (HHV-8).

Characteristics of HIV

Transmission of the disease is usually through infec-tion with blood or semen containing the HIV-1 virus orthe related HIV-2. Sexual transmission occurs via mu-

cosal surfaces followed by spread throughout the lymphatic system. HIV-1/2 are members of thelentivirus group, which are budding viruses (figure13.6b) whose genome is relatively complex and tightlycompressed. The many virion proteins (figure 13.6a)are generated by RNA splicing and cleavage by theviral protease.

The infection of cells by HIV

Infection takes place when envelope glycoproteingp120 of HIV binds avidly to cell-surface CD4 mole-cules on helper T-cells, macrophages, DCs and mi-croglia. Dendritic cells populate the human mucosaand project their dendrites through the epithelial cellsso that they are directly exposed on the mucosal sur-face. The binding to the CD4 molecule initiates the fusion of gp41 on the viral membrane to various


b VIRUS BUDDING FROM T-CELL

MAJOR CORE PROTEINREVERSE TRANSCRIPTASE

NUCLEOPROTEIN

RNA

gp120

gp41

a HIV-1 STRUCTURE

c

Cell activation

HOST DNAIntegration

dsDNA HIV RNA

Transcription

Reverse transcription

TranslationssRNA

BuddingEntry

CD4

Fusion

Specific binding

Release

FREE VIRUS

HIV

CCR5/CXCR4

CELL

Figure 13.6 Characteristics of the HIV-1 acquired immunodefi-ciency syndrome (AIDS) virus. (a) HIV-1 structure. (b) Electron mi-crograph of mature and budding HIV-1 particles at the surface of

human phytohemagglutinin (PHA) blasts. (c) Intracellular life cycleof human immunodeficiency virus (HIV). (Photograph kindly sup-plied by Dr C. Upton and Professor S. Martin.)

chemokine co-receptors on the host cell (figure 13.6c).(It is worth noting that a new very successful anti-HIVdrug (Enfuvirtide) acts by specifically inhibiting thisfusion.) Early in infection the viruses utilize the CCR5co-receptor present on memory T-cells, macrophagesand DCs, and later infect resting T-cells using theCXCR4 co-receptor. It is interesting that mutations orcomplete absence of the CCR5 receptor have been de-scribed in 1% of Caucasians, and are associated withincreased resistance to infection. Such mutations havenot been described in people of African or Japanese descent.

Human immunodeficiency virus is an RNA retro-virus, which utilizes a reverse transcriptase to convertits genetic RNA into the corresponding DNA. This isintegrated into the host genome where it can remain la-tent for long periods (figure 13.6c). Stimulation of la-tently infected T-cells or macrophages activates HIVreplication through an increase in the intracellular con-centration of NFkB (nuclear factor-kappa B) dimers,which bind to consensus sequences in the HIV en-hancer region. This initiation of HIV gene transcrip-tion occurs when the host cells are activated bycytokines or by specific antigen. It is significant thattumor necrosis factor-a (TNFa), which upregulatesHIV replication through this NFkB pathway, is presentin elevated concentrations in the plasma of HIV-infected individuals, particularly in the advancedstage when they are infected with multiple organisms.Perhaps also, the more rapid progression of HIV infection in Africa may be linked to activation of theimmune system through continual microbial insult.

The AIDS infection depletes helper T-cells

Natural history of the disease

The sequence of events following HIV-1 infection ischarted in figure 13.7. The virus normally enters thebody by infecting Langerhans’ cells in the rectal orvaginal mucosa and then moves to local lymph nodeswhere it replicates. The virus is then disseminated by aviremia, which is associated with an acute early syn-drome of fever, myalgia and arthralgia. A dominantnucleocapsid viral antigen, p24, can be detected in theblood during this phase. This phase is eventually con-trolled by activation of CD8 T-cells and by circulatingantibodies to p24 and the envelope proteins gp120 andgp41. The efficacy of cytotoxic T-cells in controlling thevirus is seen by a decline in level of virus, associatedwith a rebound of CD4+ cells. This immune responseleads to sequestration of HIV in lymphoid tissue,which becomes the major reservoir of HIV during thenext stage of the disease, which is regarded as the clini-cally latent phase. Within the lymphoid follicles infectious virus becomes concentrated in the form ofimmune complexes held by follicular dendritic cells(FDCs). Although only low levels of virus are pro-duced during the latent phase, destruction of CD4+

cells continues within the lymphoid tissue. Eventuallycirculating CD4 T-cell numbers fall progressively,but it may take many years before CD4 cell loss leads tothe chronic progressive phase of the disease. The pa-tient is now wide open to life-threatening infectionswith normally nonpathogenic (opportunistic) agents.


Time after infection

Bloo

d le

vels

ACUTE VIRALSYNDROME

ASYMPTOMATIC ACQUIREDIMMUNODEFICIENCY SYNDROME

YearsMonths 1 2 3 4 5 6

Anti-p24

Anti-gp120

CD4

CTL

Viralload

Figure 13.7 The natural history of HIV-1infection. The changes in p24 antigen andantibody shown late in the disease are seenin some but not all patients. CTL, cytotoxicT-lymphocyte.

Mechanisms of CD4 cell depletion

The major immunologic feature of AIDS is the destruc-tion of CD4 cells, a phenomenon that cannot be solelyexplained by the direct cytopathic effect of HIV. Thereis certainly no shortage of hypotheses to account forCD4 depletion with the corresponding change in theCD4 : CD8 ratio. These include:1 A direct cytopathic effect by the virus either on sin-gle cells or through the formation of multinucleatesyncytia between infected and noninfected CD4 cells.2 Human immunodeficiency virus sensitizes CD4+ Tcells for activation-induced apoptosis. Stimulation ofCD4 T-cells in HIV-infected individuals leads to apop-tosis. This may explain the deterioration that followsother infections and the subsequent cytokine produc-tion that is so common in these patients. There is someevidence that HIV-infected cells are particularly sensi-tive to Fas-induced apoptosis, and this may relate tothe expression of Fas on infected or uninfected T-cellsinduced by gp120 and tat proteins produced by HIV.3 Cytotoxic CD8 cells are crucial in reducing viral loadearly in the HIV infection. They expand rapidly in in-fected patients and may play an important role inlysing infected CD4-positive cells.4 Antibodies against HIV proteins may bind to infected cells which may then be destroyed by antibody-dependent cellular cytotoxicity (ADCC).5 Human immunodeficiency virus infection is oftenassociated with defective production or maturation ofnew T-cells from the thymus or bone marrow.It is likely that a combination of several of these mecha-nisms ultimately act to tip the balance in favor of thevirus.

Human immunodeficiency virus has strategies thatallow it to evade the immune response

Human immunodeficiency virus employs a number ofstrategies to evade immune attack but most importantis the high mutation rate of the virus that allows it toevade specific antibodies and cytotoxic T-cells. In factimmune responses against the virus may cause selection pressure that favors survival of the most genetically variable virus strains. Also, by depletingCD4 cells, especially HIV specific T-cells, HIV infectionresults in a severely compromised immune system. Inaddition, an HIV protein called Nef has the ability todownregulate the expression of MHC class 1 mole-

cules, a process that could impair cytotoxic activity byCD8 cells.

Laboratory diagnosis of AIDS

Patients with HIV infection will demonstrate the pres-ence of viral antibodies within a few weeks of infec-tion. CD4 levels decrease rapidly after infection as isshown by a reversal of the normal CD4 :CD8 ratio.Quantitative HIV-RNA plasma viral load measure-ment is now routinely employed to evaluate and mon-itor patients with HIV infection. It includes measuringthe baseline of viral load prior to initiation of antiretro-viral therapy and assessing the efficacy of antiviraldrug treatment. Quantitative HIV-RNA measure-ments are also the best indicator of the development ofAIDS-defining opportunistic infections and subse-quent progression of the disease. Associated with thedecline in CD4 cells many tests of CMI will show de-fects, including the delayed hypersensitivity skin re-sponse to recall antigens.

Treatment of HIV disease

Therapeutic options for treating HIV are expandingwith the development of numerous new drugs. Themanagement of patients, however, has become highlycomplex due to the emergence of drug resistance andrecognition of long-term toxicity of antiretroviralagents. Prolonged survival depends on the use of anti-retroviral agents, immunization and chemoprophy-laxis, and aggressive treatment of infectious andmalignant complications when they occur. An increas-ing number of antiretroviral agents have been devel-oped and these include both nonnucleoside andnucleoside reverse transcriptase inhibitors and pro-tease inhibitors. Usually these are given as triple-drugtherapy (HAART or highly active antiretroviral drugtherapy) to reduce the development of mutated formsof the virus. Although the optimal time to initiate thesetherapies is controversial, they should always be usedin symptomatic patients and in any infected personwith a CD4 count below 500 mm–3 or with a viral load>50000 copies/ml. The major drawback of thesedrugs is their enormous expense, which limits theiruse in those communities of the world where they aremost needed. The ideal solution is to produce both pro-phylactic and therapeutic vaccines against HIV, butdespite a huge effort an effective vaccine remainselusive.



REVISION

Primary immunodeficiency states• These occur in the human, albeit somewhat rarely, as aresult of a defect in almost any stage of differentiation inthe whole immune system.• Rare X-linked mutations produce disease in males.• Defects in phagocytic cells, the complement pathwaysor the B-cell system lead in particular to infection with bacteria, which are disposed of by opsonization andphagocytosis.• Patients with T-cell deficiencies are susceptible to virus-es and fungi, which are normally eradicated by cell-mediated immunity (CMI).

Deficiencies of innate immune mechanisms• Chronic granulomatous disease results from mutationsin the NADPH (nicotinamide adenine dinucleotide phos-phate) oxidase of phagocytic cells.• Chediak–Higashi leukocytes contain giant lysosomalgranules which interfere with their function.• Leukocyte adhesion deficiency involves mutations inthe CD18 subunit of b2-integrins.• Deficiency of one of the early components of the classi-cal complement pathway may result in immune complexdisease.• Deficiency of C3 results in severe recurrent pyogenic infections.• Deficiency of the late complement components resultsin increased susceptibility to recurrent Neisseria infections.• Lack of C1 inhibitor leads to hereditary angioedema.• Deficiencies in C1, C4 or C2 are associated with systemiclupus erythematosus (SLE) -like syndromes.

Primary B-cell deficiency• Congenital X-linked agammaglobulinemia (XLA) (Bruton), involving differentiation arrest at the pre-Bstage, is caused by mutations in the Bruton’s tyrosine kinase (Btk) gene.• Patients with immunoglobulin A (IgA) deficiency aresusceptible to infections of the respiratory and gastroin-testinal tract.• In common variable immunodeficiency the B-cells failto secrete antibody, probably due to abnormal interactionswith T-cells.• Mutations in the T-cell CD40L (CD40 ligand) gene pro-vide the basis for hyper-IgM syndrome.

Primary T-cell deficiency• DiGeorge syndrome results from failure of thymic development.

Primary T-cell dysfunction• This group of patients have circulating T-cells, but theirfunction is abnormal due a variety of genetic defects.

Severe combined immunodeficiency (SCID)• Fifty to sixty percent of the patients with SCID have mu-tations in the gene for the g chain common to receptors forIL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.• Many SCID patients have a genetic deficiency of thepurine degradation enzymes adenosine deaminase(ADA) and purine nucleoside phosphorylase (PNP),which leads to accumulation of toxic products. Patientswith ADAdeficiency are being corrected by gene therapy.• The bare lymphocyte syndrome is due to defective ex-pression of major histocompatibility complex (MHC) classII molecules.

Other combined immunodeficiency disorders• Wiskott–Aldrich males have a syndrome characterizedby thrombocytopenia, eczema and immunodeficiency.• Defective DNA repair mechanisms are found in pa-tients with ataxia telangiectasia.

Recognition of immunodeficiencies• Humoral immunodeficiency can be assessed initiallyby quantitation of immunoglobulins in the blood.• B- and T-cell numbers can be enumerated by flow cyto-metric analysis.

Secondary immunodeficiency• Immunodeficiency may arise as a secondary conse-quence of malnutrition, lymphoproliferative disorders,agents such as X-rays and cytotoxic drugs, and viral infections.

Acquired immunodeficiency syndrome (AIDS)• AIDS results from infection by the RNA retrovirusesHIV-1 and HIV-2.• Human immunodeficiency virus (HIV) infects T-helpercells through binding of its envelope gp120 to CD4 withthe help of a cofactor molecule, CXCR4 and other similarcytokine receptors. It also infects macrophages, microglia,T-cell-stimulating dendritic cells (DCs) and follicular den-dritic cells (FDCs), the latter through a CD4-independentpathway.• Within the cell, the RNA is converted by the reversetranscriptase to DNA, which can be incorporated into thehost’s genome where it lies dormant until the cell is acti-vated by stimulators such as tumor necrosis factor-a

FURTHER READING

Ballow, M. (2002) Primary immunodeficiency disorders: Antibodydeficiency. Journal of Allergy and Clinical Immunology, 109, 581–91.

Buckley, R.H. (2002) Primary cellular immunodeficiencies. Journal ofAllergy and Clinical Immunology, 109, 74–57.

Buckley, R.H. (2000) Primary immunodeficiency diseases due to de-fects in lymphocytes. New England Journal of Medicine, 343,1313–24.

Cohen, O.J. (1997) Host factors in the pathogenesis of HIV disease.Immunological Reviews, 159, 31–48.

Conley, M.E. (ed.) (2003) Section on immunodeficiency. CurrentOpinion in Immunology, 15, 567–98.

Gandhi, R.T. & Walker, B.D. (2002) Immunologic control of HIV-1.Annual Review of Medicine, 53, 149–72.

Lekstrom-Himes, J.A. & Gallin, J.I. (2000) Immunodeficiency dis-eases caused by defects in phagocytes. New England Journal of Medicine, 343, 1703–14.

Ochs, H.D., Smith, C.I.E. & Puck, J.M. (eds) (2000) Primary Immunod-eficiency Diseases — A Molecular and Genetic Approach. Oxford Uni-versity Press, New York & Oxford.

Stebbing, J., Gazzard, B. & Douek, D.C. (2004) Where does HIV live?New England Journal of Medicine, 350, 1872–80.


(TNFa) which increase NFkB (nuclear factor-kappa B) levels.• There is usually a long asymptomatic phase after theearly acute viral infection has been curtailed by an im-mune response, and the virus is sequestered to the FDC inthe lymphoid follicles where it progressively destroys theDC meshwork.• Adisastrous fall in CD4 cells destroys cell-mediated de-fenses and leaves the patient open to life-threatening infec-tions through opportunist organisms such as Pneumocystiscarinii and cytomegalovirus.• There is a tremendous battle between the immune sys-tem and the virus, with extremely high rates of viral de-struction and CD4 T-cell replacement.

• CD4 T-cell depletion may eventually occur as a result ofdirect pathogenicity, apoptosis, antibody-dependent cel-lular cytotoxicity (ADCC), direct cytotoxicity by CD8 cellsor disruption of normal T-cell production.• AIDS is diagnosed in an individual with opportunisticinfections, by low CD4 but normal CD8 T-cells in blood,poor delayed-type skin tests, positive tests for viral anti-bodies and p24 antigen, lymph node biopsy and isolationof live virus or demonstration of HIV genome by poly-merase chain reaction (PCR).


148

INAPPROPRIATE IMMUNE RESPONSES CANLEAD TO TISSUE DAMAGE

When an individual has been immunologicallyprimed, further contact with antigen leads to sec-ondary boosting of the immune response. However,the reaction may be excessive and lead to tissue dam-age (hypersensitivity). It should be emphasized thatthe mechanisms underlying these inappropriate reac-tions are those normally employed by the body in com-bating infection, as discussed in Chapter 11. Coombsand Gell defined four types of hypersensitivity. TypesI, II and III depend on the interaction of antigen withhumoral antibody, whereas type IV involves T-cellrecognition. Because of the longer time course this has in the past been referred to as “delayed-type hypersensitivity.”

TYPE I: ANAPHYLACTIC HYPERSENSITIVITY

Type 1 allergic disease, often referred to as atopic dis-ease, is a group of conditions occurring in people witha hereditary predisposition to produce immunoglobu-lin E (IgE) antibodies against common environmentalantigens (allergens). These conditions include some ofthe commonest causes of ill health including allergicrhinitis, asthma and atopic eczema.

Type I allergic reactions are due to activation of Th2 cells and the overproductionof IgE antibodies

The reason for this overproduction of IgE antibodies incertain individuals is unclear. There is increasing evi-dence that lack of exposure to bacteria which stimulateTh1 responses in early life favors the Th2 phenotypewith subsequent development of atopy. This “hygienehypothesis” could explain the lower incidence of aller-gic disease in individuals who farm or live in ruralcommunities where they are presumably exposed toorganisms from livestock. Allergic reactions are ini-tiated by a limited number of allergens which are de-posited in low doses on mucosal epithelium or skin.This is a particularly efficient way of activating Th2cells and inducing IgE responses. Allergic individualsnot only have larger numbers of allergen-specific Th2cells in their blood, but these cells produce greateramounts of interleukin-4 (IL-4) per cell than Th2 cellsfrom normal people. As with other antigens, allergensare processed by Langerhans’ or other antigen-presenting cells and are proteolytically cleaved intosmall peptides and presented to uncommitted Th0cells. In the case of an allergic response, these cells nowdifferentiate into Th2 lymphocytes, which release various cytokines that inhibit Th1 cell activation andupregulate IgE production. These cytokines, particu-larly IL-4 and IL-13 together with signals delivered

CHAPTER 14

Hypersensitivity

Inappropriate immune responses can lead to t issuedamage, 148

Type I: Anaphylactic hypersensitivity, 148Type I allergic reactions are due to activation of Th2 cells and

the overproduction of IgE antibodies, 148There is a strong genetic component to allergic diseases, 149Clustering of IgE receptors on mast cells through cross-linking

triggers anaphylaxis, 149Atopic allergy, 150

Type II : Antibody-dependent cytotoxichypersensitivity, 154Type II reactions between members of the same species

(alloimmune), 154

Autoimmune type II hypersensitivity reactions, 156Anti-receptor autoimmune diseases, 156Type II drug reactions, 156

Type II I : Immune complex-mediated hypersensitivity, 157Inflammatory lesions due to locally formed complexes, 157Disease resulting from circulating complexes, 158

Type IV: Cell-mediated (delayed-type)hypersensitivity, 160Tissue damage produced by type IV reactions, 161

by the B-cell surface molecule CD40, induce isotypeswitching in newly generated IgM-bearing B-cellsfrom m to e resulting in subsequent production of IgE.

There is a strong genetic component to allergic diseases

Genetic susceptibility to allergic reactions has not beenclearly defined but high levels of IgE are noted in certain allergic (atopic) families. Human leukocyte antigen (HLA) linkage is associated with allergic re-sponses to a number of allergens including ragweedand house dust mite. A further major genetic locus,which regulates serum IgE levels, has been identifiedon chromosome 5q in the region containing the genesfor IL-4, IL-5 and IL-9, cytokines that are important inregulating IgE synthesis. Polymorphism of the IL-9gene is associated with asthma, as is a locus on chromo-some 11q13 where the gene encodes the b chain of theIgE receptor.

Clustering of IgE receptors on mast cells throughcross-linking triggers anaphylaxis

Mast cells display a high-affinity receptor (FceRI) forthe Ce2 :Ce3 junction region of IgE Fc, a propertyshared with their circulating counterpart, the basophil.

Cross-linking of receptor-bound IgE antibodies bya multivalent antigen will trigger release of the inflam-matory mediators responsible for the acute allergic re-action through aggregation of these receptors (figure14.1). Activation is rapidly followed by the breakdownof phosphatidylinositol to inositol triphosphate (IP3),

the generation of diacylglycerol (DAG) and an increase in intracytoplasmic free calcium. This bio-chemical cascade allows the granules to fuse with theplasma membrane and release their preformed mediators into the surrounding tissue. It also results in synthesis of lipid mediators including a series ofarachidonic acid metabolites formed by the cyclo-oxygenase and lipoxygenase pathways, and produc-tion of a variety of cytokines which are responsible forthe late phase of the acute allergic reaction.

Mast cell and basophil mediators

Mast cell degranulation is the major initiating event ofthe acute allergic reaction. The preformed mediatorsreleased from the granules include histamine, heparin,tryptase, neutral protease and various eosinophil andneutrophil chemotactic factors. Histamine itself is responsible for many of the immediate symptoms ofallergic reactions including bronchoconstriction, vasodilatation, mucus secretion and edema caused byleakage of plasma proteins from small vessels. Theseeffects can all be reproduced and visualized in the immediate skin test where allergen injected into theskin produces a characteristic wheal and flare reaction with redness, edema and pruritus (itchiness) (cf. figure 14.5a). Tryptase released by mast cells activates receptors on endothelial cells that selectively attracteosinophils and basophils.

Activation of the mast cells also results in the liberation of newly formed lipid mediators, which include the leukotrienes LTB4, LTC4 and LTD4, theprostaglandin D2 (PGD2) and platelet activating factor(PAF). Prostaglandin D2, the leukotrienes and PAF are highly potent bronchoconstrictors, which also increase vascular permeability and are chemotactic for inflammatory cells.

The late phase of the allergic reaction is mediated by cytokines

Mast cells and basophils are responsible for significantproduction of proinflammatory cytokines includingtumor necrosis factor (TNF), IL-1, IL-4 and IL-5, andchemokines such as MIP-1a and MIP-1b. Within 12 hof an acute allergic reaction a late-phase reaction occurs, which is characterized by a cellular infiltrate of CD4+ cells, monocytes and eosinophils. These cells will themselves release a variety of Th2-type cytokines, especially IL-4 and IL-5, which are respon-sible for further inflammation at the site of the allergen.Macrophages in particular can be activated through anFceRII receptor leading to significant release of TNF

CHAPTER 14—Hypersensitivity 149

Degranulation

MASTCELL

IgERECEPTOR

DIVALENTDNP HAPTENIgE

ANTI-DNP

Figure 14.1 Clustering of immunoglobulin E (IgE) receptors by divalent hapten (used as a model for multivalent antigen).

and IL-1. This late reaction, therefore, resembles a de-layed hypersensitivity reaction because of the infiltra-tion of T-cells and the effects of the cytokines. Adifferentiating feature, however, is the presence ofeosinophils and Th2 cells in the late phase of the aller-gic response and their absence from delayed hypersen-sitivity reactions.

The sudden degranulation of mast cells thereforeleads to release of a complex cascade of mediators,which generally results in the symptoms of acute allergic reactions. Under normal circumstances, these mediators help to orchestrate the development of a de-fensive acute inflammatory reaction. When there is amassive release of these mediators under abnormalconditions, as in atopic disease, their bronchoconstric-tive and vasodilatory effects predominate and becomedistinctly threatening.

Eosinophils are prominent in the allergic reaction

The preferential accumulation of eosinophils is a char-acteristic feature of allergic diseases and is due to theproduction of eosinophil chemoattractants from mastcells. These include eotaxin and RANTES (regulatedupon activation, normal T-cell expressed and secreted). Interleukin-5, which is produced by Th2cells, is important in eosinophil activation and also inhibits the natural apoptosis of eosinophils which regulates their lifespan. Under the influence of thesecytokines, eosinophils adhere to vascular endotheli-um via b2-integrins and move through to the site of theallergic reaction. The eosinophil granules release verybasic, highly charged polypeptides. These includemajor basic protein and eosinophil peroxidase, whichcause tissue damage including destruction of the respiratory epithelium in asthmatics. Eosinophils are also an important source of leukotrienes, PAF, cytokines including IL-3 and IL-5, and a variety ofeosinophil chemoattractants which further amplifythe eosinophil response.

Atopic allergy

Examples of clinical responses to inhaled allergens

Allergic rhinitis. Nearly 10% of the population suffer toa greater or lesser degree from allergies involving lo-calized IgE-mediated anaphylactic reactions to extrin-sic allergens such as grass pollens, animal danders, thefeces from mites in house dust (figure 14.2) and so on.The most common manifestation is allergic rhinitis,often known as hay fever. The target organs are the

mucus membranes of the nose and eyes (figure 14.3)and the allergic inflammation results in congestion,itchiness and sneezing, which is so characteristic ofthis condition. An increasing number of causative allergens have now been cloned and expressed including Der p1 and Der p2 from house dust mitesand Fel d1 from cat dander. Exposure to these andother allergens produces the typical symptoms withinminutes.

Asthma. Asthma is a chronic disease characterized byincreased responsiveness of the tracheobronchial treeto a variety of stimuli resulting in reversible airflowlimitation and inflammation of the airways. It isthought that exposure to indoor allergens early in in-fancy predisposes to the disease regardless of a familyhistory, but the development of asthma is influencedby multiple genetic and environmental factors.Bronchial biopsy and lavage of asthmatic patients reveals involvement of mast cells, eosinophils andmacrophages as the major mediator-secreting effectorcells, while T-cells provide the microenvironment re-quired to sustain the inflammatory response which isan essential feature of the histopathology (figure 14.4).Cytokines released by all these infiltrating cells giverise to allergic inflammation resulting in edema of theairway wall, mucus hypersecretion, variable airflowobstruction and bronchial hyper-responsiveness.There is also a repair-type response involving the


Figure 14.2 House dust mite —a major cause of allergic disease.The electron micrograph shows the rather nasty looking mite,graced by the name Dermatophagoides pteronyssinus, and fecal pelletson the bottom left, which are the major source of allergen. The bicon-cave pollen grains (top left), shown for comparison, indicate the sizeof particles which can become airborne and reach the lungs. The miteitself is much too large for that. (Reproduced courtesy of Dr E.Tovey.)

production of fibroblast growth factor, transforminggrowth factor-b (TGFb) and platelet-derived growthfactor (PDGF), which lead to fibrosis and hypertrophyof smooth muscle that further narrows the airways.

Anaphylaxis. The earliest accounts of inappropriate re-sponses to foreign antigens relate to anaphylaxis. Thephenomenon can be readily reproduced in guinea pigswhich, like humans, are a highly susceptible species. Asingle injection of 1 mg of an antigen such as egg albu-min into a guinea pig has no obvious effect. However,if the injection is repeated 2–3 weeks later, the sensi-tized animal reacts very dramatically with the symp-toms of generalized anaphylaxis; almost immediatelythe guinea-pig begins to wheeze and within a few minutes dies from asphyxia. Examination shows intense constriction of the bronchioles and bronchi andgenerally there is contraction of smooth muscle and dilatation of capillaries. Similar anaphylactic reactionscan occur in human subjects and comprise laryngealedema, hypotension and lower-airway obstruction.They may be produced by peanuts, shellfish or otherfood allergens, or may follow wasp and bee stings orinjections of penicillin or other drugs in sensitized in-dividuals. In many instances only a timely injection ofepinephrine to counter the hypotension, the smoothmuscle contraction and capillary dilatation, can pre-vent death.

Latex allergy. This is a hypersensitivity reaction to pro-teins contained in latex, the name given to the sap ofthe rubber tree. It is often seen in health-care workersand others who have significant exposure to rubber


Mechanism

ALLERGENIgE

SYNTHESISMAST-CELLACTIVATION

LeukotrienesEotaxin

RANTES etc

ALLERGICRHINITIS ASTHMA

URTICARIA

FOODALLERGY

Treatment Allergenavoidance Hyposensitization

Mediatorantagonists

Latephase

inhibitors1

ATOPICECZEMA

LOCALANAPHYLAXIS

CHRONICINFLAMMATION

Recruitment of eosinophils + Th2 cells

HISTAMINELEUKOTRIENES ETC

Macrophageactivation

MAb anti-IgE

Trigger cellstabilization

MAb anti-IgE

2 3 4 5

MC/Mf

Figure 14.3 Atopic allergies: sites of local responses and possibletherapies. Events and treatments relating to local anaphylaxis in

green and to chronic inflammation in red. Mf, macrophage; MC,mast cell; IgE, immunoglobulin E.

VASODILATATIONSMOOTH MUSCLEHYPERTROPHY

DESQUAMATIONOF

EPITHELIUM

MUCUS PLUG

INFILTRATION OFEDEMATOUS MUCOSA& SUBMUCOSA WITH

EOSINOPHILS & T-CELLS

BASEMENT-MEMBRANETHICKENING

MUCUS GLANDHYPERPLASIA

Figure 14.4 Pathologic changes in asthma. Diagram of cross-section of an airway in severe asthma.

gloves or other rubber-containing materials. The acuteallergic reaction may present with skin rashes, itchingor redness of the eyes, nasal symptoms or coughing,wheezing and shortness of breath. In severe cases ana-phylactic shock may occur. Aerosols of latex may occurwhen rubber gloves containing powders are removedbecause the latex can bind to the powder and causeacute respiratory symptoms in allergic individuals. Aswith other allergies, avoidance of the allergen is cru-cial. In the case of hospital workers this may entailavoiding all contact with airway masks and straps,anesthesia bags, chest tubes, catheter bags and manyother rubber-containing products in the home. There issome evidence that latex-allergic individuals may develop symptoms from various fruits, especially avocado and banana, which contain proteins thatcross-react with latex.

Food allergy

Awareness of the importance of IgE sensitization tofood allergens in the gut has increased dramatically sothat allergy to peanuts or peanut butter is necessitatingmajor changes in school lunch programs. Many foodshave been incriminated, especially nuts, shellfish, milkand eggs, and food additives such as sulfiting agents.Sensitization to egg white and cows’ milk may evenoccur in early infancy through breast-feeding, withantigen passing into the mother’s milk. Contact of thefood with specific IgE on mast cells in the gastrointesti-nal tract may produce local reactions such as diarrheaand vomiting. It may also allow the allergen to enterthe body by changing gut permeability through medi-ator release resulting in systemic reactions includingskin eruptions (urticaria), bronchospasm and anaphy-lactic shock.

Clinical tests for allergy

Sensitivity is normally assessed by the response to in-tradermal challenge with antigen. The release of hista-mine and other mediators rapidly produces a whealand flare reaction at the site (figure 14.5a), maximalwithin 30 min and then subsiding. This immediate re-action may be followed by the late-phase reaction,which sometimes lasts for 24 h and is characterized bya dense infiltration of eosinophils and T-cells and ismore edematous than the early reaction. The similarityto the histopathology of the inflammatory infiltrate in chronic asthma is obvious, and these late-phase reactions can also be seen following challenge of thebronchi and nasal mucosa of allergic subjects.

Allergen-specific serum IgE can also be measured by

an ELISA type test, the results of which correlate wellwith skin test results.

Therapy

If one considers the sequence of reactions from initial exposure to allergen right through to the production ofatopic disease, it can be seen that several points in thechain provide legitimate targets for therapy (figure 14.3).Avoidance of contact with environmental allergens isoften impractical, but it is possible to avoid contact withincriminating animals, drugs or ingested allergens.

Modulation of the immunologic response. Attempts to desensitize patients immunologically by repeatedsubcutaneous injections of allergen can lead to worth-while improvement in certain clinical situations. Thisis thought to be due to the activation of Th1-type cellsrather than Th2, resulting in production of increasingamounts of IgG rather than IgE. The IgG antibody willcompete for antigen with IgE and will divert the aller-gen from contact with tissue-bound IgE. Other strate-gies attempt to inhibit the binding of IgE to its mast cellreceptor using either small blocking peptides or a hu-manized monoclonal anti-IgE antibody (Xolair). It isnoteworthy that this antibody cannot trigger life-threatening anaphylaxis by cross-linking mast cellbound IgE because the epitope is masked throughcombination with the receptor.

Mast cell stabilization. At the drug level, much relief hasbeen obtained with agents such as inhalant isoprenalineand sodium cromoglycate, which render mast cells re-sistant to triggering. Sodium cromoglycate blocks chlo-ride channel activity and maintains cells in a normalresting physiological state, which probably accounts forits inhibitory effects on a wide range of cellular func-tions, such as mast cell degranulation, eosinophil andneutrophil chemotaxis and mediator release, and reflexbronchoconstriction. Some or all of these effects are re-sponsible for its anti-asthmatic actions.

Mediator antagonism. Histamine H1-receptor antago-nists have for many years proved helpful in the symp-tomatic treatment of allergic disease. For asthma thelong-acting inhaled b2-agonists such as salmeterol andformoterol, which are bronchodilators, protect againstbronchoconstriction for over 12 h. Potent leukotrieneantagonists such as Pranlukast also block constrictorchallenges and show striking efficacy in some patients.Theophylline has been used in the treatment of asthma for more than 50 years and remains the singlemost prescribed drug for asthma worldwide. As aphosphodiesterase (PDE) inhibitor it increases intra-


cellular cAMP (cyclic adenosine monophosphate)thereby causing bronchodilatation, inhibition of IL-5-induced prolongation of eosinophil survival and prob-ably suppression of eosinophil migration into thebronchial mucosa. Newer approaches to therapy in-clude the use of leukotriene receptor antagonists, anti-bodies against IL-4 or the IL-4 receptor, antibodies toendothelial cell adhesion molecules and inhibition ofeosinophil activity using antibodies against IL-5.

Attacking chronic inflammation. The triggering of macrophages through allergen interaction with surface-bound IgE is clearly a major initiating factorfor late reactions, as discussed above. Resistance to this stimulus can be very effectively achieved with corticosteroids. These drugs play a major role in thetreatment of allergic disease by suppressing the tran-scription of multiple inflammatory genes, especiallythose encoding cytokine production.


(a)

(d)

(e)(f)

(b)

(c)

Figure 14.5 Hypersensitivity reactions.Type I (a) Skin prick tests with grass pollen allergen in a patient

with typical summer hay fever. Skin tests were performed 5 h (left)and 20 min (right) before the photograph was taken. The tests on theright show a typical end-point titration of a type I immediate whealand flare reaction. The late-phase skin reaction (left) can be clearlyseen at 5 h, especially where a large immediate response has pre-ceded it. Figures for allergen dilution are given. (b) An atopic eczemareaction on the back of a knee of a child allergic to rice and eggs.

Type III (c) Facial appearance in systemic lupus erythematosus(SLE). Lesions of recent onset are symmetrical, red and edematous.They are often most pronounced on the areas of the face which re-ceive most light exposure, i.e. the upper cheeks and bridge of thenose, and the prominences of the forehead. (d) Histology of acute

inflammatory reaction in polyarteritis nodosa associated with im-mune complex formation with hepatitis B surface (HBs) antigen. Avessel showing thrombus (Thr) formation and fibrinoid necrosis(FN) is surrounded by a mixed inflammatory infiltrate, largely polymorphs.

Type IV (e) Mantoux test showing cell-mediated hypersensitivityreaction to tuberculin, characterized by induration and erythema. (f)Type IV contact hypersensitivity reaction to nickel caused by theclasp of a necklace. ((a), (b) and (e) kindly provided by Professor J.Brostoff; (c) by Dr G. Levene; (d) by Professor N. Woolf; (f) repro-duced from British Society for Immunology teaching slides withpermission of the Society and Dermatology Department, LondonHospital.)

TYPE II : ANTIBODY-DEPENDENT CYTOTOXICHYPERSENSITIVITY

This form of hypersensitivity is due to an abnormal an-tibody directed against a cell or a tissue. Such an anti-body attack may activate the complement cascadecausing target cell destruction by direct membranedamage (figure 14.6). Alternatively, antibody may op-sonize cells which are then removed by phagocytes.Opsonization may occur directly through the Fc re-ceptor or by immune adherence where C3b on theantigen–antibody complex attaches to the CR1 recep-tor on the phagocytic cell. Target cells coated with lowconcentrations of IgG antibody can also be killed “non-specifically” through an extracellular nonphagocyticmechanism involving nonsensitized leukocytes,which bind to the target by their specific receptors for

the Cg2 and Cg3 domains of IgG Fc (figure 14.7). Thisso-called antibody-dependent cellular cytotoxicity(ADCC) may be exhibited by both phagocytic cells,such as polymorphs and monocytes, and by non-phagocytic natural killer (NK) cells. Functionally, thisextracellular cytotoxic mechanism would be expectedto be of significance where the target is too large for in-gestion by phagocytosis; for example, large parasitesand solid tumors.

Type II reactions between members of the samespecies (alloimmune)

Transfusion reactions

Of the many different polymorphic constituents of thehuman red cell membrane, ABO blood groups form


COMPLEMENT C1423

C3

C3

Ab

Ab

Ab MACCOMPLEMENT C56789

Adherence to FcgR and phagocytosis Adherence to C3bR and phagocytosis Lysis

Extracellularcytotoxic attackby NK-cells andmyeloid cells(ADCC)

C3b

Figure 14.6 Antibody-dependent cytotoxic hypersensitivity (typeII).Antibodies directed against cell surface antigens cause cell deathnot only by C-dependent lysis but also by Fcg and C3b adherence re-

actions leading to phagocytosis, or through nonphagocytic extracel-lular killing by NK and myeloid cells (antibody-dependent cellularcytotoxicity (ADCC)).

ba

Fc RECEPTOR

K

Fc

KILL

ANTIBODY-COATEDTARGET CELL

Figure 14.7 Killing of antibody (Ab) -coated target by antibody-dependent cellular cytotoxicity (ADCC). Fcg receptors bind the ef-fector to the target, which is killed by an extracellular mechanism.Human monocytes and interferon g (IFNg) -activated neutrophilskill Ab-coated tumor cells using their FcgRI and FcgRII receptors;

natural killer (NK) cells bind to their targets through FcgRIII recep-tors. (a) Diagram of effector and target cells. (b) Electron micrographof attack on Ab-coated chick red cell by a mouse NK cell showingclose apposition of effector and target and vacuolation in the cyto-plasm of the latter. ((b) Courtesy of Dr P. Penfold.)

the dominant system. The antigenic groups Aand B arederived from H substance by the action of glycosyl-transferases encoded by A or B genes respectively. Individuals with both genes (group AB) have the twoantigens on their red cells, while those lacking thesegenes (group O) synthesize H substance only. Anti-bodies to A or B occur when the antigen is absent fromthe red cell surface; thus a person of blood group Awillpossess anti-B and so on. These isohemagglutinins areusually IgM and probably belong to the class of “natur-al antibodies”; they would be boosted through contactwith antigens of the gut flora which are structurallysimilar to the blood group carbohydrates, so that theantibodies formed cross-react with the appropriate redcell type. If an individual is blood group A, they wouldbe tolerant to antigens closely similar to A and wouldonly form cross-reacting antibodies capable of aggluti-nating B red cells. Similarly an O individual wouldmake anti-A and anti-B (table 14.1). A transfusion ofmismatched blood would result in the transfused redcells being coated by the isohemagglutinins and destroyed by complement activation or phagocytic activity giving rise to severe transfusion reactions.

Rhesus (Rh) incompatibility

The Rh blood groups form the other major antigenicsystem, the RhD antigen being of the most conse-quence for isoimmune reactions. A mother who isRhD-negative (i.e. dd genotype) can readily be sensi-tized by red cells from a baby carrying RhD antigens(DD or Dd genotype). This sensitization occurs mostoften at the birth of the first child when a placentalbleed can release a large number of the baby’s erythro-cytes into the mother. The antibodies formed are pre-dominantly of the IgG class and are able to cross theplacenta in any subsequent pregnancy. Reaction withthe D-antigen on the fetal red cells leads to the latter’sdestruction through opsonic adherence, giving rise tohemolytic disease of the newborn (figure 14.8). For this reason RhD-negative mothers are now treatedprophylactically with low doses of avid IgG anti-D atthe time of birth of the first child. This coats the baby’sred cells in the mother’s circulation promoting theirphagocytosis and preventing sensitization.

Organ transplant rejection

Hyperacute graft rejection mediated by preformed antibodies in the graft recipient is a classic example ofan attack by antibody on target cells. Recipients maydevelop such antibodies as a result of previous bloodtransfusions or failed transplants or multiple pregnan-cies. Following attachment of the blood supply to thenew graft these antibodies bind to donor endothelialantigens resulting in thrombosis of vessels in the graftand very rapid hyperacute rejection. Fortunately this complication is not commonly seen, as patientsawaiting transplantation are routinely checked for thepresence of antibodies.


BLOOD GROUP(PHENOTYPE) GENOTYPE ANTIGEN

SERUMANTIBODY

A AA,AO A ANTI-B

B BB,BO B ANTI-A

AB AB A and B NONE

O OO HANTI-AANTI-B

Table 14.1 ABO blood groups and serum antibodies.

a bSENSITIZATION HEMOLYTIC DISEASE c

BIRTH

Bleed

RhD– MOTHER

IgGANTI-D

Bleed

IgGAnti-D

PROPHYLAXIS WITH IgG ANTI-D

RhD– MOTHER RhD– MOTHER

RhD+

2nd babyRhD+

st baby1RhD+

st baby1

Figure 14.8 Hemolytic disease of the new-born due to rhesus (Rh) incompatibility.(a) RhD-positive red cells from the first babysensitize the RhD-negative mother. (b) The mother’s immunoglobulin G (IgG)anti-D crosses the placenta and coats theerythrocytes of the second RhD-positivebaby causing type II hypersensitivity hemolytic disease. (c) Immunoglobulin Ganti-D given prophylactically at the firstbirth removes the baby’s red cells throughphagocytosis and prevents sensitization ofthe mother.

Autoimmune type II hypersensitivity reactions

A variety of organ-specific autoimmune diseases re-sult from antibodies directed against various cell or tissue antigens. Autoantibodies to the patient’s ownred cells are produced in autoimmune hemolytic anemia. Red cells coated with these antibodies have ashortened half-life, largely through their adherence tophagocytic cells in the spleen. Antibodies to plateletsresult in autoimmune thrombocytopenia, and theserum of patients with Hashimoto’s thyroiditis con-tains antibodies which in the presence of complementare directly cytotoxic to thyroid cells. In Goodpasture’ssyndrome (included here for convenience), antibodiesto the basement membranes of kidney glomeruli andlung alveoli are present. Biopsies show these anti-bodies together with complement components boundto the basement membranes, where the action of the full complement system leads to serious damage(figure 14.9a).

Anti-receptor autoimmune diseases

Autoantibodies directed against cellular receptorsmay give rise to disease by either blocking or depletingthe receptor from the cell surface or by activating thereceptor. As will be discussed more fully in Chapter 17,myasthenia gravis is a disorder due to an abnormal antibody directed against the acetylcholine receptorsresulting in profound muscular weakness. In Graves’disease (thyrotoxicosis), however, the abnormal anti-body stimulates the receptor for thyroid-stimulatinghormone (TSH) resulting in uncontrolled productionof thyroid hormones.

Type II drug reactions

Drugs may become coupled to body components andthereby undergo conversion from a hapten to a fullantigen that may sensitize certain individuals. If IgEantibodies are produced, anaphylactic reactions canresult. In some circumstances, particularly with topi-cally applied ointments, cell-mediated hypersensi-tivity may be induced. In other cases where coupling toserum proteins occurs, the possibility of type III im-mune complex-mediated reactions may arise. In thepresent context we are concerned with those instanceswhere the drug appears to form an antigenic complexwith the surface of a formed element of the blood and evokes the production of antibodies which are cytotoxic for the cell–drug complex. When the drug iswithdrawn, the sensitivity is no longer evident.

Examples of this mechanism have been seen in the hemolytic anemia sometimes associated with contin-ued administration of chlorpromazine or phenacetin,in the agranulocytosis associated with the taking ofaminopyrine or of quinidine, and the now classic situa-tion of thrombocytopenic purpura which may be pro-duced by Sedormid, a sedative of yesteryear. In thelatter case, freshly drawn serum from the patient willlyse platelets in the presence, but not in the absence, ofSedormid; inactivation of complement by preheatingthe serum at 56°C for 30 min abrogates this effect.


(a)

(b)

Figure 14.9 Glomerulonephritis: (a) due to linear deposition of antibody to glomerular basement membrane, here visualized bystaining the human kidney biopsy with a fluorescent anti-immunoglobulin G (IgG); and (b) due to deposition of antigen–antibody complexes, which can be seen as discrete masses lining theglomerular basement membrane following immunofluorescentstaining with anti-IgG. Similar patterns to these are obtained with afluorescent anti-C3. (Photographs kindly supplied by Dr S. Thiru.)

TYPE II I : IMMUNE COMPLEX-MEDIATEDHYPERSENSITIVITY

Under a number of circumstances the body may be ex-posed to an excess of antigen over a protracted period.The union of such antigens with the subsequentlyformed antibodies forms insoluble complexes at fixedsites within the body where they may well give rise toacute inflammatory reactions (figure 14.10). Whencomplement is fixed, the anaphylatoxins C3a and C5awill cause release of mast cell mediators resulting in in-creased vascular permeability. These same chemotac-tic factors and those released by mast cells will lead toan influx of polymorphonuclear leukocytes, which at-tempt to phagocytose the immune complexes. This inturn results in the extracellular release of the poly-morph granule contents, particularly when the com-plex is deposited on a basement membrane and cannotbe phagocytosed (so-called “frustrated phagocyto-sis”). The proteolytic enzymes (including neutral proteinases and collagenase), kinin-forming enzymes,polycationic proteins and reactive oxygen and nitrogen intermediates which are released from thepolymorph will damage local tissues and intensify the inflammatory responses. Under appropriate conditions, platelets may be aggregated with two consequences: they provide yet a further source of vasoactive amines and they may also form mi-crothrombi, which can lead to local ischemia. Insolublecomplexes taken up by macrophages cannot readily bedigested and provide a persistent activating stimulusleading to release of the cytokines IL-1 and TNF, reac-tive oxygen intermediates (ROIs) and nitric oxide(NO) (figure 14.10) which further damage the tissue.

The outcome of the formation of immune complexes

in vivo depends not only on the absolute amounts ofantigen and antibody, but also on their relative propor-tions which govern the nature of the complexes andhence their distribution within the body. Between anti-body excess and mild antigen excess, the complexesare rapidly precipitated and tend to be localized to thesite of introduction of antigen, whereas in moderate togross antigen excess, soluble complexes are formed.These small complexes containing C3b bind by im-mune adherence to CR1 complement receptors on the human erythrocyte and are transported to fixedmacrophages in the liver where they are safely inacti-vated. If there are defects in this system, for exampledeficiencies in classical complement pathway compo-nents or perhaps if the system is overloaded, then theimmune complexes are free in the plasma and wide-spread disease involving deposition in the kidneys,joints and skin may result.

Inflammatory lesions due to locally formed complexes

The Arthus reaction

Maurice Arthus found that injection of soluble antigenintradermally into hyper-immunized rabbits withhigh levels of antibody produced an erythematous andedematous reaction reaching a peak at 3–8 h and thenusually resolving. The lesion was characterized by anintense infiltration with polymorphonuclear leuko-cytes. The injected antigen precipitates with antibodyand binds complement. Using fluorescent reagents,antigen, Ig and complement components can all bedemonstrated in this lesion. Anaphylatoxin is soongenerated and causes mast cell degranulation,


Attract polymorphs

ANTIGEN–ANTIBODY COMPLEX

Platelet aggregation Macrophageactivation

Complement activation

Anaphylatoxin

MICRO-THROMBI

VASOACTIVEAMINE RELEASE

RELEASE PROTEOLYTICENZYMES & POLYCATIONIC

PROTEINS

RELEASE IL-1TNF, ROIs

NO.

RELEASEMAST CELLMEDIATORS

Figure 14.10 Type III immune complex-mediated hypersensitivity. IL, interleukin;NO, nitric oxide; ROIs, reactive oxygen intermediates; TNF, tumor necrosis factor.

influx of polymorphs with release of polymorph granules, and local tissue injury. Local intravascularcomplexes will also cause platelet aggregation and vasoactive amine release resulting in erythema andedema.

Reactions to inhaled antigens

Intrapulmonary Arthus-type reactions to exogenousinhaled antigen appear to be responsible for the condi-tion of hypersensitivity pneumonitis. The severe respi-ratory difficulties associated with farmer’s lung occurwithin 6–8 h of exposure to the dust from moldy hay.These patients are sensitized to thermophilic actino-mycetes which grow in the moldy hay, and extracts ofthese organisms give precipitin reactions with the sub-ject’s serum and Arthus reactions on intradermal injec-tion. Inhalation of bacterial spores present in dust fromthe hay introduces antigen into the lungs and a com-plex-mediated hypersensitivity reaction ensues. Simi-lar situations arise in pigeon-fancier’s disease, wherethe antigen is probably serum protein present in the

dust from dried feces and in rat handlers sensitized torat serum proteins excreted in the urine (figure 14.11).There are many other quaintly named cases of extrinsicallergic alveolitis resulting from continual inhalationof organic particles; for example, cheese washer’s dis-ease (Penicillium casei spores), furrier’s lung (fox furproteins) and maple bark stripper’s disease (spores ofCryptostroma). Although the initial damage in the lungis due to localized immune complexes, subsequent in-filtration of macrophages and T-cells will result in therelease of a variety of proinflammatory cytokineswhich produce further tissue damage.

Disease resulting from circulating complexes

Serum sickness

Injection of relatively large doses of foreign serum (e.g.horse antidiphtheria) used to be employed for varioustherapeutic purposes. Horse serum containing spe-cific antibodies is still used therapeutically (such as for the treatment of snake bite) but immune complex


c

DL C

O m

l CO

/min

/mm

Hg

Mar10

1973

15

20

25

28

Apr May Jun Jul Aug Sep Oct Nov Dec

WORK EXPOSURE Predicted value=32.2 ml

(a) (b)

Figure 14.11 Extrinsic allergic alveolitis due to rat serum proteinsin a research assistant handling rats (type III hypersensitivity).Typical systemic and pulmonary reactions on inhalation and posi-tive prick tests were elicited by rat serum proteins; precipitinsagainst serum proteins in rat urine were present in the patient’sserum. (a) Bilateral micronodular shadowing during acute episodes.(b) Marked clearing within 11 days after cessation of exposure torats. (c) Temporary fall in pulmonary gas exchange measured byDLco (gas transfer, single breath) following a 3-day exposure to ratsat work (arrowed). (From K.B. Carroll, J. Pepys, J.L. Longbottom,D.T.D. Hughes & H.G. Benson (1975) Clinical Allergy 5, 443; figureskindly provided by Professor J. Pepys.)

disease is nowadays more likely to be seen in patientstreated with monoclonal antibodies originating inmice.

Some individuals receiving foreign serum will syn-thesize antibodies against the foreign protein, givingrise to a condition known as “serum sickness,” whichappears about 8 days after the injection. It results from the deposition of soluble antigen–antibody com-plexes, formed in antigen excess, in small vesselsthroughout the body. The clinical manifestations in-clude a rise in temperature, swollen lymph nodes, ageneralized urticarial rash and painful swollen jointsassociated with low serum complement levels andtransient albuminuria. To be pathogenic, the com-plexes have to be of the right size—too big and they aresnapped up smartly by the macrophages of themononuclear phagocyte system; too small and theyfail to induce an inflammatory reaction. Even whenthey are the right size, it seems that they will only local-ize in vessel walls if there is a change in vascular per-meability. This may come about through release of5-hydroxytryptamine (5HT; serotonin) from plateletsreacting with larger complexes or through an IgE orcomplement-mediated degranulation of basophilsand mast cells to produce histamine, leukotrienes and

PAF. The effect on the capillaries is to cause separationof the endothelial cells and exposure of the basementmembrane to which the appropriately sized complex-es attach and attract neutrophils that give rise to thevasculitis so typical of immune complex-mediateddisease. The skin, joints, kidneys and heart are particu-larly affected. As antibody synthesis increases, antigenis cleared and the patient normally recovers.

Immune complex glomerulonephritis

Many cases of glomerulonephritis are associated withcirculating complexes. These are retained in or on theendothelial side of the glomerular basement mem-brane (figure 14.12) where they build up as “lumpy”granules staining for antigen, Ig and complement. Theinflammatory process damages the basement mem-brane, causing leakage of serum proteins and conse-quent proteinuria. Because serum albumin moleculesare small they appear in the urine even with just minordegrees of glomerular damage. Figure 14.9b depictsDNA/anti-DNA/complement deposits in the kidneyof a patient with systemic lupus erythematosus (SLE).Similar immune complex disease may follow variousbacterial infections with certain strains of so-called


1 2 3 4 5

Vasoactivemediators Adherence Platelet

microthrombus

Polymorphenzymerelease

GLOMERULARCAPILLARY LUMEN

LARGE

SMALL

Immunecomplexes

URINARY SPACE

EPITHELIAL CELL

BASEMENT MEMBRANE

ENDOTHELIAL CELL

Figure 14.12 Deposition of immune complexes in the kidneyglomerulus. (1) Complexes induce release of vasoactive mediatorsfrom basophils and platelets which cause (2) separation of endothe-lial cells, (3) attachment of larger complexes to exposed basementmembrane, while smaller complexes pass through to epithelial side;(4) complexes induce platelet aggregation; (5) chemotactically at-

tracted neutrophils release granule contents in “frustrated phagocy-tosis” to damage basement membrane and cause leakage of serumproteins. Complex deposition is favored in the glomerular capillarybecause it is a major filtration site and has a high hydrodynamic pres-sure. Deposition is greatly reduced in animals depleted of plateletsor treated with vasoactive amine antagonists.

“nephritogenic” streptococci, in chronic parasitic in-fections such as quartan malaria, and in the course ofchronic viral infections.

Deposition of immune complexes at other sites

The favored sites for immune complex deposition arethe skin, joints and kidney. The vasculitic skin rasheswhich are a major feature of serum sickness are alsocharacteristic of systemic and discoid lupus erythe-matosus (figure 14.5c), where biopsies of the lesions re-veal amorphous deposits of Ig and C3 at the basementmembrane of the dermal–epidermal junction. Thenecrotizing arteritis produced in rabbits by experi-mental serum sickness closely resembles the histologyof polyarteritis nodosa, where immune complexescontaining the hepatitis B surface (HBs) antigen of thehepatitis B virus are present in the lesions (figure14.5d). In some instances drugs such as penicillin be-come antigenic after conjugation with body proteins

and form complexes that mediate hypersensitivity re-actions. The choroid plexus, being a major filtrationsite, is also favored for immune complex deposition,and this could account for the frequency of central nervous disorders in SLE. Similarly, in subacute sclerosing panencephalitis, deposits containing Ig andmeasles antigen may be found in neural tissue.

TYPE IV: CELL-MEDIATED (DELAYED-TYPE)HYPERSENSITIVITY

This form of hypersensitivity results from an exagger-ated interaction between antigen and the normal cell-mediated immune mechanisms. T-cell responses maybe autoreactive or directed against microorganisms,transplants or against fixed antigens such as chemicalsfound on the skin in cases of contact dermatitis. Fol-lowing earlier priming, memory T-cells recognize theantigen together with class II major histocompatibilitycomplex (MHC) molecules on an antigen-presenting


CYTOTOXIC T-CELL

ACTIVATEDMACROPHAGE

VIRALLY-INFECTEDCELL

Cytotoxicity

Cytotoxicity?

SENSITIZEDT-HELPER

Chronicgranuloma

Tissuedamage

IL-4, IL-5

IFN�, GM-CSF

EPITHELIOID CELL

GIANT CELL

EOSINOPHIL

Antigenic

stimulation

PROTEASES, TNFROIs, NO.

Tissuedamage

TCR ab

TCR gd

Fusion

Figure 14.13 The cellular basis of type IVhypersensitivity. GM-CSF, granulocyte-macrophage colony-stimulating factor;IFNg, interferon g; IL, interleukin; NO·, nitric oxide; ROIs, reactive oxygen inter-mediates; TCR, T-cell receptor; TNF, tumornecrosis factor.

cell, and are stimulated into blast cell transformationand proliferation. The stimulated T-cells release anumber of proinflammatory cytokines which functionas mediators of the ensuing hypersensitivity response,particularly by attracting and activating macrophagesand cytotoxic T-cells (figure 14.13).

Perhaps the best-known example is the Mantoux re-action obtained by injection of tuberculin into the skinof an individual in whom previous infection with themycobacterium has induced a state of cell-mediatedimmunity (CMI). The reaction is characterized by ery-thema and induration (figure 14.5e), which appearsonly after several hours (hence the term “delayed”)and reaches a maximum at 24–48 h. Histologically, theearliest phase of the reaction is seen as a perivascularcuffing with mononuclear cells followed by a more ex-tensive exudation of mono and polymorphonuclearcells. The latter soon migrate out of the lesion leavingbehind a predominantly mononuclear cell infiltrateconsisting of lymphocytes and cells of the monocyte–macrophage series. This contrasts with the essentially“polymorph” character of the Arthus reaction.

Tissue damage produced by type IV reactions

Infections

The development of a state of cell-mediated hypersen-sitivity against intracellular bacteria is probably re-sponsible for the lesions associated with bacterialallergy such as the cavitation and caseation seen inhuman tuberculosis, and the granulomatous skin lesions found in patients with the borderline form ofleprosy. Chronic infection with these organisms leadsto continual release of cytokines from sensitized T-lymphocytes. The macrophage accumulation and acti-vation that follows is responsible for tissue injury dueto release of ROIs and NO. Macrophages may differen-tiate into epithelioid and giant cells, which with theproliferating lymphocytes and fibroblasts form thestructure termed a chronic granuloma. This repre-sents an attempt by the body to wall off a site of persis-tent infection (figure 14.13).

The skin rashes in smallpox and measles and the lesions of herpes simplex may be largely attributed todelayed-type allergic reactions with extensive damageto virally infected cells by cytotoxic T-cells. Cell-mediated hypersensitivity has also been demonstra-

ted in pathologic lesions of the fungal diseases candi-diasis, dermatomycosis, coccidioidomycosis and his-toplasmosis, and in the parasitic disease leishmaniasis.

Contact dermatitis and atopic eczema

Contact hypersensitivity in the skin is often producedby foreign materials capable of binding to body con-stituents, possibly surface molecules of the Langer-hans’ cell, to form new antigens. This may occur inpeople who become sensitized while working withchemicals such as picryl chloride and chromates, orwho repeatedly come into contact with the substanceurushiol from the poison ivy plant. p-Phenylene diamine in certain hair dyes, neomycin in topically applied ointments, and nickel salts formed from arti-cles such as nickel jewellery clasps (figure 14.5f), canprovoke similar reactions. Eczema is not a specific dis-ease but is skin inflammation induced by atopic aller-gens, irritants or contact allergens which produceT-cell mediated skin damage. The epidermal route ofinoculation tends to favor the development of a T-cell response through processing by class II-rich dendriticLangerhans’ cells, which migrate to the lymph nodesand present antigen to T-lymphocytes. These then mi-grate back to the skin where they release interferon g(IFNg) that upregulates Fas on keratinocytes. These inturn become susceptible to apoptosis induced by acti-vated T-cells which express FasL (Fas ligand). Thus,these delayed-type reactions are characterized by amononuclear cell infiltrate peaking at 12–15 h, accom-panied by edema of the epidermis with microvesicleformation. As would be expected, effective therapy forcontact dermatitis and eczema should be directed atsuppressing T-cell function. Topical corticosteroids arethe most potent anti-inflammatory drugs but a num-ber of trials have now shown the effectiveness of oralcyclosporin in treating these disorders.

Other examples

Delayed hypersensitivity contributes significantly tothe prolonged reactions which result from insect bites.In numerous organ-specific autoimmune diseases,such as type I diabetes, cell-mediated hypersensitivityreactions undoubtedly provide the major engine fortissue destruction. The immunopathogenesis of thesedisorders will be discussed in Chapter 17.



REVISION

Excessive stimulation of the normal effector mechanismsof the immune system can lead to tissue damage, and wespeak of hypersensitivity reactions, of which several typescan be distinguished.

Type I: Anaphylactic hypersensitivity• Anaphylaxis involves contraction of smooth muscleand dilatation of capillaries.• This depends upon the reaction of antigen with specificimmunoglobulin E (IgE) antibody bound through its Fc tothe mast cell.• Cross-linking and clustering of the IgE receptors leadsto release from the granules of mediators including histamine, leukotrienes and platelet activating factor(PAF), plus eosinophil and neutrophil chemotactic factors and the cytokines interleukin-3 (IL-3), IL-4, IL-5and granulocyte-macrophage colony-stimulating factor (GM-CSF).• Interleukin-4 is involved in the isotype switch to IgE.

Atopic allergy• Atopy stems from an excessive IgE response to extrinsicantigens (allergens) which leads to local anaphylactic reac-tions at sites of contact with allergen.• Hay fever and extrinsic asthma represent the most com-mon atopic allergic disorders resulting from exposure toinhaled allergens.• Serious prolongation of the response to allergen iscaused by T-cells of Th2-type, which recruit tissue-damaging eosinophils through release of IL-5. This Th2bias is reinforced by nitric oxide (NO·) produced by cytokine-stimulated airway epithelial cells.• Many food allergies involve type I hypersensitivity.• Strong genetic factors include the propensity to makethe IgE isotype.• The offending antigen is identified by intradermal pricktests giving immediate wheal and erythema reactions, byprovocation testing and also by an ELISAtype test.• Where possible, allergen avoidance is the best treatment.• Symptomatic treatment involves the use of long-actingb2-agonists and newly developed leukotriene antagonists.Chromones, such as sodium cromoglycate, block chloridechannel activity thereby stabilizing mast cells and inhibit-ing bronchoconstriction. Theophylline, the single mostprescribed drug for asthma, is a phosphodiesterase (PDE)inhibitor which raises intracellular calcium; this causesbronchodilatation and inhibition of IL-5 effects on

eosinophils. Chronic asthma is dominated by activatedTh2 cells and is treated with topical steroids, supple-mented where necessary by long-acting b2-agonists andtheophylline.• Courses of antigen injection may desensitize by forma-tion of blocking IgG or IgA antibodies or through T-cellregulation.

Type II: Antibody-dependent cytotoxic hypersensitivity• This involves the death of cells bearing antibody at-tached to a surface antigen.• The cells may be taken up by phagocytic cells to whichthey adhere through their coating of IgG or C3b, or theymay be lysed by the operation of the full complement system.• Cells bearing IgG may also be killed by polymorphs andmonocytes or by natural killer (NK) -cells through the extracellular mechanism of antibody-dependent cellularcytotoxicity (ADCC).• Examples are: transfusion reactions, hemolytic diseaseof the newborn through rhesus (Rh) incompatibility, antibody-mediated graft destruction, autoimmune reac-tions directed against the formed elements of the bloodand kidney glomerular basement membranes, and hyper-sensitivity resulting from the coating of erythrocytes orplatelets by a drug.

Type III: Immune complex-mediated hypersensitivity• This results from the effects of antigen–antibody com-plexes through (i) activation of complement and attractionof polymorphonuclear leukocytes which release tissue-damaging mediators on contact with the complex, and (ii) aggregation of platelets to cause microthrombi and vasoactive amine release.• Where circulating antibody levels are high, the antigenis precipitated near the site of entry into the body. The reac-tion in the skin is characterized by polymorph infiltration,edema and erythema maximal at 3–8 h (Arthus reaction).• Examples are: farmer’s lung, pigeon-fancier’s diseaseand pulmonary aspergillosis, where inhaled antigens provoke high antibody levels; reactions to an abrupt increase in antigen caused by microbial cell death duringchemotherapy for leprosy or syphilis; and an element ofthe synovial lesion in rheumatoid arthritis.• In relative antigen excess, soluble complexes are formedwhich are removed by binding to the CR1 (C3b) receptors on red cells. If this system is overloaded or if the classicalcomplement components are deficient, the complexes

FURTHER READING

Erb, K.J. (1999) Atopic disorders: Adefault pathway in the absence ofinfection? Immunology Today, 20, 317–22.

Geha, R.S. (2003) Section on allergy and hypersensitivity. CurrentOpinion in Immunology, 15, 603–46.

Haeney, M., Chapel, H., Snowden, N. & Misbah, S. (1999) Essentials of

Clinical Immunology, 4th edn. Blackwell Science, Oxford. [A verybroad account of the diseases involving the immune system. Goodillustration by case histories and the laboratory tests available:also MCQs.]

Trautmann, A., Akdis, M., Brocker, E.B. et al. (2001) New insights into the role of T cells in atopic dermatitis and allergic contact dermatitis. Trends in Immunology, 22, 530–2.


circulate in the free state and are deposited under circum-stances of increased vascular permeability at certain pre-ferred sites—kidney glomerulus, joints, skin and choroidplexus.• Examples are: serum sickness following injection oflarge quantities of foreign protein; glomerulonephritis as-sociated with systemic lupus erythematosus (SLE) or in-fections with streptococci, malaria and other parasites;neurological disturbances in SLE and subacute sclerosingpanencephalitis; polyarteritis nodosa linked to hepatitis Bvirus; and hemorrhagic shock in dengue viral infection.

Type IV: Cell-mediated or delayed-type hypersensitivity• This is based upon the interaction of antigen withprimed T-cells and represents tissue damage resultingfrom inappropriate cell-mediated immunity (CMI) reactions.• A number of soluble cytokines including interferon g(IFNg) are released which activate macrophages and ac-count for the events that occur in a typical delayed hyper-sensitivity response such as the Mantoux reaction totuberculin; i.e. the delayed appearance of an indurated

and erythematous reaction that reaches a maximum at24–48 h and is characterized histologically by infiltrationwith mononuclear phagocytes and lymphocytes.• Continuing provocation of delayed hypersensitivity by persisting antigen leads to formation of chronic granulomas.• Th2-type cells producing IL-4 and IL-5 can also producetissue damage through their ability to recruit eosinophils.• CD8 T-cells are activated by class I major histocompati-bility antigens to become directly cytotoxic to target cellsbearing the appropriate antigen.• Examples are: tissue damage occurring in bacterial (tuberculosis, leprosy), viral (smallpox, measles, herpes),fungal (candidiasis, histoplasmosis) and parasitic (leish-maniasis, schistosomiasis) infections; contact dermatitisfrom exposure to chromates and poison ivy; insect bites;and psoriasis.


164

GRAFT REJECTION IS IMMUNOLOGIC

The replacement of diseased organs by a transplant ofhealthy tissue has long been an objective in medicinebut has been frustrated to no mean degree by the unco-operative attempts by the body to reject grafts fromother individuals. Before discussing the nature andimplications of this rejection phenomenon, it would behelpful to define the terms used for transplants be-tween individuals and species:Autograft —the tissue is grafted back onto the original

donor.Isograft —graft between syngeneic individuals (i.e. of

identical genetic constitution) such as identicaltwins or mice of the same pure line strain.

Allograft—graft between allogeneic individuals (i.e.members of the same species but different geneticconstitution), e.g. human to human and one mousestrain to another.

Xenograft —graft between xenogeneic individuals (i.e.of different species), e.g. pig to human.

It is with the allograft reaction that we are most con-cerned. The most common allografting procedure is blood transfusion where the unfortunate conse-quences of mismatching are well known. Consider-able attention has been paid to the rejection of solidgrafts such as skin, and the sequence of events is worth

describing. After suturing the allogeneic skin in place,the graft becomes vascularized within a few days, butbetween the third and ninth day the circulation gradu-ally diminishes and there is increasing infiltration ofthe graft bed with lymphocytes and monocytes butvery few plasma cells. Necrosis begins to be visiblemacroscopically and within a day or so the graft issloughed completely (figure M15.1.1).

First and second set rejection

As would be expected in an immunological reaction,second contact with antigen would produce a more ex-plosive event than the first. Indeed the rejection of asecond graft from the same donor is much accelerated(Milestone 15.1). In this second set rejection the initialvascularization is poor and may not occur at all. Thereis rapid invasion by polymorphonuclear leukocytesand lymphoid cells, including plasma cells, andthrombosis and acute cell destruction can be seen by3–4 days. Second set rejection is not the fate of all subse-quent allografts but only of those derived from theoriginal donor or a related strain (figure M15.1.2).Grafts from new donors are rejected as first set reac-tions. Rejection therefore has all the hallmarks of animmunological response in that it shows both memoryand specificity.

Chapter 15

Transplantation

Graft rejection is immunologic, 164First and second set rejection, 164

Consequences of major histocompatibil i ty complex(MHC) incompatibil i ty, 165Class II MHC differences produce a mixed lymphocyte reaction

(MLR), 165The graft-vs-host (g.v.h.) reaction, 165

Mechanisms of graft rejection, 166Allograft recognition, 166Lymphocytes mediate acute rejection, 167There are different forms of graft rejection, 167

The prevention of graft rejection, 168Matching tissue types on graft donor and recipient, 168

Agents producing general immunosuppression, 168Is xenografting a practical proposition?, 171Clinical experience in graft ing, 171

Privileged sites, 171Kidney grafts, 171Thoracic organ transplantation, 172Liver and intestine transplantation, 172Hematopoietic stem cell transplantation, 172Other organs, 173

Association of HLA type with disease, 173Association with immunologic diseases, 173

Reproductive immunology, 174The fetus is a potential allograft, 174

CHAPTER 15—Transplantation 165

Milestone 15.1—The Immunologic Basis of Graft Rejection

Hospital in Boston, and Hamburger in Paris, who graftedkidneys between dizygotic twins using sublethal X-irradi-ation. The key breakthrough came when Schwartz andDamashek’s report on the immunosuppressive effects ofthe antimitotic drug 6-mercaptopurine was applied inde-pendently by Calne and Zukowski in 1960 to the prolonga-tion of renal allografts in dogs. This was followed very

The field of transplantation owes a tremendous debt to SirPeter Medawar, the outstanding scientist who kick-startedand inspired its development. Even at the turn of the cen-tury it was an accepted paradigm that grafts between un-related members of a species would be unceremoniouslyrejected after a brief initial period of acceptance (figureM15.1.1). That there was an underlying genetic basis forrejection became apparent from Padgett’s observations inKansas City in 1932 that skin allografts between familymembers tended to survive for longer than those betweenunrelated individuals, and J.B. Brown’s critical demon-stration in St Louis in 1937 that monozygotic (i.e. genetical-ly identical) twins accepted skin grafts from each other.However, it was not until Medawar’s research in the earlypart of the Second World War, motivated by the need totreat aircrew with appalling burns, that rejection was laidat immunology’s door. He showed that a second graftfrom a given donor was rejected more rapidly and morevigorously than the first, and further that an unrelatedgraft was rejected with the kinetics of a first-set reaction(figure M15.1.2). This second-set rejection is character-ized by memory and specificity and thereby bears the hall-marks of an immunologic response. This of course waslater confirmed by transferring the ability to express a second set reaction with lymphocytes.

The message was clear: to achieve successful transplan-tation of tissues and organs in the human, it would be nec-essary to overcome this immunogenetic barrier. Limitedsuccess was obtained by Murray, at the Peter Bent Brigham

Figure M15.1.1 Rejection of CBA skin graft by strain A mouse.(a) Ten days after transplantation; discolored areas caused by de-struction of epithelium and drying of the exposed dermis. (b)Thirteen days after transplantation; the scabby surface indicatestotal destruction of the graft. (Photographs courtesy of ProfessorL. Brent.)

(a) (b)

CONSEQUENCES OF MAJORHISTOCOMPATIBILITY COMPLEX (MHC) INCOMPATIBILITY

Class II MHC differences produce a mixedlymphocyte reaction (MLR)

When lymphocytes from individuals of different classII haplotype are cultured together in vitro, blast celltransformation and mitosis occurs, the T-cells of eachpopulation of lymphocytes reacting against MHCclass II complexes on the surface of the other popu-lation (see “allograft recognition” below). This constitutes the MLR. The responding cells belong pre-dominantly to a population of CD4+ T-lymphocytesand are stimulated by the class II molecules present

mostly on B-cells, macrophages and, especially, den-dritic antigen-presenting cells. For many years theMLR was employed in transplantation laboratories to determine the degree of compatibility between individuals. With the introduction of very accuratemolecular human leukocyte antigen (HLA) testing theMLR is no longer routinely performed.

The graft-vs-host (g.v.h.) reaction

When competent T-cells are transferred from an HLA-incompatible donor to an immunosuppressed recipi-ent who is incapable of rejecting them, the grafted cellssurvive and have time to recognize the host antigensand react immunologically against them. Instead ofthe normal transplantation reaction of host against

(continued)

graft, we have the reverse, the so-called graft-vs-host (g.v.h.) reaction. In the human, fever, anemia,weight loss, rash, diarrhea and splenomegaly are ob-served, with cytokines, especially tumor necrosis fac-tor (TNF), being thought to be the major mediators ofpathology. The greater the transplantation antigen dif-ference, the more severe that reaction. Graft-vs-hostmay therefore be observed in immunologically anergicsubjects receiving bone marrow grafts, for example forcombined immunodeficiency (see p. 142) or for the re-establishment of bone marrow in subjects whose ownmarrow has been destroyed by massive doses ofchemotherapy used to treat malignant disease (figure15.1).

MECHANISMS OF GRAFT REJECTION

Allograft recognition

Remember, we defined the MHC by its ability to pro-voke the most powerful rejection of grafts betweenmembers of the same species. It transpires that normalindividuals have a very high frequency of alloreac-


B2 LOST (SCAR)C DEGENERATING

A MERGING WITH HOST

a

1ST SET2ND SET

bDAY 2 DAY 6 DAY 10

NORMAL APPEARANCEOF SKIN GRAFTS

B2 DEGENERATINGA & C NORMAL

AB2

C

AB2

C

A

C

MEDIAN GRAFT SURVIVAL 6.0 ± 0.8 10.4 ± 1.1 p<0.01

Days after grafting

AUTOGRAFTS

% G

raft

surv

ival

50

100

4 8 12 16

rapidly by Murray’s successful grafting in 1962 of an unre-lated cadaveric kidney under the immunosuppressiveumbrella of azathioprine, the more effective derivative of6-mercaptopurine devised by Hutchings and Elion.

This story is studded with Nobel Prize winners. Readersinterested in the historical aspects will gain further insight

into the development of this field and the minds of the scientists who gave medicine this wonderful prize in P.I.Terasaki’s (ed.) (1991) History of Transplantation; Thirty-fiveRecollections. UCLA Tissue Typing Laboratory, Los Ange-les, CA.

Figure M15.1.2 Memory and specificity in skin allograft rejec-tion in rabbits. (a) Autografts and allografts from two unrelateddonors, B and C, are applied to the thoracic wall skin of rabbit Awhich has already rejected a first graft from B (B1). While the auto-graft Aremains intact, graft C seen for the first time undergoes first

set rejection, whereas a second graft from B (B2) is sloughed off veryrapidly. (b) Median survival times of first and second set skin allo-grafts showing faster second set rejection. (From P.B. Medawar(1944) Journal of Anatomy, 78, 176.)

T-CELLS

a b

g.v.h.g.v.h.

Chemo-therapy

SCID

Figure 15.1 Graft-vs-host (g.v.h.) reaction. When competent T-cellsare inoculated into a host incapable of reacting against them, thegrafted cells are free to react against the antigens on the host’s cellswhich they recognize as foreign. The ensuing reaction may be fatal. Two of many possible situations are illustrated. (a) A patientwith severe combined immunodeficiency (SCID) receives a graftfrom an HLA incompatible donor. (b) A leukemia patient post-chemotherapy receives a bone marrow transplant from an allogeneic donor.

tive cells (i.e. cells that react with allografts), whichpresumably accounts for the intensity of MHC-mismatched rejection. Whereas merely a fraction of 1%of the normal T-cell population is specific for a givensingle peptide, upwards of 10% of the T-cells react withalloantigens. The reason for this is that very large numbers of T-cells, each specific for a foreign peptidepresented in the groove of self MHC molecules, maycross-react with an allogeneic MHC molecule contain-ing host peptides in its groove. Such a complex mimicsself MHC with foreign peptide in its groove and, be-cause the T-cell repertoire is recognizing large num-bers of previously unseen foreign antigens, largenumbers of recipient T-cells are activated. This is theso-called “direct pathway.” In addition, T-cells mayrecognize some allogeneic peptides presented on selfMHC molecules, a process called the “indirect path-way.” T-cells recognizing peptides derived from graftproteins are present in low frequency comparable tothat observed with any foreign antigen. Nonetheless, agraft, which has been in place for an extended period,will have the time to expand this small population significantly so that later rejection will depend progressively on this pathway.

Lymphocytes mediate acute rejection

A primary role of lymphoid cells in first set rejectionwould be consistent with the histology of the early re-action showing infiltration by mononuclear cells espe-cially CD8+ T-cells, with very few polymorphs orplasma cells (figure 15.2). Activated CD8+ cytotoxiccells proliferate and form specific alloreactive cloneswhich recognize class I alloantigens on the graft. Theythen mount a cytotoxic reaction against graftparenchymal and endothelial cells by releasing per-forin, granzyme and toxic cytokines such as TNFa.CD4+ cells also play a major role in graft rejection bythe secretion of the cytokines which mediate delayedhypersensitivity reactions. These include interferon g(IFNg), which activates and recruits macrophages andupregulates MHC antigen expression on the graft, in-terleukin-2 (IL-2), which promotes T-cell proliferationand CD8+ cell activation and lymphotoxin, which is directly cytotoxic to allogeneic cells.

There are different forms of graft rejection

Hyperacute rejection is the most dramatic form ofgraft rejection, occurring within minutes of transplan-tation. It is due to the presence in the recipient of preformed antibodies directed against donor HLA orABO antigens. Such antibodies will attach to the

endothelial cells of the donor organ, fix complementand cause endothelial damage. This will initiate bloodclotting and produce aggregation of platelets whichcontributes to the vascular occlusion, seen in renaltransplantation as glomerular microthrombi. Fortu-nately, by ABO matching and by performing cross-matches in which patients are screened forpre-existing antibodies, hyperacute rejection is nolonger a major problem in most transplant centers. It isworth pointing out that, because humans have a vari-ety of natural antibodies against animal tissue, hypera-cute rejection is the major hurdle to xenogeneictransplants.

Acute early rejection, which occurs up to 10 daysafter transplantation, is characterized by a dense cellu-lar infiltration (figure 15.2) and rupture of peritubularcapillaries. This is a cell-mediated hypersensitivity reaction mainly involving an attack by CD8+ cells ongraft cells whose MHC antigen expression has beenupregulated by IFNg.

Acute late rejection, which occurs from 11 days on-wards in patients suppressed with prednisone andazathioprine, is probably caused by the binding of immunoglobulin (presumably antibody) and comple-ment to the arterioles and glomerular capillaries,where they can be visualized by immunofluorescenttechniques. These immunoglobulin deposits on thevessel walls induce platelet aggregation in theglomerular capillaries leading to acute renal shut-down (figure 15.3). The possibility of damage to antibody-coated cells through antibody-dependentcell-mediated cytotoxicity must also be considered.

Chronic or late rejection may occur months or yearsafter the initial transplant depending on the success ofimmunosuppressive therapy. The main pathologic


Figure 15.2 Acute rejection of human renal allograft showingdense cellular infiltration of interstitium by mononuclear cells. (Pho-tograph courtesy of Drs M. Thompson and A. Dorling.)

feature is vascular injury and occlusion of the vessels.This is due to proliferation of smooth muscle cells, andaccumulation of T-cells and macrophages in the intimaof the vessel wall. The cause of this graft arteriosclero-sis is predominantly activation of macrophages in thevessel wall with the release of proinflammatory cy-tokines and various smooth muscle growth factors.There is also evidence of a humoral component in thatdeposits of antibody directed against donor tissue, orantigen–antibody complexes have been detected invascular endothelial cells and may give rise to occlu-sion of the vessels. It has been noticed that chronic rejection is more prominent in patients who initiallyshowed even mild acute rejection and also in thosewho have chronic viral infections, especially with cytomegalovirus.

The complexity of the interaction of cellular and

humoral factors in graft rejection is presented in figure15.4.

THE PREVENTION OF GRAFT REJECTION

Matching tissue types on graft donor and recipient

Improvements in operative techniques and the use of drugs have greatly diminished the effects of mis-matching HLA specificities on solid graft survival.Nevertheless for the best graft survival a high degreeof matching, especially at the MHC class II loci, is indi-cated (figure 15.5). The consensus is that matching atthe DR loci is of greater benefit than the B loci, which inturn are of more relevance to graft survival than the Aloci. Bone marrow grafts, however, require a high de-gree of compatibility, which is now afforded by thegreater accuracy of the modern DNAtyping methods.

Because of the many thousands of different HLAphenotypes (figure 15.5), it is usual to work with a largepool of potential recipients so that when graft materialbecomes available the best possible match can bemade. The position will be improved when the pool ofavailable organs can be increased through the devel-opment of long-term tissue storage banks, but tech-niques are not good enough for this at present. Bonemarrow cells fortunately can be kept viable even afterfreezing and thawing. With a paired organ such as thekidney, living donors may be used and siblings pro-vide the best chance of a good match (figure 15.5).

Agents producing general immunosuppression

The use of agents that nonspecifically interfere withthe induction or expression of the immune response(figure 15.6) can control graft rejection. Because theseagents act on various parts of the cellular immune re-sponse, patients on immunosuppressive therapy tendto be susceptible to infections and more prone to thedevelopment of lymphoreticular cancers, particularlythose with a known viral etiology.

Immunosuppressive drugs

Many of the immunosuppressive drugs now em-ployed were first used in cancer chemotherapy be-cause of their toxicity to dividing cells. Aside from thecomplications of blanket immunosuppression men-tioned above, these antimitotic drugs are especiallytoxic for cells of the bone marrow and small intestineand must therefore be used with great care.

For many years the major drugs used for immuno-


PP

gbm

Figure 15.3 Acute late rejection of human renal allograft showingplatelet (P) aggregation in a glomerular capillary (gbm) induced bydeposition of antibody on the vessel wall (electron micrograph).(Photograph courtesy of Professor K. Porter.)


(b) NATURAL KILLERCELL

(c) COMPLEX ARMEDNK-CELL

NK NK

T

TARGET

Mφ

(a) SENSITIZEDT-CELL

(h) ACTIVATEDMACROPHAGE

(g) COMPLEMENT

Mφ /P

(e) Phagocytosis ofopsonized target

(± C')

(d) ANTIBODY-DEPENDENTCELLULAR

CYTOTOXICITY

(f) Plateletadherence

IFNγ

IFNγ

Mφ /P/NK

ANTIBODY ANTIGEN Fc binding sitePROCESSED ANTIGEN

Figure 15.4 Mechanisms of target cell de-struction. (a) Direct killing by cytotoxic T-cells (Tc) and indirect tissue damagethrough release of cytokines from delayed-type hypersensitivity T-cells. (b) Killing bynatural killer (NK) cells enhanced by inter-feron (IFN). (c) Specific killing by immunecomplex-armed NK cell. (d) Attack by anti-body-dependent cellular cytotoxicity. (e)Phagocytosis of target coated with anti-body. (f) Sticking of platelets to antibodybound to the surface of graft vascular en-dothelium leading to formation of mi-crothrombi. (g) Complement-mediatedcytotoxicity. (h) Activated macrophages.Mf, macrophage; P, polymorph.

HLA-LOCUS HLA TISSUE TYPE (ALLELIC FORMS)

DP1–6

DQ2,4,5,6,7,8,9

DR1,4,7,8,9,10,11,12,13,14,15,16,17,18

B7,8,13,15,18,27,35,37–42,44,46–65,73,78

A1,2,3,11,23–26,29–34,36,43,66,68,69,74

Cw1–8

DP

DQ

DR

B

A

C

PARENTS

CHILDREN’S HAPLOTYPES

a b c d

b db ca da c

Figure 15.5 Polymorphic HLA specifici-ties and their inheritance. The complex lieson chromosome 6. Since there are severalpossible alleles at each locus, the probabili-ty of a random pair of subjects from the gen-eral population having identical HLAspecificities is very low. However, there is a1 : 4 chance that two siblings will be identi-cal in this respect because each group ofspecificities on a single chromosome formsa haplotype which will be inherited en bloc,giving four possible combinations of pater-nal and maternal chromosomes. Parent andoffspring can only be identical if the motherand father have one haplotype in common.

suppression were azathioprine (Imuran), which inhibits the synthesis of nucleic acid, cyclophos-phamide, which acts on DNA preventing correct du-plication during cell division, and corticosteroids suchas prednisolone. The latter intervenes at many pointsin the immune response, affecting lymphocyte recircu-lation and the generation of cytotoxic effector cells. In addition, their outstanding anti-inflammatory potency rests on features such as inhibition of neutrophil adherence to vascular endothelium andsuppression of monocyte/macrophage functions including release of proinflammatory cytokines. Cor-ticosteroids form complexes with intracellular recep-tors, which then bind to regulatory genes and blocktranscription of TNF, IFNg, IL-1, IL-2, IL-3 and IL-6, i.e.they block expression of cytokines.

Several new agents (figure 15.6) are having a dra-matic effect in human transplantation. They includemycophenolic acid, which has a similar but more effec-tive mode of action to azathioprine. The fungalmetabolites cyclosporin and FK506 (Tacrolimus) complex with proteins termed immunophilins, and

this complex interferes with calcineurin which is cru-cial for the transcription of IL-2 in activated T-cells. Inaddition they also block the synthesis of other cy-tokines and thereby interfere with activated CD4+

helper function. Rapamycin (Sirolimus) is a macrolidelike FK506, but in contrast acts to block signals inducedby combination of cytokines with their receptors suchas IL-2R, IL-4R, IL-10R and IL-15R.

These drugs are used not only for prophylaxis andtreatment of transplant rejection, but also in a widerange of disorders due to T-cell-mediated hypersen-sitivity. Indeed, the benefits of cyclosporin in diseasessuch as idiopathic nephrotic syndrome, type 1 insulin-dependent diabetes, Behçet’s syndrome, active Crohn’sdisease, aplastic anemia, severe corticosteroid-dependent asthma and psoriasis have confirmed thepathogenic role of the cellular immune system.

Targeting lymphoid populations

Anti-CD3 monoclonal antibodies (OKT-3) are in wide-spread use as anti-T-cell reagents to successfully re-


ANTI:TCRCD3

CD4/8CD40

CD45RBLFA-1

ICAM-1

Activationby antigen

Therapeuticmonoclonals

T-cell

Drugs & othertreatments

STEROID

CTLA-4-FcgFUSION PROTEIN

U.V.

CYCLOSPORIN

FK506

STEROID

IL-2response

DNAsynthase

RAPAMYCIN AZATHIOPRINE (6MP)METHOTREXATE

MIZORBINEMYCOPHENOLIC

ACIDBREQUINAR

Mitosis

CYCLOPHOSPHAMIDEX-RAYS

IL-2R

Cytokinesynthesis IL-2

G0 G0 G1 S G2/M G1/0

ANTI-IL-2R (CD25)± TOXINS

Figure 15.6 Immunosuppressive agents used to control graft re-jection. These drugs act at many different points in the immune re-sponse. Simultaneous treatment with agents acting at sequentialstages in development of the rejection response would be expected

to synergize strongly and this is clearly seen with cyclosporin and rapamycin. ICAM-1, intercellular adhesion molecule-1; IL, inter-leukin; LFA-1, lymphocyte function-associated antigen-1; TCR, T-cell receptor.

verse acute graft rejection. Their benefits were initiallyconstrained by their immunogenicity but this problemhas been circumvented by “humanizing” the antibody.The antibody suppresses T-cell-mediated rejection bybinding to a subunit of the CD3 molecule, and interfer-ing with CD3–T-cell receptor complex-mediated acti-vation of T-cells. Unfortunately a major side effect is acytokine release syndrome, which manifests clinicallyas a variable constellation of symptoms includingfever, chills, tremor, nausea and vomiting, diarrhea,rash, and especially pulmonary edema.

The IL-2 receptor, expressed by activated but notresting T-cells, represents another potential target forblocking the immune response. A humanized versionof a murine monoclonal anti-IL-2R antibody (Da-clizumab or Basiliximab) has, in association with otherimmunosuppressive drugs, been shown to reduce thefrequency of acute kidney rejection. Attention is nowturning to the use of anti-adhesion molecule mono-clonal antibodies such as an anti-LFA (lymphocytefunction-associated antigen), as immunosuppressanttherapy for transplanted patients.

IS XENOGRAFTING A PRACTICALPROPOSITION?

Because the supply of donor human organs for trans-plantation lags seriously behind the demand, a wide-spread interest in the feasibility of using animal organshas emerged. Of even greater practical use is the possi-ble transplantation of animal cells and tissues to treatdisease. Transplants of animal pancreatic islet cellscould cure diabetes, and implants of neuronal cellswould be useful in Parkinson’s disease and other braindisorders. Pigs are more favored than primates asdonors, both on grounds of ethical acceptability andthe hazards of zoonoses, although these animals havebeen shown to harbor endogenous retroviruses thatcan infect human cells in vitro. The first hurdle to beovercome is hyperacute rejection due to the presencein all humans of xenoreactive natural antibodies.These activate complement in the absence of regula-tors of the human complement system such as CD46,CD55 and CD59 (cf. p. 137), which precipitates the hyperacute rejection phenomenon.

The next crisis is acute vascular rejection occurringwithin 6 days as antibodies are formed to the xeno-antigens and attack donor endothelial cells. This re-sults in damage to the vessel walls and intravascularthrombosis.

Even as the immunologic problems are being over-come, the question of whether animal viruses might

infect humans and cause man-made pandemics (xeno-zoonosis), still needs to be considered.

CLINICAL EXPERIENCE IN GRAFTING

Privileged sites

The vast majority of corneal grafts survive without theneed for immunosuppression. This is due to uniquequalities of the cornea that interfere with the inductionand expression of graft rejection. These include the ab-sence of donor-derived antigen-presenting cells in thecorneal graft which also has the ability to deflect thesystemic immune response from a Th1 to a Th2 path-way. It has also been shown that the corneal allograftexpresses FasL (Fas ligand) that attaches to Fas result-ing in apoptosis of attacking T-cells.

Kidney grafts

Renal transplantation is now the treatment of choicefor end-stage renal failure. With improvement in pa-tient management there is a high rate of survival, andoutcomes for transplant recipients continue to im-prove with a 5-year patient survival of around 80%when the kidney is derived from a deceased donor (fig-ure 15.7) and 90% when it is obtained from a livingdonor. Matching at the HLA-DR locus has a strong ef-fect on graft survival, but in the long term (5 years ormore) the desirability of reasonable HLA-B, and to a lesser extent HLA-A, matching also becomes apparent.

When transplantation is performed because of im-mune complex-induced glomerulonephritis, the im-munosuppressive treatment used may help to prevent


100

80

60

40

20

0

Perc

enta

ge s

urvi

val

Years

0 1 2 3 4 5 6 7 8 9 10

First grafts (877)

Patients (877)

Figure 15.7 Actuarial survival of primary cadaveric kidney graftsin 877 patients treated at the Oxford Transplant Centre with tripletherapy of cyclosporin, azathioprine and prednisolone.

a similar lesion developing in the grafted kidney. Patients with glomerular basement membrane anti-bodies (e.g. Goodpasture’s syndrome) are likely to de-stroy their renal transplants unless first treated withplasmapheresis and immunosuppressive drugs.

The number of requests for simultaneouskidney–pancreas transplants continues to rise as pa-tients with type 1 diabetes have noted the benefits ofthis type of therapy. Regrettably there are inadequatetissues for all the patients requiring them.

Thoracic organ transplantation

The overall 1-year survival figure for heart transplantshas moved up to over the 70% mark. More specificmodalities of immunosuppression continue to de-crease the impact of acute and chronic rejection andimmunosuppression related side effects. Full HLAmatching is of course not practical, but a single mis-match at the DR locus gave 90% survival at 3 yearscompared with a figure of 65% for two DR mismatches.Aside from the rejection problem, it is likely that thenumber of patients who would benefit from cardiac replacement is much greater than the number dyingwith adequately healthy hearts. More attention willhave to be given to the possibility of xenogeneic graftsand mechanical substitutes.

The number of lung transplants also continues to increase. The current indications for single lung transplantation include restrictive lung disease, emphysema, pulmonary hypertension and other nonseptic, end-stage pulmonary disease. Indicationsfor bilateral sequential lung transplantation includecystic fibrosis and patients in whom there is chronic in-fection with end-stage pulmonary failure includingpatients with bronchiectasis. There are still severalproblems, however, that need to be worked out beforelung transplantation will offer consistent and long-term relief of symptoms and increased longevity forthe vast majority of patients. The problem of chronicrejection, as manifested by obliterative bronchiolitis isa major concern. There is now some progress in the useof single lobes from living related donors for certaingroups of patients, most notably, patients with cysticfibrosis. There is also considerable interest in investi-gating the establishment of chimerism with conse-quent induction of tolerance where recipients ofcadaveric tissues are also given donor bone marrow atthe time of their transplant. These individuals seem todevelop some degree of chimerism and show a trendtoward donor specific nonreactivity and increasingsurvival.

Liver and intestine transplantation

Survival rates for orthotopic liver grafts continue toimprove and this is now the second most commontransplant after renal transplantation. An increasingnumber of these have been grafts of a portion of theliver from living donors. To improve the prognosis ofpatients with inoperable primary hepatic or bile ductmalignancies, transplantation of organ clusters withliver as the central organ may be employed. These in-clude liver and pancreas, or liver, pancreas, stomachand small bowel or even colon. As with other trans-plants combined immunosuppressive therapy attack-ing several facets of the rejection process are mosteffective. Over 700 intestine transplants have been per-formed with an overall 5-year patient survival of 50%.

Hematopoietic stem cell transplantation

Patients with certain immunodeficiency disorders andaplastic anemia are obvious candidates for treatmentwith bone marrow stem cells, as are leukemia patientstreated radically with marrow ablative chemoradio-therapy to eradicate the neoplastic cells. The source ofthese stem cells may be bone marrow, umbilical cordblood or peripheral blood following the administra-tion of granulocyte colony-stimulating factor (G-CSF)to increase the number of stem cells. The major prob-lems still restricting the use of this form of therapy are infectious complications, veno-occlusive disease ofthe liver and g.v.h. disease. To reduce the use of thehighly toxic myeloablative regimens thought to be es-sential for eradication of malignant cells in the marrow,nonmyeloablative conditioning regimens are nowbeing used. The success of this approach depends on agraft-vs-malignancy effect provided by the allogeneictransplant. This allows for safer conditioning of the pa-tient, fewer side effects with equivalent results andfewer leukemic relapses.

Graft-vs-host disease is a major problem in bonemarrow grafting

Graft-vs-host disease resulting from recognition of recipient antigens by allogeneic T-cells in the bonemarrow inoculum represents a serious, sometimesfatal complication. The incidence of g.v.h. disease is re-duced if T-cells in the grafted marrow are first purgedwith a cytotoxic cocktail of anti-T-cell monoclonals.Two forms of the disease are recognized, an acute formwhich occurs in the first 2–3 months after transplanta-tion and a chronic form which usually develops after 3


months. The acute form presents with severe dermati-tis, hepatitis and enteritis due to production of in-flammatory cytokines by activated donor T-cellsrecognising recipient HLA. Some of these patients canbe successfully treated with anti-IL-2 receptor mono-clonal antibody and anti-TNF monoclonal antibody.The chronic g.v.h. reaction has a clinical picture similarto that seen in the autoimmune disease scleroderma.There is increased collagen deposition in the skin re-sulting from stimulation of fibroblast collagen produc-tion by cytokines such as IL-1, IL-4 and TNFa.

Successful results with bone marrow transfers re-quire highly compatible donors if g.v.h. reactions are tobe avoided. Siblings offer the best chance of finding amatched donor (figure 15.5). Several methods havebeen used to deplete donor T-cells from the graft and,although this reduces the incidence of g.v.h., there is ahigher incidence of leukemia relapse secondary to thedecrease in the graft-vs-leukemia effect.

Other organs

It is to be expected that improvement in techniques ofcontrol of the rejection process will encourage trans-plantation in several other areas such as in diabetes,where the number of transplants recorded is risingrapidly with a success rate of around 40%. The 5-yearsurvival rate of 47% for lung transplants is still lessthan satisfactory and one looks forward to the success-ful transplantation of skin for lethal burns.

Reports are coming in of experimental forays intothe grafting of neural tissues. Mutant mice with de-generate cerebellar Purkinje cells which mimic thehuman condition, cerebellar ataxia, can be restored byengraftment of donor cerebellar cells at the appropri-ate sites. Clinical trials with transplantation of humanembryonic dopamine neurons to reverse the neurolog-ical deficit in Parkinson’s disease have been severelyhampered by the excessive death of the grafted cells.

ASSOCIATION OF HLA TYPE WITH DISEASE

Association with immunologic diseases

There are numerous examples of diseases that are asso-ciated with a specific HLAgenotype (table 15.1). Asig-nificant association between a disease and a given HLAspecificity does not imply that we have identified thedisease susceptibility gene, because there may be evenbetter correlation with another HLA gene in linkagedisequilibrium with the first. Linkage disequilibriumdescribes a state where closely linked genes on a chro-

mosome tend to remain associated rather than beinggenetically randomized throughout the population.

Many, but not all, of the HLA-linked diseases are au-toimmune in nature and the reasons for this are notclear. Most of these diseases are associated with specif-ic class II MHC genes suggesting that they control thetype of reaction which will result from the presentationof autoantigens or other causative antigens to T-cells.Alternatively the MHC complex with particular selfpeptides could positively or negatively select either reactive or suppressive T-cells. Another possibility isthat HLA antigens might affect the susceptibility of acell to viral attachment or infection, thereby influenc-ing the development of autoimmunity to associatedsurface components.

Insulin-dependent diabetes mellitus is associatedwith DQ8 and DQ2 but the strongest susceptibility isseen in the DQ2/8 heterozygote. The fact that twogenes are necessary to determine the strongest sus-


DR11DR17DR17DQ8DQ2/8DQ6DR17DR2DR4DR8DR17DR17

DR2,DQ6DQ6DR17DQ2DR2

a

DISEASE HLAALLELE

RELATIVERISK

Hashimoto’s diseasePrimary myxedemaGraves' diseaseInsulin-dependent diabetes

Addison's disease (adrenal)Goodpasture's syndromeRheumatoid arthritisJuvenile rheumatoid arthritisSjögren's syndromeChronic active hepatitis (autoimmune)Multiple sclerosisNarcolepsyDermatitis herpetiformisCeliac diseaseTuberculoid leprosy

3.25.73.714200.26.313.15.88.19.713.9

1238

56.43.68.1

Class II associated

c

Subacute thyroiditisPsoriasis vulgarisIdiopathic hemochromatosisMyasthenia gravis

B35Cw6A3B8

13.713.38.24.4

Other class I associations

b Class I, HLA-B27 associated

Ankylosing spondylitisReiter’s diseasePost-salmonella arthritisPost-shigella arthritisPost-yersinia arthritisPost-gonococcal arthritisUveitisAmyloidosis in rheumatoid arthritis

B27B27B27B27B27B27B27B27

87.437.029.720.717.614.014.68.2

Table 15.1 Association of HLA with disease. (Data mainly fromL.P. Ryder, E. Andersen & A. Svejgaard (1979) Tissue Antigens(Suppl.) and E. Thorsby (1995) The Immunologist, 3, 51.)

ceptibility and the universality of this finding in allpopulations studied implies that the DQ moleculesthemselves, not other molecules in linkage disequilib-rium, are primarily involved in disease susceptibility.Possession of some subtypes of DQ6 gives a dominant-ly protective effect (table 15.1).

DR4 and, to a lesser extent, DR1 are risk factors forrheumatoid arthritis in white Caucasians. Analysis of DR4 subgroups, and of other ethnic populationswhere the influence of DR4 is minimal, has identified aparticular linear sequence from residues 67–74 as thedisease susceptibility element, and the variations observed are based on sharing of this sequence withother HLA-DR specificities. This stretch of amino acidsis highly polymorphic and forms particular pockets inthe peptide-binding cleft. As in diabetes, the DR2 alleleis under-represented and DR2-positive patients haveless severe disease, implying that a DR2-linked genemight be protective in some way.

There is a very strong association of HLA-B27 withankylosing spondylitis. Approximately 95% of pa-tients are of B27 phenotype as compared with around5% in controls. The incidence of B27 is also markedlyraised in other conditions accompanied by sacroiliitis,for example Reiter’s disease, acute anterior uveitis,psoriasis and other forms of infective sacroiliitis suchas Yersinia, gonococcal and Salmonella arthritis. Onesuggestion is that a bacterial peptide may cross-reactwith a B27-derived sequence and provoke an autoreac-tive T-cell response.

Deficiencies in C4 and C2, which are MHC class IIImolecules, clearly predispose to the development ofimmune complex disease. This could be due to abnor-mal clearance of immune complexes in the absence of aclassical complement pathway.

REPRODUCTIVE IMMUNOLOGY

The fetus is a potential allograft

One of the mysteries of immunology is why a fetus thatcarries paternal MHC is not normally rejected by themother. In the human hemochorial placenta, maternalblood with immunocompetent lymphocytes does circulate in contact with the fetal trophoblast and maternal responses to fetal antigens do result in theproduction of antibodies to the father’s MHC. Theseantibodies do not produce damage to the maternal tro-phoblast because of the presence there of inhibitors ofcomplement proteins. Some of the many speculationsto explain how the fetus avoids allograft rejection aresummarized in figure 15.8.

Awell-documented factor is the lack of both conven-tional class I and class II MHC antigens on the placen-

tal villous trophoblast. This makes the trophoblast re-sistant to attack by maternal T-cells. There is also theunique expression of the nonclassical HLA-G proteinon the extravillous cytotrophoblast. This is a minimal-ly polymorphic class I MHC protein that protects thetrophoblast from killing by uterine natural killer (NK)cells, by binding to inhibitory NK cell receptors.

There is now evidence that the placenta itself mayproduce inhibitory cytokines such as IL-10, transform-ing growth factor-b (TGFb) and IL-4 which promoteTh2 responses and inhibit Th1 responses. Other factorsthat may protect the fetus are the presence of FasLat the trophoblast maternal–fetal interface, and thesuppression of T-cell activity through tryptophandegradation brought about by the catabolic enzymeindoleamine 2,3-dioxygenase present in trophoblastcells and macrophages.


UTERUS

MOTHER

Antigen sink absorbingMHC antibodies on

placental cells

Absence ofclassical

MHC class I& class IILocal nonspecific

immunosuppressione.g. TGFb, IL-10,

tryptophan breakdown

Physical barrier

Complementcontrol proteins

Resistanceto cytotoxicity

HLA-G

PLACENTA

EXTRAVILLOUSCYTOTROPHOBLAST

SYNCYTIO-TROPHOBLAST

CYTOTROPHOBLAST

EXTRAEMBRYONICMESODERM

FasL

Figure 15.8 Mechanisms postulated to account for the survival ofthe fetus as an allograft in the mother. FasL, Fas ligand; IL, inter-leukin; MHC, major histocompatibility complex; TGFb, transform-ing growth factor-b.


REVISION

Graft rejection is an immunologic reaction• It shows specificity, the second set response is brisk, it ismediated by lymphocytes, and antibodies specific for thegraft are formed.

Consequences of major histocompatibility complex (MHC)incompatibility• Class II MHC molecules provoke a mixed lymphocytereaction (MLR) of proliferation and blast transformationwhen genetically dissimilar lymphocytes interact.• Class II differences are largely responsible for the reac-tion of tolerated grafted lymphocytes against host antigen(graft-vs-host (g.v.h.) reaction).

Mechanisms of graft rejection• CD8 lymphocytes play a major role in the acute early re-jection of first set responses.• The strength of allograft rejection is due to the surpris-ingly large number of allospecific precursor cells. Thesederive mainly from the variety of T-cells which recognizeallo-MHC plus self peptides plus a small number whichdirectly recognize the allo-MHC molecule itself; later re-jection increasingly involves allogeneic peptides present-ed by self MHC.

Different forms of graft rejection exist• Preformed antibodies cause hyperacute rejection with-in minutes.• Acute graft rejection is mediated mainly by cytotoxic T-cells.• Chronic or late rejection is due to release of proinflam-matory cytokines by macrophages in the vessel wall. Thisarteriosclerosis may also be due to antibody directedagainst the donor or to immune complex deposition.

Prevention of graft rejection• Rejection can be minimized by cross-matching donorand graft for ABO and MHC tissue types.• Rejection can be blocked by agents producing generalimmunosuppression such as antimitotic drugs like aza-thioprine, or anti-inflammatory steroids. Cyclosporin and FK506 prevent interleukin-2 (IL-2) production, andrapamycin blocks signal transduction triggered by activa-tion of the IL-2 receptor.• Anumber of T-cell specific monoclonal antibodies, suchas anti-CD3 and anti-IL-2R, are useful in controlling graftrejection.

Xenografting• The major hurdle to the use of animal organs is hypera-cute rejection due to the presence of xenoreactive cross-reacting antibodies in the host.

Clinical experience in grafting• Cornea and cartilage grafts are avascular and compara-tively well tolerated.• Kidney grafting gives excellent results, although im-munosuppression must normally be continuous.• High success rates are also being achieved with heart,liver and, to a lesser extent, lung transplants.• Bone marrow grafts for immunodeficiency and aplasticanemia are accepted from matched siblings but it is diffi-cult to avoid g.v.h. disease with allogeneic marrow. Stemcells may be obtained from umbilical cord blood or fromperipheral blood, especially after administration of granu-locyte colony-stimulating factor (G-CSF).• There are two forms of g.v.h disease, an acute form withsevere skin, liver and bowel involvement and a chronicform which resembles scleroderma.

Association of HLA type with disease• HLA specificities are often associated with particulardiseases, e.g. HLA-B27 with ankylosing spondylitis, DR4with rheumatoid arthritis and DQ2 and DQ8 with type 1insulin-dependent diabetes. The reason for these diseaseassociations is not known.

The fetus as an allograft• Differences between MHC of mother and fetus may bebeneficial to the fetus but as a potential graft it must be protected against transplantation attack by the mother.• Amajor defense mechanism is the lack of classical class Iand II MHC antigens on syncytiotrophoblast and cytotro-phoblast which form the outer layers of the placenta.• The extravillous cytotrophoblast expresses a nonclassi-cal nonpolymorphic MHC class I protein, HLA-G, whichinhibits cytotoxicity by maternal natural killer (NK) cells.• The placenta produces inhibitory cytokines and otherfactors that protect against maternal T-cells.


FURTHER READING

Buckley, R.H. (2003) Transplantation immunology: Organ and bonemarrow assessment and modulation of the immune response.Journal of Allergy and Clinical Immunology, 111, S733–44.

Daar, A.S. (Chairman) (1999) Animal-to-human organ transplants —a solution or a new problem? Bulletin of the World Health Organiza-tion, 77, 54–61. [An in-depth round table discussion.]

Mellor, A.L. & Munn, D.H. (2000) Immunology at the maternal–fetalinterface: Lessons for T-cell tolerance and suppression. Annual Re-view of Immunology, 18, 367–91.

Morris, P.J. (ed.) (2000) Kidney Transplantation: Principles and Practice,5th edn. W.B. Saunders & Company, Philadelphia.

Murphy, W.J. & Blazar, B.R. (1999) New strategies for preventing

graft-versus-host disease. Current Opinion in Immunology, 11,509–15.

Niederkorn, J.Y. (1999) The immune privilege of corneal allografts.Transplantation, 67, 1503–8.

Reisner, Y. & Martelli, M.J. (1999) Stem cell escalation enables HLA-disparate haematopoietic transplants in leukaemia patients. Im-munology Today, 20, 343–7.

Tabbara, I.A. (2002) Allogeneic hematopoietic stem cell transplanta-tion: Complications and results. Archives of Internal Medicine, 162,1558–66.

Thorsby, E. (1995) HLA-associated disease susceptibility. The Immu-nologist, 3, 51–8.

Waldmann, H. (ed.) (2003) Section on transplantation. Current Opin-ion in Immunology, 15, 477–511.


177

It has long been suggested that the allograft rejectionmechanism represented a means by which the body’scells could be kept under immunologic surveillanceso that altered cells with a neoplastic potential could beidentified and summarily eliminated. For this to oper-ate, cancer cells must display some new discriminatingsurface structure which can be recognized by the im-mune system. Indeed tumor antigens can be recog-nized by raising monoclonal antibodies against themor by specific cytotoxic T-cells (Tc). Identification oftumor antigens recognized by T-cells is crucial for thefuture production of vaccines which target solid tumors.

CHANGES ON THE SURFACE OF TUMORCELLS (figure 16.1)

Virally controlled antigens

A substantial minority of tumors arise through infec-tion with oncogenic DNA viruses including Ep-stein–Barr virus (EBV) in lymphomas and humanpapilloma virus (HPV) in cervical cancers, or RNAviruses such as the human T-cell leukemia virus-1(HTLV-1). After infection, the viruses express geneshomologous with cellular oncogenes which encodefactors affecting growth, cell division and apoptosis.Failure to control these genes therefore leads poten-tially to malignant transformation. Virally derivedpeptides associated with surface major histocompati-

bility complex (MHC) on the tumor cell behave aspowerful transplantation antigens which generatespecific Tc.

Expression of normally silent genes

The dysregulated uncontrolled cell division of the can-cer cell creates a milieu in which the products of nor-mally silent genes may be expressed. Sometimes theseencode differentiation antigens normally associatedwith an earlier fetal stage. Thus, tumors may expressproteins normally expressed on fetal but not adult tissue. Such oncofetal antigens include a-fetoprotein(AFP), found on primary hepatocellular carcinomacells, and carcinoembryonic antigen (CEA) expressedby gastrointestinal and breast carcinomas. These on-cofetal antigens may also be released in various in-flammatory conditions but measurement of theirlevels in the blood may be helpful in the diagnosis ofmalignant disease and in the monitoring of progres-sion of the disease.

The majority of tumor-specific antigens are eithercompletely abnormal peptides or mutated forms ofnormal cellular proteins produced by tumor cells andcomplexed to class I MHC products. These peptides,which are not normally destined to be positioned in the surface plasma membrane, can still signal theirpresence to T-cells in the outer world by a processedpeptide/MHC mechanism. One such group of tumor-specific antigens is encoded by families of genes that

CHAPTER 16

Tumor immunology

Changes on the surface of tumor cells, 177Virally controlled antigens, 177Expression of normally silent genes, 177Mutant antigens, 178Tissue-specific differentiation antigens, 178Malignant tumors may lack class I MHC molecules, 178Changes in glycoprotein structure, 178

Immune response to tumors, 179Immune surveillance against strongly

immunogenic tumors, 179A role for acquired immune responses?, 179A role for innate immunity?, 179

Tumors develop mechanisms to evade the immune response, 180

Approaches to cancer immunotherapy, 180Antigen-independent cytokine therapy, 180Exploitation of cell-mediated immune responses, 181

Vaccination with dendrit ic cells (DCs), 182Therapy with monoclonal antibodies, 182Immunologic diagnosis of lymphoid neoplasias, 184Plasma cell dyscrasias, 184Immunodeficiency secondary to lymphoproliferative

disorders, 185

are normally silent in most normal tissues For examplethe MAGE gene (melanoma antigen gene) which codesfor proteins found on the surface of melanomas andother tumors is normally found only on human testis.Patients with these tumors often have circulating cyto-toxic T-lymphocytes (CTLs) specific for these peptides,indicating that these antigens can induce immune re-sponses and therefore could be ideal targets for im-munotherapy.

Mutant antigens

Other antigens are encoded by genes that are ex-pressed in normal cells but are mutated in tumor cells.Such mutations may change as few as one amino acid.Single point mutations in oncogenes or tumor sup-pressor genes can account for the large diversity ofantigens found on carcinogen-induced tumors. Thegene encoding the p53 cell cycle inhibitor is a hotspotfor mutation in cancer, while the oncogenic human rasgenes differ from their normal counterpart by pointmutations usually leading to single amino acid substi-tutions. Such mutations have been recorded in 40% ofhuman colorectal cancers and their preneoplastic lesions, in more than 90% of pancreatic carcinomas, inacute myelogenous leukemia and in preleukemic syn-dromes. The oncogene HER-2/neu encodes a mem-brane protein that is present on various humantumors, especially ovarian and breast carcinomas,where it could serve as a target for either monoclonalantibodies or Tc.

Tissue-specific differentiation antigens

A tumor arising from a particular tissue may expressnormal differentiation antigens specific for that tissue.For example, prostatic tumors may carry prostate-specific antigen (PSA), which is also released into theserum and can be measured as a screening test forprostate cancer. Lymphoid cells at almost any stage intheir differentiation or maturation may become malig-nant and proliferate to form a clone of cells which arevirtually “frozen” at a particular developmental stagebecause of defects in maturation. The malignant cellsbear the markers one would expect of normal lympho-cytes reaching the stage at which maturation had beenarrested. Thus, chronic lymphocytic leukemia cells resemble mature B-cells in expressing surface MHCclass II and immunoglobulin (Ig), albeit of a single idio-type in a given patient. B-cell lymphomas will expresson their surface CD19, CD20 and either k or l lightchains depending on which light chain the original cellwas carrying. Similarly, T-cell malignancies will carrythe T-cell receptor (TCR) with CD3 and other T-cell-specific cell surface antigens such as CD4, CD8, CD2,CD7 or the interleukin-2 (IL-2) receptor CD25. Usingmonoclonal antibodies directed against these specificantigens on T- or B-cells, it has been possible to classifythe lymphoid malignancies in terms of the phenotypeof the equivalent normal cell (figure 16.2).

Malignant tumors may lack class I MHC molecules

Malignant transformation of cells may be associatedwith loss or downregulation of class I MHC expressionlinked in most cases to increased metastatic potential.This presumably reflects their decreased vulnerabilityto T-cells but not natural killer (NK) cells. In breast cancer, for example, around 60% of metastatic tumorslack MHC class I.

Changes in glycoprotein structure

The chaotic internal control of metabolism within neo-plastic cells often leads to the presentation of abnormalsurface glycoproteins or glycolipids. Some of these areuseful in the diagnosis and follow-up of patients withcancer, such as CA-125 which is found in increasedamounts on the cell surface of ovarian or uterine can-cers. Similarly CA-19-9 is expressed at high levels inmost patients with gastrointestinal or breast car-cinoma. Abnormal mucin found in pancreatic andbreast tissue can have immunologic consequences andcan be recognized by CTLor by antibody.


LOSS OFORGAN-SPECIFIC

ANTIGEN

METASTATICMOLECULES

ALTEREDCARBOHYDRATE

CELL DIVISION

?

VIRAL

SILENT

MUTANTTUMOR CELL

IMMUNOLOGICALESCAPE

CHANGES

Figure 16.1 Tumor-associated surface changes. Surface carbohy-drate changes may occur during cell division. An immunoglobulinidiotype on B-cell leukemia would be a unique antigen.

IMMUNE RESPONSE TO TUMORS

Immune surveillance against stronglyimmunogenic tumors

The immune surveillance theory would predict thatthere should be more tumors in individuals whose immune systems are suppressed. This undoubtedlyseems to be the case for strongly immunogenic tumors. There is a considerable increase in skin cancerin immunosuppressed patients living in high sunshineregions and, in general, transplant patients on im-munosuppressive drugs are unduly susceptible toskin cancers, largely associated with papilloma virus,and EBV-positive lymphomas. Likewise, the lym-phomas that arise in children with T-cell deficiencylinked to Wiskott–Aldrich syndrome or ataxia telang-iectasia express EBV genes. On the other hand, there isno clear evidence that nonvirally induced, sponta-neously developing tumors, such as the common tumors in humans, have an increased incidence in im-munodeficient individuals or athymic (nude) mice.

A role for acquired immune responses?

Various immunologic effector mechanisms against tumors have been described but it is unclear which ofthese mechanisms are important as protective antitu-mor responses. Cytotoxic T-lymphocytes are thoughtto provide surveillance by recognizing and destroyingtumor cells. They employ a variety of mechanisms todestroy tumor cells including exocytosis of granulescontaining the cytotoxic effector molecules perforinand granzyme and secretion of tumor necrosis factor(TNF) which also has tumoricidal activities. Anotherand perhaps more important mechanism is based ondirect effector–target interaction between Fas on targetcells and FasL (Fas ligand) expressed on the Tc. FasLactivation of the membrane-bound Fas present on tar-get cells leads to apoptosis of the tumor cell (figure16.3). The role that CTL play in tumor immunity is un-clear, but patients with malignant disease have beenshown to have both circulating and tumor-infiltratinglymphocytes which show cytotoxicity in vitro againstthe tumor cells. The mechanisms by which tumor cellsresist this attack will be dealt with later.

A role for innate immunity?

Perhaps in speaking of immunity to tumors, one tooreadily thinks only in terms of acquired responseswhereas it is now accepted that innate mechanisms areof significance. Macrophages, which often infiltrate atumor mass, can destroy tumor cells in tissue culture

CHAPTER 16—Tumor immunology 179

EARLYT-CELLS

MATURET-CELLS

EARLYB-CELLS

MATUREB-CELLS

STEM CELLS

PLASMACELLS

CUTANEOUSLYMPHOMA

T CLLT-LYMPHOMA

MYCOSISFUNGOIDES

SEZARYSYNDROME

T ALL

T-LYMPHOBLASTICLYMPHOMA

COMMONALL PRE-B ALL

B ALLCLL

NODULAR LYMPHOMABURKITT’S LYMPHOMA

MYELOMA

Figure 16.2 Cellular phenotype of human lymphoid malignan-cies. (After M.F. Greaves & G. Janossy, personal communication.)ALL , acute lymphoblastic leukemia; CLL , chronic lymphocyticleukemia.

TUMOR CELL DESTRUCTION APOPTOSIS

TUMOR TUMOR TUMOR

FasL

Fas

TC

TNF

PERFORINGRANZYMES

Figure 16.3 Mechanisms involved in cytotoxic T-lymphocyte de-struction of tumors by cytotoxic T-cells (Tc). FasL, Fas ligand; TNF,tumor necrosis factor.

through the copious production of reactive oxygen intermediates (ROIs) and TNF. Similarly, NK cells subserve a function as the earliest cellular effectormechanism against dissemination of blood-bornemetastases. Powerful evidence implicating these cellsin protection against cancer is provided by beige micewhich congenitally lack NK cells. They die with spontaneous tumors earlier than their nondeficient littermates.

Resting NK cells are spontaneously cytolytic for cer-tain, but by no means all, tumor targets; IL-2-activatedcells (lymphokine-activated killer cells or LAK cells)display a wider lethality. As was mentioned pre-viously, recognition of class I imparts a negative inac-tivating signal to the NK cell implying conversely thatdownregulation of MHC class I, which tumors employas a strategy to escape Tc cells, would make them moresusceptible to NK attack.

Tumors develop mechanisms to evade theimmune response

Most tumors occur in individuals who are not im-munosuppressed, indicating that tumors themselveshave mechanisms for escaping the innate or acquiredimmune systems. Several such mechanisms have beensuggested (figure 16.4). Most important of these is thattumor cells have an inherent defect in antigen process-ing or presentation as they lack costimulatory mole-cules such as the B7 (CD80 and CD86) molecules. T-cellanergy occurs following antigen–MHC complexrecognition in the absence of costimulation. Tumorcells also lack other molecules important in activatingT-cells, especially MHC class II or adhesion moleculessuch as ICAM-1 (intercellular adhesion molecule-1) orLFA-3 (lymphocyte function-associated antigen-3).Furthermore, as has been previously indicated, manytumors express reduced or absent levels of class IMHC, which imparts resistance to Tc although pre-sumably increasing susceptibility to NK cells. Othertumors express functional FasL, which confers resis-tance by inducing apoptosis of autologous infiltratinglymphocytes that express Fas. Tumors themselvesmay release various immunosuppressive factors suchas transforming growth factor-b (TGFb), which is a potent immunosuppressive cytokine having effects onmany mediators of the immune response including apotent inhibitory effect on differentiation of CTL. It isalso likely that as tumors grow they tend to favor theselective outgrowth of antigen-negative variants, orthey may produce mucins that conceal or mask theirantigens so that they are not recognized by the immuneresponse.

APPROACHES TO CANCERIMMUNOTHERAPY

Although immune surveillance seems to operate onlyagainst strongly immunogenic tumors, the excitingnew information on the antigenicity of mutant andpreviously silent proteins (table 16.1), raises the possi-bility of developing effective immunotherapeutic approaches against cancer. This will almost certainlyonly succeed once the tumor load is first reduced bysurgery, irradiation or chemotherapy.

Antigen-independent cytokine therapy

Treatment with cytokines

As has been pointed out previously, the cytokine net-work is extremely complex, and administration of a cytokine designed to stimulate one branch of the


TC

TC

TC

TC

TC

CD28

TUMOR CELLS

TGFb

INHIBITION

FasL

Fas

(a)

(f)

(e)

(d)

(c)

(b)

No MHC

APOPTOSIS

MUCIN

ANTIGENVARIANT

No B7

Tumor antigen

TC

Figure 16.4 Mechanisms by which tumor cells escape destructionby the immune response. Tumor cells may (a) lack costimulatorymolecules such as B7; (b) express reduced levels of class I major his-tocompatibility complex (MHC); (c) express FasL (Fas ligand),which causes apoptosis of attacking cytotoxic T-cells (Tc); (d) pro-duce various cytokines which inhibit the immune response; (e) de-velop antigen-negative variants; (f) produce mucins which disguisetheir antigens. TGFb, transforming growth factor-b.

immune response may lead to inhibition of anotherportion of the response. In some cases administrationof systemic cytokine has produced serious side effectswith life-threatening consequences.

Interleukin-2 has been used in a number of experi-mental protocols with the idea of expanding Tc and NKcells. A number of these patients had serious side ef-fects due to the production of other cytokines, whichproduced fever, shock and a vascular leak syndrome.Another approach is to expand peripheral blood lym-phocytes with IL-2 to generate large numbers of LAKcells which can be infused back into the patient to tar-

get the tumor cells. Administration of autologous LAKcells together with high doses of IL-2 has led, in onestudy, to a considerable reduction in tumor burden inrenal cancer patients.

Tumor necrosis factor has powerful tumor-killingcapability and can cause hemorrhagic necrosis andtumor regression. Unfortunately its systemic adminis-tration is associated with very severe toxicity due to itsactivation of the cytokine cascade.

In trials using interferon a (IFNa) and IFNb, signifi-cant responses were seen in patients with various ma-lignancies, including a remarkable response rate of80–90% among patients with hairy cell leukemia andmycosis fungoides.

With regard to the mechanisms of the antitumor effects, in certain tumors IFNs may serve primarily asantiproliferative agents. In others, activation of NKcells and macrophages may be important, while aug-menting the expression of class I MHC molecules may make the tumors more susceptible to control byimmune effector mechanisms. In some circumstancesthe antiviral effect could be contributory.

For diseases like renal cell cancer and hairy cellleukemia, IFNs have induced responses in a signifi-cantly higher proportion of patients than conventionaltherapies. However, in the wider setting, most investi-gators consider that the role of IFNs will be in combina-tion therapy, for example, with active immunotherapyor with various chemotherapeutic agents.

Exploitation of cell-mediated immune responses

Immunization with viral antigens

Based on the not unreasonable belief that certain formsof cancer (e.g. lymphoma) are caused by oncogenicviruses, attempts are being made to isolate the virusand prepare a suitable vaccine from it. In fact, large-scale protection of chickens against the developmentof Marek’s disease lymphoma has been successfullyachieved by vaccination with another herpesvirus native to turkeys. In human Burkitt’s lymphoma, workis in progress to develop a vaccine to exploit the abilityof Tc cells to target the EBV-related antigens present onthe cells of all Burkitt’s tumors. Similarly in patientswith cervical cancer Tc against the causative HPV canbe successfully induced using vaccinia virus express-ing HPV genes.

Immunization with whole tumor cells

This has the advantage that we do not necessarily haveto know the identity of the antigen concerned. The dis-


Antigen Malignancy

Tumor specific

Immunoglobulin V-region

TCR V-regionMutant p21/rasMutant p53

Developmental

B-cell non-Hodgkin’s lymphoma, multiple myeloma

p210/bcr-abl fusion product

MART-1/Melan AMAGE-1, MAGE-3

GAGE familyTelomerase

T-cell non-Hodgkin’s lymphomaPancreatic, colon, lung cancerColorectal, lung, bladder, head and neck cancer

Chronic myelogenous leukemia, acute lymphoblastic leukemiaMelanomaMelanoma, colorectal, lung, gastric cancersMelanomaVarious

Human papilloma virusEpstein–Barr virus

Cervical, penile cancerBurkitt’s lymphoma, nasopharyngeal carcinoma, post-transplant lymphoproliferative disorders

Tyrosinasegp100Prostatic acid phosphataseProstate-specific antigenProstate-specific membrane antigenThyroglobulina-fetoprotein

MelanomaMelanomaProstate cancerProstate cancerProstate cancer

Thyroid cancerLiver cancer

HER-2/neuCarcinoembryonic antigenMuc-1

Breast and lung cancersColorectal, lung, breast cancerColorectal, pancreatic, ovarian, lung cancer

Viral

Tissue specific

Overexpressed

Table 16.1 Potential tumor antigens for immunotherapy. (Repro-duced with permission from L. Fong & E.G. Engleman (2000) Den-dritic cells in cancer immunotherapy. Annual Review of Immunology18, 245–73.)

advantage is that the majority of tumors are weaklyimmunogenic and do not present antigen effectively,and so cannot activate resting T-cells. Remember, thesurface MHC–peptide complex on its own is notenough and will result in T-cell anergy, which is amajor limitation to the effective development of an im-mune response. Costimulation with molecules such asB7.1 (CD80) and B7.2 (CD86) and possibly certain cy-tokines is required to push the resting T-cell into activeproliferation and differentiation. Once the T-cells are activated, accessory costimulation is no longer required due to upregulation of accessory bindingmolecules such as CD2 and LFA-1. In murine studiesvaccination with B7-transfected melanoma cells generated CD8+ cytolytic effectors which protectedagainst subsequent tumor challenge; in other words,the introduction of the costimulatory molecule B7 enhances the immunogenicity of the tumor so that theB7-transfected cells can activate resting T-cells to recognize and attack even nontransfected tumor cells(figure 16.5). Another approach is to transfect tumorcells with syngeneic MHC class II genes, to enable thetransfected cells to present endogenously encodedtumor peptides to CD4 cells. A less sophisticated butmore convenient approach involves the administra-tion of irradiated melanoma cells together with BCG(bacille bilié de Calmette–Guérin) or other adjuvant,which, by generating a plethora of inflammatory cy-tokines, increases the efficiency of presentation oftumor antigens. It would be exciting to suppose that inthe future we might transfect a tumor in situ by firinggold particles bearing appropriate gene constructssuch as B7, IFNg (to upregulate MHC class I and II),granulocyte-macrophage colony-stimulating factor(GM-CSF) and IL-2.

Therapy with subunit vaccines

The variety of potential protein targets so far identified(table 16.1) has spawned a considerable investment inclinical therapeutic trials using peptides as vaccines.Because of the pioneering work in characterizingmelanoma-specific antigens, this tumor has been thefocus of numerous studies. Encouraging results interms of clinical benefit, linked to the generation ofCTLs, have been obtained following vaccination withpeptides with or without adjuvants. The inclusion ofaccessory factors, such as IL-2 or GM-CSF, and block-ade of CTLA-4 (cytotoxic T-lymphocyte antigen-4) canbe crucial for success.

The unique idiotype on monoclonal B-cell tumors

with surface Ig offers a potentially feasible target forimmunotherapy. This form of therapy, however, re-quires the preparation of a different vaccine for eachpatient. Other tumor-specific antigens and mutantpeptide sequences are all possible candidates for im-munotherapy such as the human melanoma-specificMAGE antigenic peptides. Various forms of immu-nization are possible, including the injection of peptidealone or with adjuvant and the use of recombinant de-fective viruses carrying the sequence encoding theprotein.

VACCINATION WITH DENDRITIC CELLS (DCs)

The sheer power of the dendritic antigen-presentingcell for the initiation of T-cell responses has been thefocus of an ever-burgeoning series of immunothera-peutic strategies. These have elicited tumor-specificprotective immune responses after injection of isolatedDCs loaded with tumor lysates, tumor antigens, orpeptides derived from them. Administration of DCstransfected with tumor cell derived RNA has beenshown to induce the expansion of tumor-specific T-cells in cancer patients and considerable success hasbeen achieved in animal models and increasingly withhuman patients (figure 16.6). The copious numbers ofDCs needed for each patient’s individual therapy areobtained by expansion of CD34+ precursors in bonemarrow by culture with cytokines including GM-CSF,IL-4 and TNF. Alternatively CD14+ monocytes fromperipheral blood generate DC in the presence of GM-CSF plus IL-4. It is unclear why the administration ofsmall numbers of antigen-pulsed DCs induces specificT-cell responses and tumor regression in patients inwhom both the antigen and DCs are already plentiful.The suggestion has been made that DCs in or near malignant tissues may be ineffective, perhaps due toIL-10 secretion by the tumor.

Therapy with monoclonal antibodies

The strategies

Immunologists have for a long time been bemused by the idea of eliminating tumor cells with specific antibody or antibody linked to a killer molecule. Such immunotoxins represent a class of magic bullet in which the toxin of plant or bacterial origin is attached to an antibody or growth factor. After bind-ing to the cell surface, the toxin is internalized and


kills the cell by catalytic inhibition of protein syn-thesis. One such immunotoxin consists of anti-idiotype antibody conjugated with ricin, a toxin ofsuch devastating potency that one molecule enteringthe cell is lethal. Such immunotoxins need to have a reasonable half-life in the circulation, to be able topenetrate into tumors and not bind significantly tonontumor cells. Other practical problems include difficulties in moving the antibodies through the tortu-ous vessels found in tumors, and the development inthe tumor of mutant cells that fail to express the targetantigens. Along the same lines, internalizing a photo-

sensitizer renders the cells vulnerable to photody-namic therapy.

Radioimmunoconjugates that carry a radiationsource to the tumor site for therapy or imaging arebeing intensively developed and have two advantagesover toxins: they are nonimmunogenic and they candestroy adjacent tumor cells which have lost antigen.Radioimmunotherapy, using isotopes such as yttrium-90 (90Y), indium-111 (111In) or iodine-131 (131I) linked toantibody, delivers doses of radiation to tumor tissuewhich would be impossibly toxic with external beamsources. Results are even better when used in synergy


MHC

. . . . . . . .

RESTING T-CELL

TCR

TUMOR CELL

NO STIMULATION

RESTING T-CELL ACTIVATED T-CELL

Transfect with B7

+/or cytokine genes

CYTOKINERECEPTORCD28

B7CYTOKINE

NONTRANSFECTED TUMOR

STIMULATION ATTACK!

CD2 LFA-1

LFA-3 ICAM-1

Figure 16.5 Immunotherapy by transfec-tion with costimulatory molecules. Thetumor can only stimulate the resting T-cellwith the costimulatory help of B7 and/orcytokines such as granulocyte-macrophagecolony-stimulating factor (GM-CSF), inter-feron g (IFNg) and interleukin-2 (IL-2), IL-4and IL-7. Once activated, the T-cell with upregulated accessory molecules can now attack the original tumor lacking costimulators. ICAM-1, intercellular adhe-sion molecule-1; LFA, lymphocyte func-tion-associated antigen; MHC, majorhistocompatibility complex; TCR, T-cell receptor.

Figure 16.6 Clinical response to autologous vaccine utilizing den-dritic cells pulsed with idiotype from a B-cell lymphoma. Comput-ed tomography scan through patient’s chest (a) prevaccine and (b) 10months after completion of three vaccine treatments. The arrow in(a) points to a paracardiac mass. All sites of disease had resolved and

the patient remained in remission 24 months after beginning treat-ment. (Photography kindly supplied by Professor R. Levy from thearticle by F.J. Hsu, C. Benike, F. Fagnoni et al. (1996) Nature Medicine 2,52; reproduced by kind permission of Nature America Inc.)

(a) (b)

with chemotherapy. A potent antilymphoma drug (Zevalin or Bexaar) employs a monoclonal antibody tothe B-cell surface antigen CD20, to which is attachedradioactive 90Yor 131I. These radiolabeled monoclonalantibodies destroy B-cell lymphomas by antibody-dependent cellular cytotoxicity (ADCC) and by signaling-induced apoptosis, acting in synergy withDNA damage due to gamma-radiation. Therapy withthis agent has been spectacularly successful. Likewise,radionuclide conjugates of a humanized anti-CD33(Mylotarg) are very useful in patients with myeloidleukemia and can destroy even large tumor burdens.

Another strategy is to target growth factor receptorspresent on the surface of tumor cells. Blocking of suchreceptors should deprive the cells of crucial growthsignals. Herceptin is a monoclonal antibody that rec-ognizes and binds to the HER-2/neu protein found on30% of breast cancer cells. Because the protein is notpresent on normal cells, the antibody selectively at-tacks the tumor causing its shrinkage in a substantialproportion of cases. In a similar manner, a humanizedanti-CD52 (Campath-1H or alemtuzumab) has shownbenefit in a range of hematological tumors, and anti-bodies to the epidermal growth factor receptor (EGFR)enhanced the effects of conventional chemotherapyand radiation therapy. Another new approach is to tar-get the tumor blood supply. Tumors generally cannotgrow beyond the size of 1 mm in diameter without thesupport of blood vessels and, because the tumor is newtissue, its blood vessels will also have to be new. Thesevessels form through the process known as angiogene-sis, the sprouting of new blood vessels from existingones, but they are biochemically and structurally dif-ferent from normal resting blood vessels and so pro-vide differential targets for therapeutic monoclonalantibodies.

Genetic technology has now allowed the develop-ment of chimeric antibodies that have the variableantigen-binding region of a mouse monoclonal anti-body but with a human constant region. Taking thisone stage further, the mouse variable region frame-work regions can be replaced with human frame-works, creating the “humanized” antibodiesmentioned above (cf. figure 3.11). Both chimeric andhumanized antibodies retain the specificity of themonoclonal but are much less immunogenic. Otheroptions for immunologic attack using monoclonal an-tibodies are possible. For example, a mixture of twobispecific heteroconjugates of antitumor/anti-CD3and antitumor/anti-CD28 should act synergisticallyto induce contact between a T-cell and the tumor to ac-tivate direct cytotoxicity (figure 16.7).

Another use of monoclonal antibodies is to purgebone marrow grafts of unwanted cells in vitro in thepresence of complement from a foreign species. Thus,differentiation antigens present on leukemic cells butabsent from bone marrow stem cells can be used to prepare tumor-free autologous stem cells to restorefunction in patients treated with chemotherapy or X-irradiation (figure 16.8).

Immunologic diagnosis of lymphoid neoplasias

With the availability of a range of monoclonal anti-bodies and improvements in immuno-enzymic andflow cytometric technology, great strides have beenmade in exploiting, for diagnostic purposes, the factthat malignant lymphoid cells, especially leukemiasand lymphomas, display the markers of the normallymphocytes which are their counterparts. Thus in thediagnosis of non-Hodgkin’s lymphomas, the majorityof which are of B-cell origin, the feature that is diagnos-tic is the synthesis of monotypic Ig, i.e. of one lightchain only (figure 16.9a). In contrast, the population ofcells at a site of reactive B-cell hyperplasia will stain forboth k and l chains (figure 16.9b).

Plasma cell dyscrasias

Multiple myeloma

This is defined as a malignant proliferation of a clone ofplasma cells secreting a monoclonal Ig. The myelomaor “M” component in serum is recognized as a tightband on gel electrophoresis (figure 16.10) as all mole-cules in the clone are, of course, identical and have thesame mobility. Since Ig-secreting cells produce an ex-


SS

NK TC

F(ab')2HETEROCONJUGATE

SS

TUMORFcg -RECEPTOR

CD3/28

F(ab')2HETEROCONJUGATE

Figure 16.7 Focusing effector cells by heteroconjugates. CouplingF(ab¢)2 fragments of monoclonal antibodies specific for the tumorand an appropriate molecule on the surface of natural killer (NK) orcytotoxic T-cells (Tc), provides the specificity to bring the effectorsinto intimate contact with the target cell.

cess of light chains, free light chains are present in theplasma of multiple myeloma patients, and can be rec-ognized in the urine as Bence-Jones protein.

Waldenström’s macroglobulinemia

This disorder is produced by the unregulated prolifer-ation of cells of an intermediate appearance calledlymphoplasmacytoid cells which secrete a monoclon-al IgM, the Waldenström macroglobulin. Since the IgMis secreted in large amounts and is confined to the in-travascular compartment, there is a marked rise inserum viscosity, the consequences of which can be tem-porarily mitigated by vigorous plasmapheresis. Thedisease runs a fairly benign course and the prognosis isquite good, although the appearance of lymphoplas-macytoid tumor cells in the blood is an ominous sign.

Heavy chain disease

Heavy chain disease is a rare condition in which quan-tities of abnormal heavy chains are excreted in theurine . The amino acid sequences of the N-terminal re-gions of these heavy chains are normal, but they have adeletion extending from part of the variable domainthrough most of the CH1 region so that they lack the


PATIENT2

Radicalchemotherapyor X-irradiation

1 Bonemarrow

STEMCELL

TUMORCELL

DIFFERENTIATEDCELL

STEMCELL

3Antibody andcomplement

4Restore

Figure 16.8 Treatment of leukemias by autologous bone marrowrescue. By using cytotoxic antibodies to a differentiation antigenpresent on leukemic cells and even on other normal differentiatedcells, but absent from stem cells, it is possible to obtain a tumor-freepopulation of the latter which can be used to restore hematopoieticfunction in patients subsequently treated radically to destroy theleukemic cells.

1 1

16 82

(a)

(b)

18 1

2556

Anti Kappa

Anti

Lam

bda

Anti Kappa

Anti

Lam

bda

Figure 16.9 Use of flow cytometry to diagnose malignant lym-phoma. Cells are dispersed and stained with fluorescein-labeled antibodies against k (kappa) and l (lambda) light chains. Lym-phoma cells are monotypic and in this case stain with anti-k (a) whilenormal lymphocytes are polytypic and stain with both anti-k andanti-l (b).

cysteine required to form the disulphide bond to thelight chains.

Immunodeficiency secondary tolymphoproliferative disorders

Immunodeficiency is a common feature in patientswith lymphoid malignancies. The reasons for this arestill obscure, but it appears that the malignant cells interfere with the development of the correspondingnormal cells. Thus, in multiple myeloma the levels ofnormal B-cells and of non-myeloma Ig may be grosslydepressed and the patients susceptible to infectionwith pyogenic bacteria.


REVISION

Changes on the surface of tumor cells• Processed peptides derived from oncogenic viruses arepowerful major histocompatibility complex (MHC) —associated transplantation antigens.• Some tumors express genes that are silent in normal tis-sues; sometimes they have been expressed previously inembryonic life (oncofetal antigens).• Many tumors express weak antigens associated withpoint mutations in oncogenes such as ras and HER-2/neu.• Many tumors express normal differentiation antigensspecific for that tissue.• Tumors may lack class I MHC molecules.• Dysregulation of tumor cells frequently causes struc-tural abnormalities in surface glycoprotein or glycolipidstructures.

Immune response to tumors• T-cells generally mount effective surveillance againsttumors associated with oncogenic viruses or ultravioletinduction which are strongly immunogenic.• More weakly immunogenic tumors are not controlledby T-cell surveillance, although sometimes low-grade re-sponses are evoked.• Cytotoxic T-cells (Tc) may provide surveillance andcause tumor cell destruction or apoptosis.• Natural killer (NK) cells probably play a role in contain-ing tumor growth and metastases. They can attack MHCclass I negative tumor cells because the class I moleculenormally imparts a negative inactivation signal to NKcells.

Tumor cells have a variety of mechanisms to evade theimmune response• Tumor cells lack costimulatory molecules for antigenpresentation and this may result in T-cell anergy.• Tumors express reduced levels of MHC class I or class II.• Some tumors express FasL (Fas ligand) which causesapotosis of attacking lymphocytes with Fas on their surface.• Tumors may release inhibitory cytokines such as trans-forming growth factor-b (TGFb).• Tumors may produce mucins that mask their tumorspecific antigens.

Approaches to cancer immunotherapy• Systemic cytokine therapy may be used to stimulatespecific effector cells but is associated with severe side effects.• Interleukin-2 (IL-2)-stimulated NK cells (LAK) are active against renal carcinoma. Interferon g (IFNg) andIFNb are very effective in the T-cell disorders, hairy cellleukemia and mycosis fungoides.• Cancer vaccines based on oncogenic viral proteins canbe expected.• Immunization with tumor-specific peptides may beuseful, but a different vaccine may be required for each patient. Effective melanoma-specific antigens have beenidentified. Immunogenic potency of a tumor antigen isgreatly enhanced by dendritic cells (DCs) pulsed with theantigen.• Weakly immunogenic tumors provoke effective anti-

Figure 16.10 Myeloma paraprotein demonstrated by gel elec-trophoresis of serum. Lane 1, normal; lane 2, g-paraprotein; lane 3,near b-paraprotein; lane 4, fibrinogen band in the g-region of a plasmasample; lane 5, normal serum; lane 6, immunoglobulin (Ig) deficien-cy (low g); lane 7, nephrotic syndrome (raised a2-macroglobulin, low

albumin and Igs); lane 8, hemolysed sample (raisedhemoglobin/haptoglobin in a2 region); lane 9, polyclonal increase inIgs (e.g. infection, autoimmune disease); lane 10, normal serum. (Gelkindly provided by Mr A. Heys.)

FURTHER READING

Begent, R.H.J., Verhaar, M.J., Chester, K.A. et al. (1996) Clinical evi-dence of efficient tumor targeting based on single-chain Fv anti-body selected from a combinatorial library. Nature Medicine, 2,979–84.

Chattopadhyay, U. (1999) Tumour immunotherapy: Developmentsand strategies. Immunology Today, 20, 480–2.

Dunn, G.P., Old, L.J. & Schreiber, R.D. (2004) The three Es of cancer.Annual Review of Immunology, 22, 329–60.

Finn, O.J. (2003) Cancer vaccines: Between the idea and the reality.Nature Reviews. Immunology, 3, 630–41.

Finn, O.J. (ed.) (2004) Section on tumor immunology. Current Opin-ion in Immunology, 16, 127–62.



cancer responses if transfected with costimulatory mole-cules such as B7 or with MHC class II genes.• Vaccination with DCs loaded with tumor antigens or tumor-derived RNA yields tumor-specific immune responses.• Monoclonal antibodies conjugated to toxins or radionu-clides can target tumor cells or antigens associated withmalignancy.• Monoclonal antibodies targeting growth factor recep-tors on tumor cells or the blood supply to the tumor areuseful in treatment.• Bifunctional antibodies can bring effectors such as NKand Tc close to the tumor target.• Monoclonal antibodies attached to an isotope may beused to image tumors.

Lymphoid malignancies• The surface phenotype aids in the diagnosis and classi-fication of lymphomas and leukemias.

• Multiple myeloma represents a malignant proliferationof a single clone of plasma cells producing a single “M”band on electrophoresis.• Bence-Jones protein consists of free light chains foundin the urine of patients with multiple myeloma.• Waldenström’s macroglobulinemia is associated withlarge amounts of serum IgM causing hyperviscosity.• Malignant lymphoid cells produce secondary immun-odeficiency by suppressing differentiation of the corre-sponding normal lineage.

188

THE SCOPE OF AUTOIMMUNE DISEASES

The monumental repertoire of the adaptive immunesystem has evolved to allow it to recognize and en-snare microbial molecules of virtually any shape. In so doing it has been unable to avoid the generation of lymphocytes which react with the body’s own constituents. The term “autoimmune disease” applies to conditions where the autoimmune process con-tributes to the pathogenesis of the disease. This is different to situations where apparently harmless autoantibodies are formed following tissue damage,for example heart antibodies appearing after a my-ocardial infarction. Autoimmune diseases count

amongst the major medical problems of today’s soci-eties. There are, for example, over 6.5 million cases of rheumatoid arthritis (RA) in the USA, and type 1 diabetes is the leading cause of end-stage renal disease.

The spectrum of autoimmune diseases

These disorders may be looked upon as forming aspectrum. At one end we have “organ-specific dis-eases” with organ-specific autoantibodies. Hashi-moto’s disease of the thyroid is an example: there is aspecific lesion in the thyroid involving infiltration by mononuclear cells (lymphocytes, macrophages and

Chapter 17

Autoimmune diseases

The scope of autoimmune diseases, 188The spectrum of autoimmune diseases, 188Overlap of autoimmune disorders, 189

Nature and nurture, 190Autoimmune disorders are multifactorial, 190Genetic factors in autoimmune disease, 190Hormonal influences in autoimmunity, 192Does the environment contribute?, 192

Autoreactivity comes naturally, 193Is autoantigen available to the lymphocytes?, 194

Control of the T-helper cell is pivotal, 194Autoimmunity can arise through bypass of

T-helpers, 194Provision of new carrier determinant, 194Polyclonal activation, 194

Bypass of regulatory mechanisms results inautoimmune disease, 195Failure of Fas–FasL (Fas ligand) interaction, 195Polymorphism of CTLA-4, 195Upregulation of T-cell interaction molecules, 196Th1–Th2 imbalance with resulting overproduction of cytokines

may induce autoimmunity, 196Autoimmunity results from inadequate regulatory

cell activity, 196Tissue may be damaged both by autoantibodies and

by autoreactive T-cells, 196Pathogenic effects of humoral autoantibody, 197

Blood, 197

Thyroid, 197Muscle, 197Glomerular basement membrane (g.b.m.), 198Heart, 198Stomach, 199Other tissues, 199

Pathogenic effects of complexes with autoantigens, 199Systemic lupus erythematosus (SLE), 199

T-cell-mediated hypersensitivity as a pathogenicfactor in autoimmune disease, 200Insulin-dependent diabetes mellitus (IDDM), 200Multiple sclerosis (MS), 200Rheumatoid arthritis (RA), 200Crohn’s disease, 202Celiac disease, 202Chronic autoimmune (Hashimoto’s) thyroiditis, 202Atherosclerosis, 203Psoriasis, 203

Diagnostic value of autoantibody tests, 204Treatment of autoimmune disorders, 204

Control at the target organ level, 204Anti-inflammatory drugs, 205Immunosuppressive drugs, 205Plasmapheresis, 205T-cell control strategies, 206Other biological agents that inhibit the immune response, 207

plasma cells), destruction of follicular cells and germi-nal center formation accompanied by the productionof circulating antibodies with absolute specificity forcertain thyroid constituents (Milestone 17.1).

Moving towards the center of the spectrum are thosedisorders where the lesion tends to be localized to asingle organ but the antibodies are nonorgan-specific.A typical example would be primary biliary cirrhosiswhere the small bile ductule is the main target of inflammatory cell infiltration but the serum anti-bodies present — mainly mitochondrial — are not liver-specific.

At the other end of the spectrum are the “nonorgan-specific” or “systemic autoimmune diseases” broadly belonging to the class of rheumatologic disor-ders, exemplified by systemic lupus erythematosus(SLE), where neither lesions nor autoantibodies are

confined to any one organ. Pathologic changes arewidespread and are primarily lesions of connective tis-sue with fibrinoid necrosis. They are seen in the skin(the “lupus” butterfly rash on the face is characteristic),kidney glomeruli, joints, serous membranes and bloodvessels. In addition, the formed elements of the bloodare often affected. A bizarre collection of autoantibod-ies is found some of which react with the DNA andother nuclear constituents found in all cells of the body.

An attempt to fit the major diseases considered to beassociated with autoimmunity into this spectrum isshown in table 17.1.

Overlap of autoimmune disorders

There is a tendency for more than one autoimmunedisorder to occur in the same individual; for example,

CHAPTER 17—Autoimmune diseases 189

Milestone 17.1—The Discovery of Thyroid Autoimmunity

and the similarity of the histology (figure M17.1.1c) to thatof Rose and Witebsky’s rabbits, Roitt, Doniach and Camp-bell tested the hypothesis that the plasma cells in the glandmight be making an autoantibody to a thyroid component,so causing the tissue damage and chronic inflammatoryresponse. Sure enough, the sera of the first patients testedhad precipitating antibodies to an autoantigen in normalthyroid extracts, which was soon identified as thyroglobu-lin (figure M17.1.2).

Adams and Purves, in seeking a circulating factor that

In an attempt to confirm Paul Ehrlich’s concept of “horrorautotoxicus” —the body’s dread of making antibodies toself , Rose and Witebsky immunized rabbits with rabbitthyroid extract in complete Freund’s adjuvant. This proce-dure, which could also be carried out in other species suchas the rat, resulted in the production of thyroid autoanti-bodies and chronic inflammatory destruction of the thy-roid gland architecture (figure M17.1.1a and b).

Having noted the fall in serum gammaglobulin whichfollowed removal of the goiter in Hashimoto’s thyroiditis

(a) (b) (c)

Figure M17.1.1 Experimental autoimmune thyroiditis. (a) Con-trol rat thyroid showing normal follicular architecture. (b) Thy-roiditis produced by immunization with rat thyroid extract incomplete Freund’s adjuvant; the invading chronic inflammatorycells have destroyed the follicular structure. (Based on the experi-ments of N.R. Rose & E. Witebsky (1956) Journal of Immunology, 76,

417.) (c) Similarity of lesions in spontaneous human autoimmunedisease to those induced in the experimental model. Other fea-tures of Hashimoto’s disease such as the eosinophilic metaplasiaof acinar cells (Askenazy cells) and local lymphoid follicles are notseen in this experiment model, although the latter occur in thespontaneous thyroiditis of Obese strain chickens.

(continued)

patients with autoimmune thyroiditis (Hashimoto’sdisease or primary myxedema) have a much higher in-cidence of pernicious anemia than would be expectedin a random population matched for age and sex(10.0% as against 0.2%). Conversely, both thyroiditisand thyrotoxicosis are diagnosed in pernicious anemiapatients with an unexpectedly high frequency.

There is an even greater overlap in serological find-ings (table 17.2). Thirty percent of patients with au-toimmune thyroid disease have concomitant parietalcell antibodies in their serum. Conversely, thyroid an-tibodies have been demonstrated in up to 50% of perni-cious anemia patients. It should be stressed that theseare not cross-reacting antibodies. The thyroid-specificantibodies will not react with stomach and vice versa.When a serum reacts with both organs it means thattwo populations of antibodies are present, one withspecificity for thyroid and the other for stomach.

NATURE AND NURTURE

Autoimmune disorders are multifactorial

The breakdown of mechanisms required for maintain-ing self tolerance has a multifactorial etiology. Super-imposed upon a genetically complex susceptibility, wehave the influence of sex hormones and various envi-ronmental factors. Particularly important are micro-bial agents, which could promote the movement ofautoreactive lymphocytes into infected tissues andthere contribute to their activation.

Genetic factors in autoimmune disease

Autoimmune phenomena tend to aggregate in certainfamilies. For example, the first-degree relatives (siblings, parents and children) of patients with


Bloo

d 13

1 I: %

pre

inje

ctio

n va

lue

0

Hours after injection

5 10 15

0.5 mlSERUM FROMTHYROTOXIC

PATIENT

1.0 mU TSH

100

200

300

might be responsible for the hyperthyroidism of Graves’thyrotoxicosis, injected patient’s serum into guinea-pigswhose thyroids had been prelabeled with iodine-131 (131I),and followed the release of radiolabeled material from thegland with time. Whereas the natural thyroid-stimulatinghormone (TSH) produced a peak in serum radioactivitysome 4 h after injection of the test animal, serum from

thyrotoxic patients had a prolonged stimulatory effect(figure M17.1.3). The so-called long-acting thyroid stimula-tor (LATS) was ultimately shown to be an immunoglobu-lin G (IgG) mimicking TSH thorough its reaction with theTSH receptor but differing in its time-course of action,largely due to its longer half-life in the circulation.

Figure M17.1.2 Thyroid autoantibodies in the serum of a pa-tient with Hashimoto’s disease demonstrated by precipitationin agar. Test serum is incorporated in agar in the bottom of thetube; the middle layer contains agar only while the autoantigen ispresent in the top layer. As serum antibody and thyroid autoanti-gen diffuse towards each other, they form a zone of opaque pre-cipitate in the middle layer. Saline and kidney extract controls arenegative (Based on I.M. Roitt, D. Doniach, P.N. Campbell & R.V.Hudson (1956) Lancet, ii, 820.)

Figure M17.1.3 The long-acting thyroid stimulator in Graves’disease. Injection of thyroid-stimulating hormone (TSH) causes arapid release of iodine-131 (131I) from the pre-labeled animal thy-roid in contrast to the prolonged release which follows injection ofserum from a thyrotoxic patient. (Based on D.D. Adams & H.D.Purves (1956) Proceedings of the University of Otago Medical School,34, 11.)

Hashimoto’s disease show a high incidence of thyroid autoantibodies (figure 17.1) and of overt and subclini-cal thyroiditis. Parallel studies have disclosed similarrelationships in the families of pernicious anemia pa-tients, in that gastric parietal cell antibodies are preva-lent in relatives. There is now powerful evidence thatmultiple genetic components must be involved. Thedata on twins are unequivocal. When thyrotoxicosis ortype I diabetes (insulin-dependent diabetes mellitus,IDDM) occurs in twins there is a far greater concor-

dance rate (ie. both twins affected) in identical than innonidentical twins. Furthermore, lines of animals havebeen bred which spontaneously develop autoimmunedisease. In other words, the autoimmunity is geneti-cally programmed.

Most autoimmune diseases are multigenic with pa-tients inheriting multiple genetic polymorphismswhich may vary among different ethnic populations.Dominant amongst the genetic associations with au-toimmune diseases is linkage to the major histocom-


Hashimoto’s thyroiditisPrimary myxedemaGraves’ diseasePernicious anemiaAutoimmune atrophic gastritisAddison’s diseasePremature menopause (few cases)Male infertility (few cases)Myasthenia gravisInsulin-dependent diabetes mellitusGoodpasture’s syndromePemphigus vulgarisPemphigoidSympathetic ophthalmiaPhacogenic uveitisMultiple sclerosisAutoimmune hemolytic anemiaIdiopathic thrombocytopenic purpuraIdiopathic leukopeniaPrimary biliary cirrhosisChronic active hepatitis HBs�veUlcerative colitisSjögren’s syndromeRheumatoid arthritisSclerodermaWegener’s granulomatosisPoly/dermatomyositisDiscoid lupus erythematosusSystemic lupus erythematosus (SLE)

ORGAN SPECIFIC

NONORGANSPECIFIC

Table 17.1 Spectrum of autoimmune diseases.

% POSITIVE REACTIONS FOR ANTIBODIES TO:

THYROID* STOMACH* NUCLEI* IgG†

99.9554511 2

0–15

32891416 2

0–16

811565099

0–19

2

7575352–5

DISEASE

Hashimoto’s thyroiditisPernicious anemiaSjögren’s syndromeRheumatoid arthritisSLEControls‡

50

40

30

20

10

0

HASHIMOTORELATIVES

PERNICIOUSANEMIA

RELATIVES

MATCHEDNORMALS

%Po

sitiv

e

THYROIDANTIBODY

GASTRICANTIBODY

Figure 17.1 The high incidence of thyroid and gastric autoanti-bodies in the first-degree relatives of patients with Hashimoto’sdisease or pernicious anemia. Note the overlap of gastric and thy-roid autoimmunity and the higher incidence of gastric autoantibod-ies in pernicious anemia relatives. In general, titers were muchhigher in patients than in controls. (Data from D. Doniach & I.M.Roitt (1964) Seminars in Haematology, 1, 313.)

Table 17.2 Organ-specific and nonorgan-specific serological interrelationships inhuman disease.

*Immunofluorescence test†Rheumatoid factor classical tests‡Incidence increases with age and females > malesIgG, immunoglobulin G; SLE, systemic lupus erythematosus.

patibility complex (MHC). Numerous examples havebeen described, including the association between B27 and ankylosing spondylitis, the increased risk ofIDDM for DQ8 individuals, the higher incidence ofDR3 in Addison’s disease and of DR4 in RA (see table15.1). Some HLAmolecules are associated with protec-tion against autoimmune disease. In IDDM some mol-ecules are associated with susceptibility, others withprotection, whereas others appear neutral. Figure 17.2shows a multiplex family with IDDM in which the dis-ease is closely linked to a particular HLA-haplotype.

It is not clear how particular structural variants ofMHC molecules participate in autoimmunization. Infact the association may not be with the identified allele but with closely related polymorphic genes suchas that encoding tumor necrosis factor (TNF) or other cytokines. For example, the gene for familial hemochromatosis, a disorder that causes severe ironoverload, is tightly linked to HLA-A3 and is said to bein linkage disequilibrium with it. Similarly the associa-tion of IDDM with DR3 and DR4 is almost certainly re-lated to the linkage disequilibrium between these DRalleles and the susceptibility genes at the DQ locus. It ispossible that the MHC glycoprotein may influence theoutcome of positive and negative selection within thethymus and allow immature T-cells which are reactivewith particular self antigens, to escape negative selec-tion. It is most likely, however, that a particular allelicform of the MHC molecule may have an exceptionalcapacity to present disease-inciting peptides to cyto-toxic or helper T-cells.

Several other gene families, which encode mole-cules important in the immune response, may showpolymorphisms that increase susceptibility to autoim-mune disease. Of particular interest are allelic variantsof CTLA-4 (cytotoxic T-lymphocyte antigen-4), whichis normally an important inhibitor of T-cell activation.Polymorphisms have been shown in Graves’ disease,RA, Addison’s disease, multiple sclerosis (MS), SLEand many other autoimmune diseases. There is nowinterest in the association between SLE and polymor-phism of the Fcg receptor and the CD19 receptor normally present on human B-cells. There are also indications that genetic polymorphisms of cytokines,chemokines and their respective receptors could be linked to autoimmune disease; for example,chemokine or chemokine receptor gene polymor-phisms might be related to susceptibility or the sev-erity of MS. These polymorphisms may control thepattern of cytokine secretion or influence the balanceof Th1/Th2 subsets, which could enhance susceptibil-ity to autoimmune diseases. We should remember too,that congenital deficiencies of the early classical com-plement components predispose to vasculitis and aSLE-like syndrome.

Hormonal influences in autoimmunity

There is a general trend for autoimmune disease tooccur far more frequently in women than in men (fig-ure 17.3), probably due to differences in hormonal pat-terns. Although the reason for the higher incidence ofautoimmune disease in females is not clear, it is knownthat sex hormones can affect immune responses by modifying patterns of gene expression. Femalesgenerally produce higher levels of antibodies after immune responses which are often more likely to beproinflammatory Th1 responses. Similarly, adminis-tration of male hormones to mice with SLE reduces theseverity of disease. Pregnancy is often associated withamelioration of disease severity, particularly in RA,and there is sometimes a striking relapse after givingbirth.

Does the environment contribute?

Twin studies

Although the 50% concordance rate for the develop-ment of the autoimmune disease IDDM in identicaltwins is considerably higher than that in dizygotictwins, and suggests a strong genetic element, there isstill 50% unaccounted for. In nonorgan-specific dis-eases such as SLE there is an even lower genetic contri-


13 10 9 3

36 41

DM at 2 DM at 7

PITUITARY AND ISLETCELL ANTIBODIES

POSITIVE

3 4 5

ONSET OF DM

Age (years)

Figure 17.2 HLA-haplotype linkage and onset of insulin-dependent diabetes (DM). Haplotypes: (�) A3, B14, DR6; (�) A3,B7, DR4; ( ) A28, B51, DR4; and ( ) A2, B62, C3, DR4. Disease islinked to possession of the A2, B62, C3, DR4 haplotype. The 3-year-old brother had complement-fixing antibodies to the islet cell sur-face for 2 years before developing frank diabetes. (From A.N.Gorsuch, K.M. Spencer, J. Lister et al. (1981) Lancet, ii, 1363.)

bution with a concordance rate of only 23% in same-sex monozygotic twins. This compares with 9% insame-sex dizygotic twins. There are also many exam-ples where clinically unaffected relatives of patientswith SLE have a higher incidence of nuclear autoanti-bodies if they are household contacts than if they liveapart from the proband. Summing up, in some disor-ders the major factors are genetic, whereas in others,environmental influences seem to dominate.

Microbes

A number of autoimmune diseases following infec-tious episodes have been described, usually in geneti-cally predisposed individuals. The reasons for thispredisposition are not clear but a number of mecha-nisms have been proposed:1 Molecular mimicry in which immune responses directed against viruses or bacteria may cross-reactwith self antigens (c.f. figure 17.6b(2)). Many organ-isms have been shown to possess antigenic determi-nants that are repeated in normal human tissue. Forexample, two envelope proteins of Yersinia enterocol-itica share epitopes with the extracellular domain ofthe human thyroid-stimulating hormone (TSH) recep-tor. In rheumatic fever, antibodies produced to theStreptococcus also react with heart tissue and in the Guillain–Barré syndrome, antibodies against humangangliosides cross-react with the endotoxin of Campy-lobacter jejuni. Colon antibodies present in ulcerativecolitis have been found to cross-react with Escherichiacoli 014. There is also some evidence for the view thatantigens present on Trypanosoma cruzi may cross-reactwith cardiac muscle and peripheral nervous systemantigens and provoke some of the immunopathologiclesions seen in Chagas’ disease.

Alarge number of microbial peptide sequences hav-ing varying degrees of homology with human proteinshave been identified (table 17.3) but the mere existenceof a homology is no certainty that infection with thatorganism will necessarily lead to autoimmunity.2 Microbes may act as adjuvants. Incorporation ofmany autologous proteins into adjuvants frequentlyendows them with the power to induce autoimmunedisease in laboratory animals. This indicates that au-toreactive T-cells are normally present and that theycan be activated when presented with suitably alteredautoantigens. Microbes often display adjuvant prop-erties through their ability to impart “danger signals”which activate dendritic antigen-presenting cells.3 Microbes may activate lymphocytes polyclonally.For example, bacterial endotoxins can act by providinga nonspecific inductive signal for B-cell stimulation.The variety of autoantibodies detected in cases of infectious mononucleosis must surely be attribut-able to the polyclonal activation of B-cells by the Epstein–Barr virus (EBV) again bypassing the need forspecific T-cell help. However, unlike the usual situa-tion in human autoimmune disease, these auto-antibodies tend to be immunoglobulin M (IgM) and,normally, do not persist when the microbial compo-nents are cleared from the body. In some instances microbes can act as superantigens that link CD4+ T-cells to antigen-presenting cells (APCs) through theouter surfaces of the Vb chain and the class II MHC gly-coprotein. These may bring about the polyclonal stim-ulation of certain T-cell receptor (TCR) Vb families.

AUTOREACTIVITY COMES NATURALLY

It used to be thought that self–nonself discriminationwas a simple matter of deleting autoreactive cells in the


AUTOIMMUNE DISEASE

SLE

SCLERODERMA

POLYMYOSITIS

RHEUMATOID ARTHRITIS

SJOGREN’S SYNDROME

AUTOIMMUNE THROMBOCYTOPENIA

MYASTHENIA GRAVIS

GRAVES’ THYROTOXICOSIS

FEMALE : MALE INCIDENCE RATIO2 4 6 8 10

..

Figure 17.3 Increased incidence of autoimmune disease in females.

Microbial molecule Body component

Bacteria:Arthritogenic Shigella flexneriKlebsiella nitrogenaseProteus mirabilis ureaseMycobact. tuberculosis 65 kDa hsp

Viruses:Coxsackie BCoxsackie BEBV gp110

HBV octamerHSV glycoproteinMeasles hemagglutininRetroviral gag p32

HLA-B27HLA-B27HLA-DR4Joint (adjuvant arthritis)

MyocardiumGlutamic acid decarboxylaseRA shared Dw4 T-cell epitope

Myelin basic proteinAcetylcholine receptorT-cell subsetU-1 RNA

(E.coli DNAJ hsp)

Table 17.3 Molecular mimicry: homologies between microbesand body components as potential cross-reacting T-cell epitopes.

thymus, but it is now clear that this mechanism doesnot destroy all self-reactive lymphocytes. Immunecells that recognize self proteins do exist in normalpeople and in the vast majority of cases produce noharm. Thus autoreactive T-cells specific for self anti-gens will survive in the repertoire but are not normallyactivated. There is also a subpopulation of B-cells thatmake low-affinity IgM autoantibodies. These antibod-ies, which do not normally cause tissue damage, aredemonstrable in comparatively low titer in the generalpopulation and their incidence increases steadily withage (figure 17.4).

Is autoantigen available to the lymphocytes?

Our earliest view, with respect to organ-specific anti-bodies at least, was that the antigens were sequesteredwithin the organ and, through lack of contact with theimmune system, failed to establish immunologic toler-ance. Any mishap that caused a release of the antigenwould then provide an opportunity for autoantibodyformation. For a few body constituents this holds true.In the case of sperm, lens and heart, for example, release of certain components directly into the circula-tion can provoke autoantibodies. In general, the experience has been that injection of unmodified ex-tracts of those tissues concerned in the organ-specific autoimmune disorders does not readily elicit antibodyformation.

CONTROL OF THE T-HELPER CELL ISPIVOTAL

The message then is that we are all sitting on a mine-field of self-reactive cells, with potential access to theirrespective autoantigens, but since autoimmune dis-ease is more the exception than the rule, the body hashomeostatic mechanisms, such as regulatory T-cells,that prevent them being triggered under normal cir-cumstances. Accepting its limitations, figure 17.5 pro-vides a framework for us to examine ways in whichthese mechanisms may be circumvented to allow au-toimmunity to develop. It is assumed that the key tothe system is control of the autoreactive T-helper cellsince the evidence heavily favors the T-dependence ofvirtually all autoimmune responses; thus, interactionbetween the T-cell and MHC-associated peptide be-comes the core consideration. We start with the as-sumption that these cells are unresponsive because ofclonal deletion, clonal anergy, T-suppression or inade-quate autoantigen presentation.

AUTOIMMUNITY CAN ARISE THROUGHBYPASS OF T-HELPERS

Provision of new carrier determinant

If autoreactive T-cells are tolerized and thereby unableto collaborate with B-cells to generate autoantibodies(figure 17.6a), provision of new carrier determinants towhich no self tolerance had been established wouldbypass this mechanism and lead to autoantibody pro-duction (figure 17.6b). Modification can be achievedthrough combination with a drug (figure 17.6b(3)); forexample, the autoimmune hemolytic anemia asso-ciated with administration of a-methyldopa might beattributable to modification of the red-cell surface insuch a way as to provide a carrier for stimulating B-cells which recognize the rhesus antigen. Similarly, anew helper determinant may arise through the inser-tion of viral antigen into the membrane of an infectedcell (figure 17.6b(4)).

Polyclonal activation

As indicated above, microbes such as EBV can acti-vate B-cells in a polyclonal manner, and this has beensuggested as an important mechanism for the inductionof autoimmunity. It is, however, difficult to see how apan-specific polyclonal activation could give rise tothe patterns of autoantibodies characteristic of the dif-ferent autoimmune disorders without the operation ofsome antigen-directing factor.


ANTINUCLEARANTIBODIES

30

20

10

00–10

11–20

21–30

31–40

41–50

51–60

61–70

71–80

THYROIDANTIBODIES

Age in years

% P

ositi

ve

Figure 17.4 Incidence of autoantibodies in the general popula-tion. A serum was considered positive for thyroid antibodies if it re-acted at a dilution of 1/10 in the tanned red cell test or neat in theimmunofluorescent test, and positive for antinuclear antibodies if itreacted at a dilution of 1/4 by immunofluorescence.

BYPASS OF REGULATORY MECHANISMSRESULTS IN AUTOIMMUNE DISEASE

Failure of Fas–FasL (Fas ligand) interaction

The Fas pathway normally mediates apoptosis whichis a key mechanism in the down-regulation of immuneresponses. Failure of this interaction, resulting from ei-ther absence of Fas or of FasL, has been shown to be as-sociated with autoimmune disease due to persistenceand survival of normally deleted helper T-cells specificfor self antigens. This is seen in autoimmune lympho-

proliferative syndrome, a human genetic disorder due to a defect in the Fas protein or the Fas receptor. Interleukin-2 (IL-2) also has an important role in the apoptosis of T-cells, and animals defective in IL-2 production or which lack the IL-2 receptor fail to undergo Fas mediated apoptosis resulting in autoimmunity.

Polymorphism of CTLA-4

We have previously indicated that CTLA-4 is impor-tant in regulating T-cell activity, and animals with


AUTOANTIGEN

REGULATION

T-HELPER

AUTOIMMUNEDISEASE

T-EFFECTOR

B-CELL AUTOANTIBODY

REGULATORY BYPASS T-HELPER BYPASS

Figure 17.5 Autoimmunity arises throughbypass of the control of autoreactivity. Theconstraints on the stimulation of self-reactive helper T-cells by autoantigen canbe circumvented either through bypassingthe helper cell or by disturbance of the regu-latory mechanisms.

Tolerization

NO RESPONSE AUTOIMMUNE RESPONSE

T/B

COMPLEXED

5. PROTEIN/IDIOTYPE4. VIRUS3. DRUG2. CROSS-REACTINGANTIGEN

1. ABNORMALSELF-ANTIGEN

MEMBRANE ASSOCIATEDINTRAMOLECULAR

New carrierSelf-tolerancea b

Th

Auto-antigen

T/B T/B T/B

Th Th

Th

Figure 17.6 T-helper bypass through new carrier epitope ( ) gen-erates autoimmunity. For simplicity, processing for major histocom-patibility complex (MHC) association has been omitted from the

diagram. (a) The pivotal autoreactive T-helper is unresponsive ei-ther through tolerance or inability to see a cryptic epitope. (b) Differ-ent mechanisms providing a new carrier epitope. Th, T-helper cell.

knockout of the CTLA-4 gene develop severe autoim-mune disease. There is some evidence in humans thatpolymorphism of the CTLA-4 gene may lead to IDDMand other autoimmune problems.

Upregulation of T-cell interaction molecules

The majority of organ-specific autoantigens normallyappear on the surface of the cells of the target organ inthe context of class I but not class II MHC molecules. Assuch they cannot communicate with T-helpers and aretherefore immunologically silent. When class II genesare expressed, they endow the surface molecules withpotential autoantigenicity (figure 17.7). For example,human thyroid cells in tissue culture express surfaceHLA-DR (class II) molecules after stimulation with interferon g (IFNg), and the glands of patients withGraves’ disease (thyrotoxicosis) stain strongly withanti-HLA-DR reagents, indicating active synthesis ofclass II polypeptide chains. Inappropriate class II ex-pression has also been reported on the bile ductules inprimary biliary cirrhosis and on endothelial cells andsome b-cells in the type I diabetic pancreas.

Th1–Th2 imbalance with resultingoverproduction of cytokines may induce autoimmunity

Th1 responses appear to be involved in the pathogene-sis of a number of organ-specific autoimmune diseasesby producing large quantities of inflammatory cyto-kines. Under normal circumstances this overproduc-tion can be controlled by Th2-derived cytokines,especially IL-10, which antagonizes IL-12, a cytokinecrucial for the development of Th1 cells. If an imbal-ance exists, unregulated IL-2 and IFNg production caninitiate autoimmunity. This may occur by increasingthe concentration of processed intracellular autoanti-gens available to professional APCs and increasingtheir avidity for naive T-cells by upregulating adhe-sion molecules, or even by making previously anergiccells responsive to antigen (figure 17.7).

Autoimmunity results from inadequate regulatorycell activity

Populations of regulatory cells must hold normallypresent self-reactive T-cells in check. This function isperformed by CD4+CD25+ T-cells derived from thethymus, which suppress the proliferation of other T-cells. The importance of this subpopulation of T-cells isshown in animals depleted of this population, which

die of autoimmune disease. Furthermore in animalmodels of type I diabetes and inflammatory bowel dis-ease, transfer of normal CD4+CD25+ cells can controlthe disease.

TISSUE MAY BE DAMAGED BOTH BYAUTOANTIBODIES AND BY AUTOREACTIVE T-CELLS

The tissue damage in many autoimmune diseases isproduced both by humoral and cell mediated mecha-nisms. Autoantibodies, by activating the complementcascade may cause cytotoxicity of target tissue or they


AUTOANTIGEN

T helper

3

Non spTS

Ag spTS

Id spTS

Id spTh

MHCCLASS II

COSTIMULATOR

TARGET CELL

CYTOKINES

4

5

2

1

?

APC

5

5

Figure 17.7 Bypass of regulatory mechanisms leads to triggeringof autoreactive T-helper cells through defects in (1) tolerizability orability to respond to or induce T-regulatory cells, or (2) expression ofantigen-specific, (3) nonspecific or (4) idiotype-specific T-regulators,or through (5) imbalance of the cytokine network producing dere-pression of class II genes with inappropriate cellular expression ofclass II and presentation of antigen on target cell, stimulation of anti-gen-presenting cell (APC), and possible activation of anergic T-helper. Ag sp, antigen specific; Id sp, idiotype specific; MHC, majorhistocompatibility complex; Non sp, nonspecific; Th, T-helper cell;Ts, T-suppressor.

may promote phagocytosis of target cells or antibody-dependent cellular cytotoxicity (ADCC). Some anti-bodies initiate damage by forming immune complexesand others interfere with the physiology of the targetcell either by blocking or stimulating receptors. Au-toreactive CD8+ T-cells can produce cytolysis of tissueby perforin and granzyme-B activity, and CD4+ cellsamplify attack on the target by recruitment of inflam-matory cells and by stimulating release of their mediators.

PATHOGENIC EFFECTS OF HUMORALAUTOANTIBODY

Blood

Erythrocyte antibodies play a dominant role in the de-struction of red cells in autoimmune hemolytic ane-mia. Normal red cells coated with autoantibody have ashortened half-life essentially as a result of their adher-ence to Fcg receptors on phagocytic cells in the spleen.Lymphopenia occurring in patients with SLE and RAmay also be a direct result of antibody, since nonagglu-tinating antibodies coating these white cells have beenreported in such cases.

In Wegener’s granulomatosis antibodies to the usu-ally intracellular proteinase III, the so-called antineu-trophil cytoplasmic antibodies (ANCAs) (figure 17.8)may react with the antigen on the surface of the cell,causing activation and resulting degranulation andgeneration of reactive oxygen intermediates (ROIs).

Endothelial cell injury would then be a consequence ofthe release of superoxide anion and other ROI.

Platelet antibodies are responsible for idiopathicthrombocytopenic purpura (ITP) as IgG from a pa-tient’s serum when given to a normal individual causes a decrease in the platelet count. The transientneonatal thrombocytopenia, which may be seen in in-fants of mothers with ITP, is explicable in terms oftransplacental passage of IgG antibodies to the child.

The primary antiphospholipid syndrome is characterized by recurrent venous and arterial thromboembolic phenomena, recurrent fetal loss,thrombocytopenia and the presence of cardiolipin an-tibodies. Passive transfer of such antibodies into miceis fairly devastating, resulting in lower fecundity ratesand recurrent fetal loss. The effect seems to be medi-ated through reaction of the autoantibodies with a complex of cardiolipin and b2-glycoprotein 1, whichinhibits triggering of the coagulation cascade, but mayalso activate the endothelial cells to increase prostacy-clin metabolism, produce proinflammatory cytokinessuch as IL-6, and upregulate adhesion molecules.

Thyroid

Under certain circumstances antibodies to the surfaceof a cell may stimulate rather than destroy. This is thecase in Graves’disease, which is due to the presence ofantibodies to thyroid-stimulating hormone receptors(TSH-R) which act in the same manner as TSH. Bothstimulate the production of thyroid hormones by acti-vating the adenylate cyclase system after binding tothe TSH-R. When thyroid-stimulating immunoglobu-lins (TSIs) from a mother with Graves’ disease cross theplacenta they cause the production of neonatal hyper-thyroidism (figure 17.9), which resolves after a fewweeks as the maternal IgG is catabolized.

Thyroid-stimulating immunoglobulins act inde-pendently of the pituitary–thyroid axis and patientswith this form of hyperthyroidism will have very lowserum TSH levels. Graves’ disease is often associatedwith exophthalmos, which might be due to a cross-reaction of antibodies to a 64kDa membrane proteinpresent on both thyroid and eye muscle.

Muscle

Myasthenia gravis is due to an autoantibody directedagainst the acetylcholine receptors (ACh-R) on themotor end-plates of muscle cells. These antibodies de-plete these receptors by a combination of complement-dependent lysis of the post-synaptic membrane and by


Figure 17.8 Antineutrophil cytoplasmic antibodies (ANCAs).Left: cytoplasmic ANCA (cANCA) diffuse staining specific for pro-teinase III in Wegener’s granulomatosis. Right: perinuclear ANCA(p-ANCA) staining by myeloperoxidase antibodies in periarteritisnodosa. Fixed neutrophils are treated first with patient’s serum thenfluorescein-conjugated antihuman immunoglobulin. (Kindly pro-vided by Dr G. Cambridge.)

blocking ACh-R function. This results in inhibition of acetylcholine binding and muscular weakness. Thetransient muscle weakness seen in a proportion of babies born to mothers with myasthenia gravis is compatible with the transplacental passage of an IgGcapable of inhibiting neuromuscular transmission. Inaddition, myasthenic symptoms can be induced in ani-mals by injection of monoclonal antibodies to ACh-Ror by active immunization with the purified receptorsthemselves.

Glomerular basement membrane (g.b.m.)

Goodpasture’s syndrome is an autoimmune diseasecharacterized by progressive glomerulonephritis andpulmonary hemorrhage. As with many other autoim-mune disorders it is restricted by the MHC. Suscepti-bility is increased by HLA-DRb1*1501 and DRb1*1502.It is due to antibodies directed against the a3.a4.a5(IV)component of type IV collagen, the major componentof basement membrane in the lungs and the glomeruli.These antibodies can be detected by immunofluores-cent staining of kidney biopsies that show linear depo-sition of IgG and C3 along the basement membrane of

the glomerular capillaries (see figure 14.9a; p. 156). Ifthe antibody is eluted from a diseased kidney and in-jected into a squirrel monkey it rapidly fixes to type IVcollagen on the g.b.m. of the recipient animal and pro-duces a fatal nephritis (figure 17.10).

Heart

Neonatal lupus erythematosus is the most commoncause of permanent congenital complete heart block.Almost all cases have been associated with high mater-nal titers of anti-Ro/SS-Aantibodies. The key observa-tion is that anti-Ro binds to neonatal rather than adultcardiac tissue and alters the transmembrane action po-tential by inhibiting repolarization. ImmunoglobulinG anti-Ro reaches the fetal circulation by transplacen-tal passage but although maternal and fetal hearts areexposed to the autoantibody, only the latter is affected.

The ability of b-hemolytic streptococci to elicit cross-reactive autoantibodies which damage heart muscleunderlies the pathogenesis of acute rheumatic fever.Although antibodies to the streptolysin O exotoxin(ASO) are found in low titer in many patients follow-ing streptococcal infection, high and increasing titers


a

BABY

MOTHER

Thyroidstimulating

IgG

b

Figure 17.9 Neonatal thyrotoxicosis. (a)The autoantibodies that stimulate the thy-roid through the thyroid-stimulating hor-mone receptors are immunoglobulins G(IgG) that cross the placenta. (b) The thyrotoxic mother therefore gives birth to a baby with thyroid hyperactivity, whichspontaneously resolves as the mother’s IgGis catabolized. (Photograph courtesy of Professor A. MacGregor.)

Antibody bound tog.b.m. of kidney

Ab

Ab Ab

Ab

Antibody binds tog.b.m. of kidney

MONKEY DIES WITHGLOMERULONEPHRITIS

Elute antibodywith acid

Inject intomonkey

AbRIP

monkey patient’s

Ab

Ab Ab

Ab

Figure 17.10 Passive transfer of glomerulonephritis to a squirrelmonkey by injection of antiglomerular basement membrane (anti-g.b.m.) antibodies isolated by acid elution from the kidney of a

patient with Goodpasture’s syndrome. (After R.A. Lerner, R.J. Glascock & F.J. Dixon (1967) Journal of Experimental Medicine, 126, 989.) Ab, antibody.

are strongly suggestive of acute rheumatic fever. Al-though molecular mimicry initiates the production of cross-reacting antibodies, CD4 lymphocytes, cy-tokines and adhesion molecules also play a crucial rolein the pathogenesis of this disease.

Stomach

Pernicious anemia, which manifests as a deficiency ofvitamin B12, is an autoimmune disease of the stomachcommonly due to autoantibodies directed against gas-tric parietal cells (parietal cell antibodies). The relevantantigen on these cells is the gastric adenosine triphos-phatase (ATPase), which is the major protein of themembrane lining the secretory canaliculi of parietalcells. Patients develop an atrophic gastritis in which achronic inflammatory mononuclear invasion is associ-ated with degeneration of secretory glands and failureto produce gastric acid. In many patients additionalabnormal antibodies may be directed against the B12-binding site of intrinsic factor or to the intrinsic factor-B12 complex so blocking the uptake of B12 into thebody.

Other tissues

Many other organ-specific autoimmune diseases are associated with specific antibodies directed tothose organs. Patients with idiopathic Addison’s disease have circulating antibodies to adrenal anti-gens and the serum of patients with idiopathic hy-poparathyroidism contains antibodies to cytoplasmicantigens of parathyroid cells. Women with prematureovarian failure may show antibodies to the cytoplasmof cells of the ovary and in some infertile males, agglu-tinating antibodies cause aggregation of the spermato-zoa and interfere with their penetration into thecervical mucus. An antibody pathogenesis for pem-phigus vulgaris is favored by the recognition of an autoantigen on stratified squamous epithelial cells.Antibodies to this keratinocyte transmembrane adhe-sion molecule are easily seen on direct immunofluo-rescent microscopy of affected skin.

PATHOGENIC EFFECTS OF COMPLEXESWITH AUTOANTIGENS

Systemic lupus erythematosus (SLE)

Systemic lupus erythematosus is a relapsing and re-mitting multisystem disease where autoantibodies are formed against soluble components to which theyhave continual access. The complexes that are gener-

ated give rise to lesions similar to those occurring inserum sickness. Avariety of different autoantigens arepresent in lupus, many of them within the nucleus,with the most pathognomonic being double-strandedDNA (dsDNA). The detection of antinuclear antibod-ies (ANAs) and particularly the measurement of anti-DNA antibodies are valuable tests for the diagnosis ofSLE. Deposition of complexes is widespread, as thename implies, and lesions are often present in the kid-ney, skin, joints, muscle, lung and brain (figure 17.11).About 40% of patients eventually develop kidney in-volvement and eluates of renal tissue from patientswith lupus nephritis will show anti-dsDNA. Immuno-fluorescent staining of kidney biopsies from patientswith evidence of renal dysfunction will show the pres-ence of antigen-antibody complexes and complement.The staining pattern with a fluorescent anti-IgG oranti-C3 is punctate or “lumpy-bumpy” (see figure14.9b; p. 156), in marked contrast with the linear pat-tern caused by the g.b.m. antibodies in Goodpasture’ssyndrome (figure 14.9a; p. 156). Patients with SLE pro-duce antibodies to a variety of tissue antigens includ-ing those on red cells, platelets, lymphocytes andvarious clotting factors. During the active phase of thedisease, serum complement levels fall as componentsare binding to immune aggregates in the kidney andcirculation.

It is worth recalling that normal clearance of im-mune complexes requires an intact classical comple-ment cascade and that absence of an early complement


Figure 17.11 The “lupus band” in systemic lupus erythematosus.Left: section of skin showing slight thickening of the dermo-epidermal junction with underlying scattered inflammatory cellsand a major inflammatory focus in the deeper layers. Low powerH & E. Right: green fluorescent staining of a skin biopsy at higherpower showing deposition of complexes containing immunoglobu-lin G (anti-C3 gives the same picture) on the basement membrane atthe dermo-epidermal junction. (Kindly provided by Professor D.Isenberg.)

protein predisposes to immune complex disease.Thus, although homozygous complement deficiencyis a rare cause of SLE — the archetypal immune com-plex disorder — it represents the most powerful diseasesusceptibility genotype so far identified as more than80% of cases with homozygous C1q and C4 deficiencydevelop an SLE-like disease.

T-CELL-MEDIATED HYPERSENSITIVITY AS A PATHOGENIC FACTOR INAUTOIMMUNE DISEASE

Insulin-dependent diabetes mellitus (IDDM)

Insulin-dependent diabetes mellitus (type I diabetes)is a multifactorial autoimmune disease the major feature of which is a T-cell infiltration of the islets ofLangerhans and progressive T-cell-mediated destruc-tion of insulin-producing b-cells. Auto-reactive T-cellsspecific for b-cell antigens such as glutamic acid decar-boxylase (GAD) and insulin can be identified in theblood of newly diagnosed diabetic patients. Autoanti-bodies specific for the same antigens can also be det-ected in the serum of patients and can be used as asensitive marker to predict the risk of developing thedisease (see figure 17.2; p. 192). It is not clear if these antibodies are the result of T-cell-mediated injury or ifthey play some role in the causation of the disease.Delay in onset of disease can be achieved by early treat-ment with cyclosporin, since this drug targets T-cell cytokine synthesis so specifically. The disease has astrong genetic basis and is associated with genes within the MHC, particularly the DR3.DQ2 and theDR4.DQ8 haplotypes, and with polymorphisms with-in the insulin gene region on chromosome 11p15.5.

Multiple sclerosis (MS)

Multiple sclerosis is an autoimmune disorder of thecentral nervous system mediated by T-cells directed atcentral nervous system myelin components occurringin individuals with certain class II MHC alleles. The T-cells recognize and attack components of the axonalmyelin sheath resulting in destruction of myelin andthe underlying axon. The idea that MS could be an au-toimmune disease has for long been predicated on themorphological resemblance to experimental allergicencephalomyelitis (EAE). This is a demyelinating disease leading to motor paralysis, which can be produced by immunizing experimental animals withMBP(myelin basic protein) in complete Freund’s adju-vant or by transferring Th1-cells from an affected to a

naive animal. In human MS damage to the myelin isproduced by Th1-cytokines such as TNF and IFNgand by activated macrophages, which phagocytosemyelin constituents. In addition cytotoxic T-cells may directly damage myelin and antibodies directedagainst myelin constituents can cause demyelinationby ADCC, myelin opsonization or complement activa-tion. Antibodies against MBP and MOG (myelin oligo-dendrocyte glycoprotein) are found in these patientsand are associated with more frequent relapses and aworse prognosis. Another feature of MS is the presenceof oligoclonal Igs in the cerebrospinal fluid (CSF) of pa-tients: their specificity and significance in the patho-genesis of the disease remains unknown. Although theetiology of MS is unknown, molecular mimicry mayplay a role. It is of interest that a T-cell clone derivedfrom a patient with the disease, reacted against bothMBP and an antigen found in EBV.

Rheumatoid arthritis (RA)

Immunopathogenesis

Rheumatoid arthritis is a common destructivearthropathy of unknown etiology, strongly linked tothe MHC class II proteins HLA-DRB1*0404 and *0401.The joint changes are produced by the hyperplasia ofthe synovial cells associated with increased vascular-ity and infiltration of inflammatory cells forming a pannus overlaying and destroying cartilage and bone(figure 17.12). The infiltrating cells are primarily CD4+

T-cells, which stimulate monocytes, macrophages andmast cells to secrete IL-1, IL-6, TNF and a variety ofchemokines, which recruit neutrophils into the joints.The IL-1 and TNF in the synovium stimulate fibrob-lasts and chondrocytes to release tissue-destroyingproteolytic enzymes which lead to joint damage, andbone destruction follows the stimulation of osteoclastsby these cytokines. As the malign pannus (cover)grows over the cartilage, tissue breakdown can be seenat the margin (figure 17.12c), almost certainly as a result of the release of enzymes, ROIs and especially of IL-1, IL-6 and TNF. B-cells are also activated andplasma cells are frequently observed. Secondary lymphoid follicles with germinal centers may be pre-sent in the synovium. Immune complexes of rheuma-toid factor and IgG may initiate an Arthus reaction inthe joint space leading to an influx of polymorphs. Byreleasing elastase, collagenase and proteases thesecells add to the joint destruction by degrading pro-teoglycan in the superficial layer of cartilage (figure17.13).



RHEUMATOIDARTHRITIS

Capsule

Synovial MembraneSynovial Fluid

Articular Cartilage

4Pannus

Erosion

Figure 17.12 Rheumatoid arthritis (RA). (a) Hands of a patient withchronic RA showing classical swan-neck deformities. (b) Diagram-matic representation of a diarthrodial joint showing bone and carti-laginous erosions beneath the synovial membrane-derived pannus.

(c) Histology of pannus showing clear erosion of bone and cartilageat the cellular margin. ((a) Kindly given by Professor D. Isenberg; (c) by Dr L.E. Glynn.)

IL-1,IL-6,TNF, ROICOLLAGENASE

NEUTRALPROTEINASE

Cartilage breakdown

LOCAL STRESS?

SPEC

ULAT

ION

MICROBE? Abnormalglycosylation

IMMUNECOMPLEX

II

Loss of regulatorycontrol of auto-reactive

T-cells?

Bone erosion

SELF-ASSOCIATEDIgG ANTIGLOBULIN

COMPLEXES

CYTOKINES

SYNOVIAL LINING CELLS

PANNUS

JOINT SPACE

ACUTE INFLAMMATION

COMPLEMENT

Collagensensitization

?

BTh

IgG

Figure 17.13 Immune pathogenesis ofrheumatoid arthritis and speculation onthe induction of autoimmunity. The iden-tity of the peptide(s) associated with theclass II molecule is unknown. Green arrowsindicate stimulation. Black arrows mean“becomes” or “leads to.” IgG, im-munoglobulin G; IL, interleukin; Th, T-helper cell; TNF, tumor necrosis factor; ROI,reactive oxygen intermediate.

(a) (b)(c)

Rheumatoid arthritis can be successfully treatedwith anticytokine therapy or by blocking T-cell activation

Now that the central role of cytokines in RAis appreci-ated, a number of anticytokine therapies have been developed. These include a soluble TNF receptor-IgG1fusion protein (Etanercept) which neutralizes freeTNF, and a chimeric monoclonal antibody against TNFitself (Infliximab). By blocking TNF activity, its abilityto activate the cytokine cascade of IL-1, IL-6, IL-8 andother inflammatory cytokines is impeded, making thisa valuable adjunct to the treatment of RA. Another approach is to block the activation of CD28 on T-cellsusing a fusion protein made up of CTLA-4 and IgG1.This competes with CD28 for binding to B7.1 (CD80)and B7.2 (CD86) and prevents T-cell activation. By act-ing so early in the inflammatory cascade CTLA4Ig in-hibits the secondary activation of macrophages andB-cells and shows considerable promise in treating patients with RA.

Immunoglobulin G autosensitization and immunecomplex formation

Autoantibodies to the IgG Fc region, which is ab-normally glycosylated, are known as antiglobulins orrheumatoid factors, and are the hallmark of the dis-ease, being demonstrable in virtually all patients withRA. The majority are IgM antiglobulins, the detectionof which provides a very useful clinical test for RA.

Immunoglobulin G aggregates, presumably prod-ucts of the infiltrating plasma cells which synthesizeself-associating IgG antiglobulins, can be regularly detected in the synovial tissues and in the joint fluidwhere they give rise to typical acute inflammatory re-actions with fluid exudates.

Crohn’s disease

Crohn’s disease is a chronic granulomatous inflamma-tory bowel disease which is a complex disorder withimmunologic, environmental and genetic compo-nents. The etiology is not known but it has been suggested that it is an autoimmune disease; patientsproduce antibodies to a variety of organisms, and 70%have antibodies to the yeast Saccharomyces cerevisiae.The detection of antibodies forms a useful test in the di-agnosis of Crohn’s disease. The lesions in the bowel arecharacterized by infiltration of mononuclear cells withincreased production of inflammatory cytokines in-cluding TNF, IL-1, IL-6 and IL-12. A gene on chromo-some 16, called NOD2, confers susceptibility to the

disease and a polymorphism of this gene is an impor-tant genetic risk factor. It has been suggested that theNOD2 protein has a role in apoptosis which normallyregulates the lymphocyte populations and terminatesimmune responses. Defective apoptosis is one of thefeatures of lamina propria T-cells in patients withCrohn’s disease. This results in uncontrolled activa-tion of Th1 cells, with release of IL-12, IFNg and IL-2,and subsequent activation of monocytes, which re-spond by releasing TNF and other mediators of inflam-mation. Indeed neutralization of TNF with the TNFneutralizing antibody, Infliximab, is associated withdramatic clinical responses. This human–mousechimeric antibody not only has an antiinflammatoryeffect but may increase apoptosis of lamina propria T-cells.

Celiac disease

The normally acquired tolerance to dietary proteinsseems to break down in celiac disease. This is a condi-tion due to an inappropriate T-cell response to thegliadin fraction of ingested gluten. The disease is asso-ciated with DQ2 in over 90% of cases and with DQ8 in the rest of the cases. Ingestion of gliadin leads to infiltration of the intestinal mucosa by both CD4+ andCD8+ cells. Patients develop immune responses togliadin and to a component of the endomysium identi-fied as tissue transglutaminase (TTG). The presence ofantibodies, both IgA and IgG, against TTG are bothspecific and diagnostic for celiac disease.

Chronic autoimmune (Hashimoto’s) thyroiditis

T-cells play a crucial role in the development ofHashimoto’s thyroiditis, interacting with thyroid fol-licular cells and extracellular matrix. T-cells may be-come activated either by molecular mimicry against aninfectious agent or by interacting with class II MHCmolecules aberrantly present on thyroid cells fromthese patients. Direct killing of thyroid cells by CD8+

cells is responsible for the hypothyroidism but cytokine secretion may also play a role. Patients alsodevelop serum antibodies to thyroid peroxidase andthyroglobulin but a role for these autoantibodies in thecausation or the perpetuation of the disease has notbeen defined. Titers of these antibodies are closely associated with disease activity but it is not clearwhether immune responses to these autoantigens ini-tiate thyroiditis or are secondary to T-cell mediated tis-sue damage.

We see now that there is considerable diversity in theautoimmune response to the thyroid. This may lead to


tissue destruction, metabolic stimulation, growth pro-motion or mitotic inhibition, which in different combi-nations account for the variety of forms in which autoimmune thyroid disease presents (figure 17.14).

Atherosclerosis

Cells of the immune system and the numerous inflam-matory mediators that they produce are important inthe initiation, growth and even rupture of atheroscle-rotic plaques. The targets of the postulated auto-immune attack are epitopes produced by oxidation oflow-density lipoproteins (LDLs) which have accumu-lated in the intima of the vessel. The modified LDLsstimulate endothelial cells to develop adhesion mole-cules for lymphocytes and monocytes and also acti-vate the intimal smooth muscle to produce cytokinesincluding IL-1, TNF, IL-6, IL-18, IFNg and macrophagecolony-stimulating factor (M-CSF). Endothelial cellsrelease chemokines, which attract leukocytes to thearea, and as a result monocytes and T-cells infiltrate the vessel wall. The monocytes become activated bycytokines such as M-CSF and actively ingest the modified LDL particles becoming so-called foam cells.There is also some evidence that heat shock proteins(HSP) and especially the 60 kDa family could be theculprit autoantigens in the development of early ather-osclerosis (figure 17.15). These proteins are found bothon endothelial cells and on a number of microorgan-isms such as Chlamydia and Escherichia coli and may

facilitate immunological cross-reactions betweenpathogen and self HSP60. The ultimate result is thecontinued recruitment of T-cells and macrophages,which together form the fatty streaks which are the earliest manifestations of atherosclerosis. Further cy-tokine production causes proliferation and migrationof smooth muscle cells and atherosclerotic plaques,which can interfere with blood flow or can rupturecausing an acute coronary obstruction.

Psoriasis

Psoriasis is primarily a T-cell-mediated inflammatorydisease where keratinocyte hyperproliferation is driven by proinflammatory Th1 cytokines produced locally by infiltrating T-cells. Activated T-cells withinpsoriatic lesions elaborate many cytokines, includingIFNg, TNF, IL-1, and IL-2, but only low levels of Th2 cy-tokines such as IL-4 and IL-10 are present. The natureand location of the initiating antigen is unknown. Thecrucial role of T-cells is proven by the variety of anti-T-cell biologic agents, which are effective in treatingthese patients. These include use of CTLA-4Ig, a re-combinant fusion protein of CTLA-4 and IgG, whichblocks T-cell activation or the use of antibodies againstCD4 or against the IL-2 receptor (Daclizumab). Im-provement in clinical psoriasis has also been shownusing Etanercept, which targets TNF, and Efalizumab,which targets the adhesion molecule LFA-1. Evenmore exciting is the use of recombinant human IL-4 to


PRIMARYMYXEDEMA

Destructiveimmune response

HASHIMOTO’S THYROIDITIS

GRAVES' DISEASE

Ab a

ffecti

nggr

owth

Ab affecting

metabolism

NON-GOITROUS TOXICOSIS

AUTO

IMM

UNE

COLL

OID

GOITE

R

'HAS

HITO

XICO

SIS'

HASHIMOTO’S PERSISTENT GOITER

Figure 17.14 Relationship of different autoimmune responses tothe circular spectrum of autoimmune thyroid disease. Hashitoxi-cosis refers to a thyroid which shows Hashimoto’s thyroiditis andthyrotoxicosis simultaneously. (Courtesy of Professors D. Doniachand G.F. Bottazzo.) Ab, antibody.

Figure 17.15 Expression of heat-shock protein 60 in an earlyhuman arteriosclerotic lesion. Frozen, unfixed, 4 mm thick section of a fatty streak (= early lesion) of a human carotid artery stained inindirect immunofluorescence with a monoclonal antibody to heat-shock protein 60 and a fluorescein-labeled second antibody. Astrongreaction with endothelial cells as well as cells infiltrating the intima,including foam cells, is evident. (Original magnification ¥400.)(Photograph kindly provided by Professor G. Wick.)

convert the Th1 to a Th2 response resulting in signifi-cant clinical improvement.

DIAGNOSTIC VALUE OF AUTOANTIBODYTESTS

Serum autoantibodies frequently provide valuable di-agnostic markers, and the salient information is sum-marized in table 17.4. These tests may prove of value inscreening for people at risk, for example relatives ofpatients with autoimmune diseases such as diabetes,

thyroiditis patients for gastric autoimmunity and viceversa, and ultimately the general population if the sociological consequences are fully understood andacceptable.

TREATMENT OF AUTOIMMUNE DISORDERS

Control at the target organ level

The major approach to treatment, not unnaturally, in-volves manipulation of immunologic responses (fig-


Distinction from colloid goiter, thyroid cancer and subacute thyroiditisThyroidectomy usually unnecessary in Hashimoto goiter

Tests +ve in 99% of cases.

High titers of cytoplasmic Ab indicate active thyroiditis and tendency to post-operativemyxedema: anti-thyroid drugs are the treatment of choice although HLA-B8 patientshave high chance of relapse

Help in diagnosis of latent PA, in differential diagnosis of non-autoimmunemegaloblastic anemia and in suspected subacute combined degeneration of the cord

Insulin Ab early in disease. GAD Ab standard test for IDDM. Two or more autoAb seenin 80% of new onset children or prediabetic relatives but not in controls

Distinction from tuberculous form

When positive suggests associated thymoma (more likely if HLA-B12), positive in >80%

Different fluorescent patterns in the two diseases

Distinction from other forms of anemia

Distinction from other forms of obstructive jaundice where test rarely +veRecognize subgroup within cryptogenic cirrhosis related to PBC with +ve mitochondrial Ab

Smooth muscle Ab distinguish from SLEType 1 classical in women with Ab to nuclei, smooth muscle, actin andasialoglycoprotein receptor. Type 2 in girls and young women with anti-LKM-1 (cyt P450)

High titer indicative of bad prognosis

Prognosis of rheumatoid arthritis

DNA antibodies present in active phase. Ab to double-stranded DNA characteristic; highaffinity complement-fixing Ab give kidney damage, low affinity CNS lesions

Thrombosis, recurrent fetal loss and thrombocytopenia

Characterization of the disease

Antiserine protease closely associated with disease; treatment urgent

Hashimoto’s thyroiditis

Primary myxedema

Thyrotoxicosis

Pernicious anemia

Insulin-dependentdiabetes mellitus (IDDM)

Idiopathic adrenal atrophy

Myasthenia gravis

Pemphigus vulgaris andpemphigoid

Autoimmune hemolyticanemia

Sjögren’s syndrome

Primary biliary cirrhosis

Active chronic hepatitis

Rheumatoid arthritis

SLE

Scleroderma

Wegener ’s granulomatosis

Thyroid

Thyroid

Thyroid

Stomach

Pancreas

Adrenal

Muscle

ACh receptor

Skin

Erythrocyte (Coombs’ test)

Salivary duct cells, SS-A, SS-B

Mitochondrial

Smooth muscle anti-nuclearand 20% mitochondrial

Antiglobulin,e.g. SCAT andlatex fixationAntiglobulin + raisedagalacto-Ig

High titer antinuclear, DNA

Phospholipid

Nucleolar

Neutrophil cytoplasm

DISEASE ANTIBODY COMMENT

Table 17.4 Autoimmunity tests and diagnosis.

Ab, antibody; CNS, central nervous sytem; GAD, glutamic acid decarboxylase; Ig, immunoglobulin; PA, pernicious anemia; SLE, systemiclupus erythematosus.

ure 17.16). However, in many organ-specific diseases,metabolic control is usually sufficient, for examplethyroxine replacement in primary myxedema, insulinin juvenile diabetes, vitamin B12 in pernicious anemiaand anti-thyroid drugs for Graves’ disease. Xenograftsof genetically engineered fetal or neonatal pig tissuesuch as islet cells are under study, and stem cells arebeing differentiated in culture to produce a variety oftissues that could be used to replace destroyed tissues.

Anti-inflammatory drugs

Corticosteroids have long been used to treat auto-immune diseases as they not only suppress various aspects of the immune response but also control the inflammatory lesions and particularly the influx ofneutrophils and other phagocytic cells. Patients withsevere myasthenic symptoms respond well to highdoses of steroids, and the same is true for serious casesof other autoimmune disorders such as SLE and immune complex nephritis. In RA, steroids are very effective and accelerate the induction of remission.

Selectins, leukocyte integrins and adhesion moleculeson endothelial cells appear to be downregulated as aresult of treatment and this seriously impedes the in-flux of inflammatory cells into the joint.

Immunosuppressive drugs

Because it blocks cytokine secretion by T-cells, cy-closporin is an anti-inflammatory drug and, since cytokines like IL-2 are also obligatory for lymphocyteproliferation, cyclosporin is also an antimitotic drug. Itis of proven efficacy in a variety of autoimmune dis-eases, as are the conventional nonspecific antimitoticagents such as azathioprine, cyclophosphamide andmethotrexate, usually given in combination withsteroids. The general immunosuppressive effect ofthese agents, however, places the patients at muchgreater risk from infections.

Plasmapheresis

Successful results have been obtained in a number of


TOTAL LYMPH NODEIRRADIATION

ANTIGEN &IDIOTYPE INDUCEDT-SUPPRESSION

CYCLOSPORIN

THYMUS GRAFTTHYMUS FACTORS

METABOLICCONTROL

CELL-MEDIATEDHYPERSENSITIVITY

METABOLIC DEFECT

ANTIBODY

STRUCTURAL DEFECT

INFLAMMATORYTISSUE DAMAGE

MECHANICAL ORTISSUE GRAFT

ANTI-INFLAMMATORYDRUGS

Maturation

ActivationProliferation

Differentiation

ANTI-CLASS IIANTI-CD4

ANTI-IL-2R

ANTIGEN/PEPTIDEINDUCEDTOLERANCE‘MAGIC BULLET’

ANTI-MITOTICDRUGS

STEMCELL

B/T

BLASTS

EFFECTORS

Ts

BONE MARROWGRAFT

i.v. POOLED Ig

PLASMAPHERESIS

DELIVERY OFGROWTH FACTORS

TRANSFECTION WITHPROTECTIVE GENES

T, B OR SUBSETDEPLETION

Figure 17.16 The treatment of autoim-mune disease. Current conventional treat-ments are in dark orange; some feasibleapproaches are given in lighter orangeboxes. (In the case of a live graft, bottomright, the immunosuppressive therapyused may protect the tissue from the au-toimmune damage which affected theorgan being replaced.) Ig, immunoglobu-lin; IL, interleukin; i.v., intravenous; Ts, T-suppressor.

autoimmune diseases, especially when the treatmenthas been applied in combination with antimitoticdrugs. However, plasma exchange to remove the abnormal antibodies and lower the rate of immunecomplex deposition in SLE provides only temporarybenefit.

T-cell control strategies

Arange of options is presented in figure 17.17.

Idiotype control with antibody

The powerful immunosuppressive action of anti-idiotype antibodies suggests that it may be useful incontrolling autoantibody production. In a number ofautoimmune diseases intravenous injection of Igpooled from many normal donors is of benefit and insome conditions is the treatment of choice. The pooledIg is not only a source of antiidiotype antibodies, whichcan interact with the idiotype of autoantibodies, but itcontains antibodies against numerous cytokines andalso has significant anti-inflammatory effects. The ef-fects of intravenous Ig reflect the numerous functionsof natural antibodies in maintaining immune home-ostasis in healthy individuals.

T-cell vaccination

If we regard autoreactive T-cells as tissue-destroyingpathogens, it becomes clear that these T-cells if ren-dered inactive may be employed as vaccines to preventand perhaps treat the disease. Administration of suchautologous T-cells may induce the regulatory networkto specifically control destructive T-cells, and such anapproach has been successfully employed to controlEAE in experimental animals. Trials in MS using a TCRpeptide vaccine embodying the Vb5.2 sequence expressed on T-cells specific for MBP show promise(figure 17.17(5)).

Manipulation of regulatory mediators

In most solid organ-specific autoimmune diseases it isthe Th1 cells that are pathogenic, and attempts toswitch the phenotype to Th2 should be beneficial (fig-ure 17.17(1)). Administration of IL-4, which deviatesthe immune response from Th1 to Th2, has beenshown in EAE to downregulate or inhibit the destruc-tive T-cell process. Interferon b inhibits the synthesis ofIFNg and has a number of suppressive effects on T-cell-mediated inflammation. These regulatory activitiesprovide the basis for the therapeutic use of IFNb in MS,

where it significantly reduces both the frequency andthe severity of the clinical exacerbations and is con-sidered the first drug proven to improve the naturalcourse of the disease.

Manipulation by antigen

The object is to present the offending antigen in suffi-cient concentration and in a form that will occupy theantigen-binding cleft on MHC molecules to preventbinding by the autoantigen. One strategy is to designhigh-affinity peptide analogs differing by only oneamino acid, which bind to the appropriate MHC mole-cule and antagonize the response to autoantigen (fig-ure 17.17(4)). Since we express several different MHCmolecules, this should not impair microbial defensesunduly.

We have already noted that, because the mucosalsurface of the gut is exposed to a horde of powerfullyimmunogenic microorganisms, it has been importantfor the immune defenses of the gut to evolve mecha-nisms which deter Th1-type responses. This objectiveis attained by the stimulation of cells which release immunosuppressive cytokines. Thus, feeding anti-gens should tolerize Th1 cells and this has proved to be a successful strategy for blocking EAE and other experimental autoimmune diseases. Regrettably, at-tempts to induce oral tolerance in humans have notmet with success.


5. Vaccinate withidiotype

1. Delete, suppressor switch T-cellor block IL-2R

2. Block normalsignal transduction

CD3

T-CELL

TCR

ANTIGEN/PEPTIDE

ANTIGEN-PRESENTING CELL

CD4

3. Tolerize

6. Delete Vα orVβ family

4. Antagonize

7. Block MHC

8. Transfect withimmunomodulator

MHC

Figure 17.17 Strategic options for therapy based on T-cell target-ing. IL, interleukin; MHC, major histocompatibility complex; TCR,T-cell receptor.

The tolerogen can also be delivered by inhalation of peptide aerosols and this could be a very attractiveway of generating antigen-specific T-cell suppression.Intranasal peptides have been used successfully toblock various experimental autoimmune diseases,and treatment can be effective even after induction ofdisease.

Other biological agents that inhibit the immuneresponse

A variety of monoclonal antibodies have been pro-duced which, based upon the “magic bullet” concept,selectively home onto lymphocytes bearing specificsurface receptors. A number of these antibodies andother forms of biological inhibitors are being tested forthe treatment of autoimmune diseases. Their mecha-nisms of action include:• Blockade of TNF with a specific monoclonal anti-body (Infliximab) or a TNF receptor-IgG1 fusion protein (Etanercept).• Blockade of IL-1 receptors with a recombinant IL-1-receptor antagonist.• Blockade of leukocyte recruitment to tissues using

chemokine receptor or chemokine antagonists. Thisform of therapy could target inflammation in an organspecific manner. For example CTACK (CCL27) and itsreceptor CCR10 are important for the homing of T-cellsinto the skin but not into other organs. Blocking of thisresponse could be useful in psoriasis and should sparethe rest of the immune system.• CTLA-4Ig, a recombinant fusion protein consistingof CTLA-4 linked to the constant region of IgG, whichblocks T-cell activation and turns down primary andsecondary antibody responses.• Blockade of CD4 with a fully human anti-CD4 monoclonal antibody.• Blockade of the IL-2 receptor CD25 (Daclizumab).Such an antibody targets only activated T-cells anddoes not affect the overall immune responsiveness ofthe recipient.• Blockade of adhesion molecules such as LFA-1, annexin-1 and VAP-1 (vascular adhesion protein-1).• Antibodies against the CD40 ligand.• Inhibitors of cell signaling pathways.• Stem cell transplantation, with a view to restoringhomeostasis and regulatory cells.


REVISION

The immune system balances precariously between effec-tive responses to environmental antigens and regulatorycontrol of potentially suicidal attack against self molecules.

The scope of autoimmune diseases• Autoimmunity is associated with certain diseaseswhich form a spectrum. At one pole, exemplified byHashimoto’s thyroiditis, the autoantibodies and the lesions are organ-specific with the organ acting as the target for autoimmune attack; at the other pole are thenonorgan-specific or systemic autoimmune diseases suchas SLE, where the autoantibodies have widespread reactivity and the lesions resemble those of serum sick-ness relating to deposition of circulating immune complexes.• There is a tendency for organ-specific disorders such as thyroiditis and pernicious anemia to overlap in givenindividuals, while overlap of rheumatologic disorders isgreater than expected by chance.

Genetic and environmental influences• Multifactorial genetic factors increase the likelihood

of developing autoimmune disease: these include HLAtissue type, predisposition to aggressive autoimmunityand the selection of potential autoantigens.• Females have a far higher incidence of autoimmunitythan males, perhaps due to hormonal influences.• Twin studies indicate a strong environmental influencein many disorders; both microbial and nonmicrobial fac-tors have been suspected.• Microbes may initiate autoimmune disease by a num-ber of mechanisms, including molecular mimicry, or byacting as adjuvants or superantigens, or as polyclonal acti-vators of the immune response.

Autoreactivity comes naturally• Autoantigens are, for the most part, accessible to circu-lating lymphocytes which normally include autoreactiveT- and B-cells. These autoreactive cells are controlled byregulatory CD4+CD25+ T-cells and absence of this popula-tion results in autoimmunity.

The T-helper is pivotal for control• It is assumed that the key to the system is control of autoreactive T-helper cells which are normally unrespon-

(continued)


sive because of clonal deletion, clonal anergy, T-suppression or inadequate autoantigen processing.

Autoimmunity can arise through bypass of T-helpers• Abnormal modification of the autoantigen throughsynthesis or breakdown, combination with a drug orcross-reaction with exogenous antigens, can provide newcarrier determinants, which can activate T-cells.• B-cells and T-cells can be stimulated directly by poly-clonal activators such as EBV or superantigens.• Failure of the Fas–FasL (Fas ligand) interaction can re-sult in survival of normally deleted T-cells. Interleukin-2(IL-2) besides its important activation function is also crucial for apoptosis, and absence of IL-2 or its receptormay lead to autoimmunity.

Autoimmunity can arise through bypass of regulatorymechanisms• The derepression of class II genes could give rise to inap-propriate cellular expression of class II so breaking the “silence” between cellular autoantigen and autoreactiveT-inducer.

• Th1–Th2 imbalance may result in overproduction of inflammatory cytokines.

Pathogenic effects of humoral autoantibody• Direct pathogenic effects of human autoantibodies toblood, surface receptors and several other tissues are listedin table 17.5.• Passive transfer of disease is seen in “experiments of nature” in which transplacental passage of maternal immunoglobulin G (IgG) autoantibody produces a comparable but transient disorder in the fetus.

Pathogenic effects of complexes with autoantigens• Immune complexes, usually with bound complement,appear in the kidneys, skin and joints of patients with sys-temic lupus erythematosus (SLE), associated with lesionsin the corresponding organs.

T-cell-mediated hypersensitivity as a pathogenic factor• Type 1 diabetes mellitus (IDDM) has a strong geneticbasis linked to MHC class II genes. T-cell destruction of insulin-producing cells is the basis of the disease but

DISEASE AUTOANTIGEN LESION

Autoimmune hemolytic anemia

Lymphopenia (some cases)

Idiopathic thrombocytopenic purpura

Wegener ’s granulomatosis

Anti-phospholipid syndrome

Male infertility (some cases)

Pernicious anemia

Hashimoto’s disease

Primary myxedema

Graves' disease

Goodpasture’s syndrome

Myasthenia gravis

Lambert–Eton syndrome

Acanthosis nigricans (type B) and ataxiatelangiectasia with insulin resistance

Atopic allergy (some cases)

Congenital heart block

Celiac disease

Red cell

Lymphocyte

Platelet

PMN proteinase ΙΙΙ

Cardiolipin/β2−glycoprotein1complex

Sperm

H+/K+-ATPase, gastrin receptorIntrinsic factor

Thyroid peroxidase surface antigen

TSH receptor

TSH receptor

Glomerular basement membrane

Acetylcholine receptor

Presynaptic Ca channel

Insulin receptor

β-Adrenergic receptors

Ro/SS-A

Endomysium

Erythrocyte destruction

Lymphocyte destruction

Platelet destruction

PMN induced endothelial injury

Recurrent thromboembolic phenomena

Agglutination of spermatozoa

Block acid productionBlock vitamin B12 uptake

Cytotoxic effect on thyroid cells in culture

Blocking of thyroid cell

Stimulation of thyroid cell

Complement-mediated damage to basement membrane

Blocking and destruction of receptors

Neuromuscular defect

Blocking of receptors

Blocking of receptors

Distort fetal cardiac membrane action potential

Small intestinal inflammation

Table 17.5 Direct pathogenic effects of humoral antibodies.

ATPase, adenosine triphosphatase; PMN, polymorphonuclear neutrophil; TSH, thyroid-stimulating hormone.


various autoantibodies are also present.• Multiple sclerosis (MS) is due to demyelination pro-duced by cytokines and activated macrophages and T-cells. Similarity to experimental autoimmune en-cephalomyelitis, a demyelinating disease induced by immunization with myelin in complete Freund’s adju-vant, suggests that autoimmunity forms the basis of thedisease.• Activated T-cells and macrophages are abundant in therheumatoid synovium where they give rise to inflamma-tion in the joint space through the release of IL-1, IL-6,tumor necrosis factor (TNF), prostaglandin E2 (PGE2), collagenase, neutral proteinase and reactive oxygen inter-mediates (ROIs).• Synovial lining cells grow as a malign pannus whichproduces erosions in the underlying cartilage and bone.• Rheumatoid arthritis (RA) can be successfully treatedwith anti-TNF therapy or by competing with CD28 activa-tion of T-cells using CTLA-4Ig. The presence of autoanti-bodies to IgG, known as rheumatoid factor, are useful inmaking a diagnosis of RA.

• The lesions of Crohn’s disease are characterized by infil-trating mononuclear cells and the presence of cytokines,suggesting a T-cell basis for the disorder. Neutralization ofTNF results in clinical improvement.• Celiac disease is due to T-cell activation against thegliadin fraction of gluten found in wheat products. Anti-tissue transglutaminase antibodies are diagnostic of celiacdisease.• Hashimoto’s thyroiditis is produced by cytotoxic T-cells and cytokines. The role of antibodies to thyroid com-ponents is unclear but these antibodies are useful in thediagnosis of chronic autoimmune thyroiditis.• Atherosclerosis is initiated by the movement ofmononuclear cells into the vessel wall. The target antigenmay be modified low-density lipoproteins or heat shockproteins. Cytokines produced by mononuclear cells and endothelial cells cause proliferation of smooth musclecells and atherosclerotic plaques.• Psoriasis results from keratinocyte hyperproliferationinduced by Th1 cytokines. Anti-T-cell biologic agents areuseful in the treatment.

DIFFERENCES

ORGAN-SPECIFIC (e.g. THYROIDITIS,GASTRITIS, ADRENALITIS)

NONORGAN-SPECIFIC (e.g. SYSTEMICLUPUS ERYTHEMATOSUS)

Antigens only available to lymphoid system in low concentration

Antibodies and lesions organ-specific

Clinical and serologic overlap – thyroiditis, gastritis and adrenalitis

Familial tendency to organ-specific autoimmunity

Lymphoid invasion, parenchymal destruction by cell-mediatedhypersensitivity and/or antibodies

Therapy aimed at controlling metabolic deficit or tolerizing T-cells

Tendency to cancer in organ

Antigens evoke organ-specific antibodies in normal animals withcomplete Freund’s adjuvant

Experimental lesions produced with antigen in Freund’s adjuvant

Antigens accessible at higher concentrations

Antibodies and lesions nonorgan-specific

Overlap SLE, rheumatoid arthritis, and other connective tissue disorders

Familial connective tissue disease

Lesions due to deposition of antigen–antibody complexes

Therapy aimed at inhibiting inflammation and antibody synthesis

Tendency to lymphoreticular neoplasia

No antibodies produced in animals with comparable stimulation

Diseases and autoantibodies arise spontaneously in certain animals(e.g. NZB mice and hybrids)

SIMILARITIES

Circulating autoantibodies react with normal body constituentsPatients often have increased immunoglobulins in serumAntibodies may appear in each of the main immunoglobulin classes particularly IgG and are usually high affinity and mutatedGreater incidence in womenDisease process not always progressive; exacerbations and remissionsAssociation with HLASpontaneous diseases in animals genetically programmedAutoantibody tests of diagnostic value

Table 17.6 Comparison of organ-specific and nonorgan-specific diseases.

IgG, immunoglobulin G; SLE, systemic lupus erythematosus.

(continued)

FURTHER READING

Appelmelk, B.J., Faller, G., Claeys, D., Kirchner, T. & Vanden-broucke-Grauls, C.M.J.E. (1998) Bugs on trial: The case of Heli-cobacter pylori and autoimmunity. Immunology Today, 19, 296–9.

Choy, E.H.S & Panayi, G.S. (2001) Cytokine pathways and joint inflammation in rheumatoid arthritis. New England Journal of Medicine, 345, 907–16.

Davidson, A. & Diamond, B. (2001) Advances in immunology. Au-toimmune diseases. New England Journal of Medicine, 345, 340–50.

Greaves, D.R. & Channon, K.M. (2002) Inflammation and immuneresponses in atherosclerosis. Trends in Immunology, 23, 535–41.

Kazatchkine, M.D. & Kaveri, S.V. (2001) Immunomodulation of au-toimmune and inflammatory diseases with intravenous immuneglobulin. New England Journal of Medicine, 345, 747–55.

Kelly, M.A., Rayner, M.L., Mijovic, C.H. & Barnett, A.H. (2003) Mole-cular aspects of type 1 diabetes. Molecular Pathology, 56, 1–10.

Sarvetnick, N. & Ohashi, P.S. (eds) (2003) Section on autoimmunity.Current Opinion in Immunology, 15, 647–730.

Tian, J., Olcott, A., Hanssen, L., Zekzer, D. & Kaufman, D.L. (1999)Antigen-based immunotherapy for autoimmune disease: Fromanimal models to humans? Immunology Today, 20, 190–5.

Vandenbark, A.A., Chou, Y.K., Whitham, R. et al. (1996) Treatment ofmultiple sclerosis with T-cell receptor peptides. Nature Medicine, 2,1109–15.

Van Den Brande, J.M.H., Peppelenbosch, M.P. & Van Deventer, S.J.H.(2002) Treating Crohn’s disease by inducing T lymphocyte apop-tosis. Annals of the New York Academy of Science, 973, 166–80.

Walker, L.S.K. & Abbas, A.K. (2002) The enemy within: Keeping self-reactive T cells at bay in the periphery. Nature Reviews Immunology,2, 11–19.

Wicker, L. & Wekerle, H. (eds) (1995) Autoimmunity. Current Opinionin Immunology, 6, 783–852. [Several critical essays in each annualvolume.]


Diagnostic value of autoantibody tests• A wide range of serum autoantibodies now providevaluable diagnostic markers.

Treatment of autoimmune disorders• Therapy conventionally involves metabolic control andthe use of anti-inflammatory and immunosuppressivedrugs.• Plasma exchange may be of value especially in combi-nation with antimitotic drugs.• Intravenous Ig is of benefit in a number of autoimmunedisorders. This may be due to the presence of anti-idiotypeantibodies or anti-cytokine activity.• Awhole variety of potential immunologic control thera-pies are under intensive investigation. These include T-cellvaccinations, switching the immune response from Th1 toTh2, idiotype manipulations and attempts to induce

antigen-specific unresponsiveness particularly for T-cellsusing peptides.• Numerous biologic agents that inhibit the immune re-sponse are being investigated. These include blockade ofcytokines, or adhesion molecules or receptors such as theIL-2 receptor or CD40L(CD40 ligand).• Blockade of TNF is highly effective therapy for RA andother autoimmune diseases.• The accompanying comparison of organ-specific andnonorgan-specific autoimmune disorders (table 17.6)gives an overall view of many of the points raised in thischapter.


211

Glossary

acute phase proteins: Serum proteins, mostly produced in the liver,which rapidly change in concentration (some increase, some decrease) during the initiation of an inflammatory response.

adjuvant: Any substance which nonspecifically enhances the im-mune response to antigen.

affinity (intrinsic affinity): The strength of binding (affinity con-stant) between a receptor (e.g. one antigen-binding site on an anti-body) and a ligand (e.g. epitope on an antigen).

agglutination: The aggregation of particles such as red cells or bacte-ria by antibodies.

allele: Variants of a polymorphic gene at a given genetic locus.allelic exclusion: The phenomenon whereby, following successful

rearrangement of one allele of an antigen receptor gene, rearrange-ment of the other parental allele is suppressed, thereby ensuringeach lymphocyte expresses only a single specificity of antigenreceptor (although this does not occur for achains in T-cells).

allotype:An allelic variant of an antigen which, because it is not pre-sent in all individuals, may be immunogenic in members of thesame species which have a different version of the allele.

anaphylatoxin:Acomplement breakdown product (e.g. C3a, C4a orC5a) capable of directly triggering mast cell degranulation.

anaphylaxis: An often fatal hypersensitivity reaction, triggered byIgE or anaphylatoxin-mediated mast cell degranulation, leadingto anaphylactic shock due to vasodilatation and smooth musclecontraction.

anergy: Potentially reversible specific immunological tolerance inwhich the lymphocyte becomes functionally nonresponsive.

antibody-dependent cellular cytotoxicity (ADCC): A cytotoxic reaction in which an antibody-coated target cell is directly killedby an Fc receptor-bearing leukocyte, e.g. NK cell, macrophage orneutrophil.

apoptosis: A form of programmed cell death, characterized by en-donuclease digestion of DNA.

atopic allergy: IgE-mediated hypersensitivity, i.e. asthma, eczema,hayfever and food allergy.

avidity (functional affinity): The binding strength between twomolecules (e.g. antibody and antigen) taking into account the valency of the interaction. Thus the avidity will always be equal toor greater than the intrinsic affinity (see affinity).

chemokines: Afamily of structurally-related cytokines which selec-tively induce chemotaxis and activation of leukocytes. They alsoplay important roles in lymphoid organ development, cell com-partmentalization within lymphoid tissues, Th1/Th2 develop-ment, angiogenesis and wound healing.

chemotaxis: The directional migration of cells towards an attractant.complementarity determining regions (CDR): The hypervariable

amino acid sequences within antibody and T-cell receptor vari-able regions which interact with complementary amino acids onthe antigen or peptide–MHC complex.

C-reactive protein: An acute phase protein which is able to bind tothe surface of microorganisms where it functions as a stimulator of the classical pathway of complement activation, and as an opsonin for phagocytosis.

cytokines: Low molecular weight proteins that stimulate or inhibitthe differentiation, proliferation or function of immune cells.

delayed-type hypersensitivity (DTH): A hypersensitivity reactionoccurring within 48–72 h and mediated by cytokine release fromsensitized T-cells.

diversity (D) gene segments: Found in the immunoglobulin heavychain gene and T-cell receptor b and d gene loci between the V andJ gene segments. Encode part of the third hypervariable region inthese antigen receptor chains.

endotoxin: Pathogenic cell wall-associated lipopolysaccharides ofGram-negative bacteria.

epitope: That part of an antigen recognized by an antigen receptor.Fab: Monovalent antigen-binding fragment obtained following pa-

pain digestion of immunoglobulin. Consists of an intact lightchain and the N-terminal VH and CH1 domains of the heavy chain.

F(ab’)2: Bivalent antigen-binding fragment obtained followingpepsin digestion of immunoglobulin. Consists of both light chainsand the N-terminal part of both heavy chains linked by disulfidebonds.

Fas: A member of the TNF receptor gene family. Engagement of Fas(CD95) on the surface of the cell by the Fas ligand (CD178) presenton cytotoxic cells, can trigger apoptosis in the Fas-bearing targetcell.

Fc: Crystallizable, non-antigen binding fragment of an im-munoglobulin molecule obtained following papain digestion.Consists of the C-terminal portion of both heavy chains which isresponsible for binding to Fc receptors and C1q.

fluorescent antibody: An antibody conjugated to a fluorescent dyesuch as FITC.

framework regions: The relatively conserved amino acid sequenceswhich flank the hypervariable regions in immunoglobulin and T-cell receptor variable regions and maintain a common overallstructure for all V-region domains.

gammaglobulin: The serum proteins, mostly immunoglobulins,which have the greatest mobility towards the cathode during electrophoresis.

germ line: The arrangement of the genetic material as transmittedthrough the gametes.

germinal center: Discrete areas within lymph node and spleenwhere B-cell maturation and memory development occur.

graft vs host (g.v.h.) reaction: Reaction occurring when T lympho-cytes present in a graft recognize and attack host cells.

granuloma: A tissue nodule containing proliferating lymphocytes,fibroblasts, and giant cells and epithelioid cells (both derived fromactivated macrophages), which forms due to inflammation in response to chronic infection or persistence of antigen in the tissues.

granzymes: Serine esterases present in the granules of cytotoxic Tlymphocytes and NK cells. They induce apoptosis in the target cellwhich they enter through perforin channels inserted into the tar-get cell membrane by the cytotoxic lymphocyte.

haplotype: The set of allelic variants present at a given genetic region.

hapten: Alow molecular weight molecule that is recognized by pre-formed antibody but is not itself immunogenic unless conjugatedto a “carrier” molecule which provides epitopes recognized byhelper T-cells.

hematopoiesis: The production of erythrocytes and leukocytes.high endothelial venule (HEV): Capillary venule composed of spe-

cialized endothelial cells allowing migration of lymphocytes intolymphoid organs.

HLA (human leukocyte antigen): The human major histocompatibil-ity complex (MHC).

humanized antibody: A genetically engineered monoclonal anti-body of non-human origin in which all but the antigen-bindingCDR sequences have been replaced with sequences derived fromhuman antibodies. This procedure is carried out to minimize theimmunogenicity of therapeutic monoclonal antibodies.

humoral: Pertaining to extracellular fluid such as plasma andlymph. The term humoral immunity is used to denote antibody-mediated immune responses.

hybridoma: Hybrid cell line obtained by fusing a lymphoid tumorcell with a lymphocyte which then has both the immortality of thetumor cell and the effector function (e.g. monoclonal antibody secretion) of the lymphocyte.

hypervariable regions: Those amino acid sequences within the im-munoglobulin and T-cell receptor variable regions which showthe greatest variability and contribute most to the antigen or pep-tide–MHC binding site.

idiotype: The complete set of idiotopes in the variable region of anantibody or T-cell receptor which react with an anti-idiotypicserum.

idiotype network:Aregulatory network based on interactions of idio-types and anti-idiotypes present on antibodies and T-cell receptors.

immune complex: Complex of antibody bound to antigen whichmay also contain complement components.

immunofluorescence: Technique for detection of cell or tissue-associated antigens by the use of a fluorescently-tagged ligand (e.g. an anti-immunoglobulin conjugated to fluorescein isothiocyanate).

immunogen: Any substance which elicits an immune response.Whilst all immunogens are antigens, not all antigens are immuno-gens (see hapten).

immunoglobulin superfamily: Large family of proteins character-ized by possession of “immunoglobulin-type” domains ofapproximately 110 amino acids folded into two b-pleated sheets.Members include immunoglobulins, T-cell receptors and MHCmolecules.

immunotoxin: Acytotoxic agent produced by chemically coupling atoxic agent to a specific monoclonal antibody.

internal image:An epitope on an anti-idiotype which binds in a waythat structurally and functionally mimics the antigen.

isotype:An antibody constant region structure present in all normalindividuals, i.e. antibody class or subclass.

ITAM: Immunoreceptor Tyrosine-based Activation Motifs are con-sensus sequences for src-family tyrosine kinases. These motifs arefound in the cytoplasmic domains of several signaling moleculesincluding the signal transduction units of lymphocyte antigen re-ceptors and of Fc receptors.

J chain: A molecule which forms part of the structure of pentamericIgM and dimeric IgA.

joining (J) gene segments: Found in the immunoglobulin and T-cellreceptor gene loci and, upon gene rearrangement, encode part ofthe third hypervariable region of the antigen receptors.

lectins: A family of proteins, mostly of plant origin, which bind spe-cific sugars on glycoproteins and glycolipids. Some lectins are mi-togenic (e.g. PHA, ConA).

linkage disequilibrium: The occurrence of two alleles being inherit-ed together at a greater frequency than that expected from theproduct of their individual frequencies.

lipopolysaccharide (LPS): Endotoxin derived from Gram-negativebacterial cell walls which has inflammatory and mitogenic actions.

mannose binding lectin: A member of the collectin family of calcium-dependent lectins, and an acute phase protein. It acti-vates the complement system by binding to mannose residues onthe surface of microorganisms.

marginal zone: The outer area of the splenic periarteriolar lymphoidsheath (PALS) which is rich in B cells, particularly those respond-ing to thymus-independent antigens.

margination: Leukocyte adhesion to the endothelium of blood ves-sels in the early phase of an acute inflammatory reaction.

membrane attack complex (MAC): Complex of complement com-ponents C5b–C9 which inserts as a pore into the membrane of tar-get cells leading to cell lysis.

memory cells: Clonally expanded T- and B-cells produced during aprimary immune response and which are “primed” to mediate asecondary immune response to the original antigen.

MHC (major histocompatibility complex): A genetic region encod-ing molecules involved in antigen presentation to T-cells. Class IMHC molecules are present on virtually all nucleated cells and areencoded by HLA-A, B, and C in man, whilst class II MHC mole-cules are expressed on antigen-presenting cells (primarilymacrophages, B-cells and interdigitating dendritic cells) and areencoded by HLA-DR, DQ, and DP in man. Allelic differences canbe associated with the most intense graft rejection within a species.

MHC restriction: The necessity that T-cells recognize processedantigen only when presented by MHC molecules of the originalhaplotype associated with T-cell priming.

mitogen: A substance which non-specifically induces lymphocyteproliferation.

monoclonal antibody: Homogeneous antibody derived from a sin-gle B-cell clone and therefore all bearing identical antigen-bindingsites and isotype.

mucosal-associated lymphoid tissue (MALT): A system of lym-phoid tissue found in the gastrointestinal and respiratory tracts.

murine: Pertaining to mice.myeloma protein: Monoclonal antibody secreted by myeloma cells.negative selection: Deletion by apoptosis in the thymus of T-cells

which recognize self peptides presented by self MHC molecules,thus preventing the development of autoimmune T-cells. Nega-tive selection of developing B-cells is also thought to occur if theyencounter high levels of self antigen in the bone marrow.

nude mouse: Mouse which is T-cell deficient due to a homozygousgene defect (nu/nu) resulting in the absence of a thymus (and alsolack of body hair).

oncofetal antigen:Antigen whose expression is normally restricted tothe fetus but which may be expressed during malignancy in adults.

opsonin: Substance, e.g. antibody or C3b, which enhances phagocy-tosis by promoting adhesion of the antigen to the phagocyte.

opsonization: Coating of antigen with opsonin to enhance phagocytosis.pathogen-associated molecular pattern (PAMP): Molecules such as

lipopolysaccharide, peptidoglycan, lipoteichoic acids and man-nans, which are widely expressed by microbial pathogens asrepetitive motifs but are not present on host tissues. They aretherefore utilized by the pattern recognition receptors (PRRs) ofthe immune system to distinguish pathogens from self antigens.

pattern recognition receptor (PRR): Receptors on professional anti-gen-presenting cells and phagocytes which enable them to recog-nize pathogen-associated molecular patterns (PAMPs). Amongstthe large number of different PRRs are the mannose receptor andthe macrophage scavenger receptor.

perforin: Molecule produced by cytotoxic T-cells and NK cellswhich, like complement component C9, polymerizes to form apore in the membrane of the target cell leading to cell death.

PHA(phytohemagglutinin):Aplant lectin which acts as a T-cell mitogen.phagolysosome: Intracellular vacuole where killing and digestion

of phagocytosed material occurs following the fusion of a phago-some with a lysosome.

phagosome: Intracellular vacuole produced following invaginationof the cell membrane around phagocytosed material.

positive selection: The selection of those developing T-cells in thethymus which are able to recognize self MHC molecules. This occurs by preventing apoptosis in these cells.

proteasome: Cytoplasmic proteolytic enzyme complex involved inantigen processing for association with MHC.

recombination signal sequence (RSS): Conserved heptamer (7-nucleotide)-nonamer (9-nucleotide) sequences, separated by a 12or 23 base spacer, which occur 3’ of variable gene segments, 5’ and3’ of diversity gene segments, and 5’ of joining gene segments, inboth immunoglobulin and T cell receptor genes. They function asrecognition sequences for the recombinase enzymes that mediatethe gene rearrangement process involved in the generation oflymphocyte antigen receptor diversity.

respiratory burst: The increased oxidative metabolism which oc-curs in phagocytic cells following activation.

rheumatoid factor: IgM, IgG and IgAautoantibodies to IgG, particu-larly the Fc region. These antibodies are usually found in the bloodof patients with rheumatoid arthritis.

secretory component: Proteolytic cleavage product of the poly-Ig re-ceptor which remains associated with dimeric IgA in sero-mucussecretions.

somatic hypermutation: The enhanced rate of point mutation in theimmunoglobulin variable region genes which occurs followingantigenic stimulation and acts as a mechanism for increasing anti-body diversity and affinity.

superantigen:An antigen which reacts with all the T-cells belongingto a particular T-cell receptor V region family, and which thereforestimulates (or deletes) a much larger number of cells than doesconventional antigen.

TAP: The Transporters associated with Antigen Processing (TAP-1and TAP-2) are molecules which carry antigenic peptides from thecytoplasm into the lumen of the endoplasmic reticulum for incor-poration into MHC class I molecules.

T-dependent antigen: An antigen which requires helper T-cells inorder to elicit an antibody response.

thymocyte: Developing T-cell in the thymus.T-independent antigen: An antigen which is able to elicit an anti-

body response in the absence of T-cells.titer: Measure of the relative “strength” (a combination of amount

and avidity) of an antibody or antiserum, usually given as thehighest dilution which is still operationally detectable in, for example, an ELISA.

Toll-like receptors: A family of pattern-recognition receptors thatrecognize different molecules on bacterial surfaces. Activation ofthese receptors usually results in cytokine production and upreg-ulation of the adaptive immune response.

variable (V) gene segments: Genes that rearrange together with D(diversity) and J (joining) gene segments in order to encode thevariable region amino acid sequences of immunoglobulins and T-cell receptors.

MHC class II 55, 57, 57f, 58, 60natural killer cells 45, 46fT-cells 54, 60

antigen presenting cells (APCs) 62–3antigen processing 54–7, 56f

B-cells 79defects 100–1endocytosis 57endogenous 54–5, 56f, 60endosomes 57exogenous 55–7, 56f, 60invariant chain 56fproteasomes 54–5, 55f

anti-idiotype antibodies 30autoimmune disease therapy 206as control mechanism 97–8

anti-IL-2R antibody (Daclizumab/Basiliximab)171

autoimmune disease therapy 207bone marrow transplants 172psoriasis therapy 203

anti-inflammatory cytokines 91anti-inflammatory drugs 205anti-LFA-1 antibody (Efalizumab) 203antineutrophil cytoplasmic antibodies (ANCAs)

197, 197fantinuclear antibodies (ANAs) 199antiphospholipid syndrome 197, 208tantiplatelet antibodies 197antithyroid stimulating hormone receptor

antibodies 197antitumor necrosis factor antibody (Infliximab)

202, 207apoptosis 98–9

CD95–CD95L pathway (Fas ligand) 98, 99, 99fmitochondrial pathway 98, 99, 99fnatural killer cell induction 13

Artemis gene, mutations 140arthritis, post-infection 173tArthus reaction 157–8Aspergillus fumigatus

AIDS 143chronic granulomatous disease 135

asthma, allergic 36, 150–1pathological changes 151f

ataxia telangiectasia 141, 179atherosclerosis 203, 203f, 209atopic allergy 150–4, 151f, 162

anaphylaxis 151clinical tests 152eosinophils 149–50genetic component 149HLAassociation 149IgE antibodies 148–9, 149flate-phase reactions 149–50mast cells 149–50mechanisms 148–9, 151fmediators 149–50monocytes 149–50skin tests 149Th2 cells 148–9therapy 151f, 152–3type IV hypersensitivity vs 150wheal and flare reactions 152see also hypersensitivity reactions, type I;

individual diseases/disorders

213

Index

NotesPage numbers followed by f indicate figures: page

numbers followed by t indicate tables: vsindicates a comparison or differential diagnosis.

The following abbreviations have been used:HLA, human leukocyte antigenMHC, major histocompatibility complexTCR, T-cell receptor

ABO blood groups 154–5, 155tacanthosis nigricans 208tacquired immunity 17–26

cell types 20finnate immunity vs 25fspecificity 21–2tumor immunology 179see also lymphocytes, dendritic antigen- presenting

cells, vaccinationacquired immunodeficiency syndrome (AIDS) see

HIV infectionactivation-induced cell death (AICD) 98–9, 99f

see also apoptosisacute inflammation see inflammation, acuteacute phase proteins 12–13, 12t, 15Addison’s disease 199

CTLA-4 polymorphisms 192diagnosis 204tHLAassociations 173t, 192

adenosine deaminase (ADA) deficiency 140adjuvants, vaccines 132, 134affinity, antigen–antibody interactions 52agammaglobulinemia, X-linked 138, 138fagglutination assays, antibody detection 53agglutination reactions, blood typing 53agranulocytosis, drug-related 156alemtuzumab (anti-CD52; Campath-1H) 184alkaline phosphatase, neutrophil granules 3allelic exclusion 109, 110f, 111allergic rhinitis 36, 150allergy see atopic allergyallografts 164

rejection 166–7allotypes 30, 31fa1-antitrypsin 12a-fetoprotein (AFP) 177ab TCRs 42, 43faluminium compounds, as adjuvants 132alveolar macrophages 3, 4falveolitis, extrinsic allergic 158fanaphylaxis, atopic allergy 151anaphylatoxins 9anergy, T-cell development 75, 109angiogenesis, tumor immunotherapy 184ankylosing spondylitis, HLAassociations 173t,

174, 192antiacetylcholine receptor antibodies 197–8antibodies 17–18, 19, 24, 27–41, 28f, 38

activation of complement 10allotypes 30, 31fantigenic specificity 21, 24avidity 52biological function 31, 32fclasses/subclasses 31, 39class switching see class switchingcombinatorial libraries 38detection 53, 53f, 59–60

diversity 29f, 45tsee also antigen membrane receptors

engineering 37–8feedback mechanisms 97, 103idiotypes 30, 31fin hyperacute rejection 167isotypes 29–30, 31fprotease digestion 27, 28fstructure see antibodies, structure (below)sequencing 27–8VDJ recombination 29variability plots 29fviral infections 121, 122fsee also immunoglobulin(s)

antibodies, structure 17complementarity-determining regions 31constant regions 27, 29f, 31, 32fFab fragments 27, 28f, 31F(ab’)2 fragments 27, 28fFc fragments 27, 28fheavy chains see heavy chains, antibodies hypervariable regions 27, 29f, 30light chains 27, 28, 29, 29fvariable regions 3, 27, 28, 29f, 30–1, 32f, 45t

antibody-dependent cellular cytotoxicity (ADCC)23, 23f, 119, 154

autoantibodies 197FcgRIII (CD16) 34graft rejection 168fhypersensitivity see hypersensitivity reactions,

type II mechanism 154fnatural killer cells 23, 23f, 46f

anti-CD3 monoclonal antibodies (OKT-3) 170–1anti-CD4 antibody 203, 207anti-CD52 monoclonal antibody (Campath-1H:

alemtuzumab) 184antiepidermal growth factor receptor antibodies

184antigen(s) 19, 50, 59

capture 71–2, 73detection 53–4, 60extracellular 55–7, 56f, 60follicular dendritic cell sequestration 71intracellular 54–5, 56f, 60levels in immune system regulation 97, 98f, 103presentation see antigen presentation processing see antigen processing structure 50

antigen–antibody interactions 50, 51f, 52, 59cross-reactivity 52, 52fspecificity 21, 24, 52in vitro reactions 52–4, 59

antigen membrane receptors 41–9B-cell 41, 48D-region 44interchain amplification 43–4nucleotide insertion (N-region diversity) 44random VDJ recombination 43somatic hypermutation 44–5see also TCRs

antigen presentationCD1 57–8defects 100–1, 100fmacrophages 71MHC class I 54–5, 56f, 57f, 58, 60

atopic eczema 161autoantibodies 21, 196–9, 208t

antibody-dependent cellular cytotoxicity 197autoimmune disease diagnosis 204in general population 194f

autografts 164autoimmune diseases 24, 188–210

anti-receptor 156diagnosis 204t, 210

autoantibodies 204environment 192–3, 207etiology/pathogenesis see autoimmune diseases,

pathogenesis gender 192, 193tgenetic factors 190–2, 207history 189–90hormonal influences 192, 193tmultifactorial 190organ-specific 188

systemic vs 209toverlap of 189–90, 191fspectrum 191fsystemic 189

organ-specific vs 209ttherapy 204–7, 205f, 210

anti-idiotype antibodies 206anti-inflammatory drugs 205immunosuppressive drugs 205mediator manipulation 206monoclonal antibody therapy 207plasmapheresis 205–6at target organ level 204–5T-cell vaccination 206, 206ftolerogens 206–7

autoimmune diseases, pathogenesis 193–201, 195f,196f

autoantibodies 196–9, 208autoreactive T-cells 196–7, 200–4, 208–9CTLA-4 polymorphisms 196Fas/FasL failure 195–6immune complexes 199–200, 208inadequate regulation 196, 208molecular mimicry 193, 193f

see also molecular mimicry, autoimmunediseases

privileged sites 194proinflammatory cytokines 196regulatory T-cells 194self-reactive T-cell apoptosis 195–6Th1/Th2 imbalance 196T-helper cells 194–5, 195f, 196f, 207–8type II hypersensitivity reactions 156

autoimmune hemolytic anemia 156, 197, 208tdiagnosis 204tdrug-induced 194–5

autoimmune thrombocytopenia 156gender ratio 193t

azathioprine 170autoimmune disease therapy 205mechanism of action 170f

b2-microglobulin 46B7.1 (CD80)

T-cell activation 75, 78ftumor immunotherapy 182

B7.2 (CD86) 75, 78fbacille bilié de Calmette–Guérin (BCG) 130, 132tbacteria

capsules 117extracellular 117–19, 125

opsonization 117–18, 118fsecretory immune response 118–19survival strategies 117–19toxin neutralization 117

intracellular 119–20, 119f, 120f, 125toxins 58–9

bacterial/permeability increasing (BPI) factor 3bare lymphocyte syndrome 108, 140–1Basiliximab see anti-IL-2R antibody

(Daclizumab/Basiliximab)basophils 4f

anaphylatoxin production 9chemokine specificities 116t

inflammation 115ontogeny 106f

B-cell(s) 20factivation 79, 81

T-helper cells 79, 80fthymus-dependent antigens 79, 80fthymus-independent antigens 79, 79f

antigen processing 79CD19 75CD20 75complement receptors 117–18deficiencies 138–9, 142t, 146

see also individual diseases/disorderslymphomas 178

cellular phenotype 179fimmunodeficiency 141–2immunotherapy 182

maturation/proliferation 91–2, 93f, 109–11, 112memory see memory B-cellsontogeny 106fspleen 68f, 69surface receptor 41, 48T-cells vs 75, 76t, 80

BCG vaccine 130, 132tb2-agonists 152blood typing, agglutination reactions 53bone marrow, B-cell development 63bone marrow transplants

anti-IL-2R antibody 172chronic granulomatous disease 135–6graft-vs-host disease 172–3, 175leukemia therapy 185fT-cell purging 172, 184tissue matching 168

Borrelia burgdorferi vaccine 131botulism, pooled gammaglobulin 128tbreast carcinomas, gene mutations 178brequinar 170fBrucella 119Bruton’s tyrosine kinase (Btk)

B-cell activation 79defects 138

Burkitt’s lymphoma 181

C1 complement proteinclassical pathway 17–18deficiency 136–7IgG activation 33–4

C1 inhibitor (C1 inh) 116deficiency 137, 137f

C2 complement proteincleavage 9deficiency 136–7

C3a complement protein 9, 10classical complement pathway 18type III hypersensitivity 157

C3b complement proteinB-cell activation 10classical complement pathway 18production 7, 8f

C3 complement proteinalternative complement pathway 7–8cleavage 8fdeficiency 137

C3 convertasealternative complement pathway 7–8, 8classical complement pathway 17, 18

C4a complement proteindeficiency 136–7production 17–18

C4 complement proteinclassical pathway 17–18cleavage 9

C5a complement protein 9, 10type III hypersensitivity 157

C5 complement proteincleavage 9deficiency 137

C9 complement protein 10, 12CA-19-9 178CA-125 178calcineurin 77, 78fcalnexin, antigen processing 56f

INDEX214

Campath-1H (anti-CD52 monoclonal antibody:alemtuzumab) 184

Campylobacter jejuni 193Candida albicans 124

AIDS 143chronic granulomatous disease 135severe combined immunodeficiency 140

carcinoembryonic antigen (CEA) 177caspases, apoptosis 99cathepsin G

neutrophil granules 3phagocytosis 7

CC chemokines 89–90, 89tinflammation 115

C chemokines 89–90, 89tCD1 57–8, 63tCD3 complex 42–3, 44f, 63t, 75CD4+ T-cells see T-helper cellsCD8+ T-cells see cytotoxic T-cellsCD14 63t, 117CD16 (FcgRIII) 34CD19 75, 80, 141, 178, 192CD20 75, 80, 141, 178, 184CD28 63t, 75, 78fCD32 (FcgRII) 34CD34 106CD40/CD40L B-cell maturation/proliferation

91–2T-cell activation 75–6

CD40L (CD40 ligand), defects 138–9CD64 (FcgRI) 34CD95–CD95L interaction 98,99CD antigens 62, 63t, 75celiac disease 202, 208t, 209

HLAassociations 173t, 202cell-mediated hypersensitivity see hypersensitivity

reactions, type IVcell-mediated immunity 22–4, 24–5, 88–91, 94

cytokine control 88fhumoral immunity vs 128intracellular pathogens 119–20, 123parasites 123–4tumor immunotherapy 181–2viral infections 121–3, 122fsee also T-cell(s)

cell surface markers 62, 73CD antigens 62, 63t, 75detection 62

centroblasts, germinal centers 92, 93fceruloplasmin 12Chediak–Higashi disease 136cheese-washer’s disease 158chemokines 88–90, 89t

allergies 149dendritic cell production 71inflammation 114, 115leukocyte specificities 116tlymphocyte homing 64receptors 83see also individual chemokines

cholera vaccine 129chronic granulomatous disease (CGD) 135–6

characteristics 136floci 138f

chronic inflammation see inflammation, chronicchronic/late graft rejection 167–8chronic lymphocytic leukemia (CLL) 178class switching 91, 92–3, 94, 111

hypermutation 45clonal selection 19, 21fclotting system, innate immunity 11colony stimulating factors 84fcolorectal cancers 178colostrum 127combinatorial libraries, antibodies 38commensal flora, innate immunity 1, 3common variable immunodeficiency (CVID) 138complement 7–10, 15

activation in type II hypersensitivity 154alternative pathway 7–8, 8, 8f

activation 35classical pathway 17–18

activation 33–4

alternative complement pathway vs 18fdeficiency 136–7

components 10, 17see also individual complement proteins

deficiencies 136–7, 142tsee also individual diseases/disorders

functions 9–10inflammation 10–11, 11f, 15mannose-binding lectin (MBL) pathway 8–9

deficiencies 136membrane attack complex generation 9, 9f

receptors (CR) 9, 117–18regulation 8, 116tests for 141

complementarity-determining regions (CDRs) 27,29f, 30, 31

congenital complete heart block 198constant regions, antibodies 27, 29f, 31, 32fcontact dermatitis 161control mechanisms (immune system) 97–104

antibody feedback 97, 103antigen levels 97, 98f, 103idiotype network 97–8, 98f, 103see also individual systems

corticosteroidsapplications 153, 161, 205immunodeficiency 141immunosuppression 170, 170f

countercurrent electrophoresis 53C-reactive protein (CRP) 12Crohn’s disease 202, 209cross-reactivity, antigen–antibody interactions 52,

52fCryptococcus neoformans 124, 143CTLA-4

blockade, tumor immunotherapy 182polymorphisms 192, 196T-cell activity 75, 77, 78, 78f, 99–100

CTLA-4Ig 202, 203, 207CX3C chemokines 89–90, 89tCXC chemokines 89t, 115cyclophosphamide 170, 170f

autoimmune disease therapy 205cyclosporin 170, 170f

autoimmune disease therapy 205type IV hypersensitivity reaction therapy 161

cytokine release syndrome 171cytokines 82–92, 94

anti-inflammatory 91biological functions 83, 84t

cell-mediated immunity regulation 88finflammation 11, 115range of action 82T-cell proliferation 86–7, 87fviral infections 122–3

disease associationsasthma 150–1atopic allergy 149–50psoriasis 203

gene organization 83, 85immunosuppressive 100Janus kinase (JAK)-STAT signaling pathway

83membrane forms 82network interactions 83, 85, 85f, 86fparasitic infections 123–4production 84t

natural killer cells 13T-cells 82T-helper cells 22

Ras-MAP kinase signaling pathway 83receptors 82–3tumor immunotherapy 180–1see also individual cytokines

cytomegalovirus 128, 128t, 143cytotoxic drugs, immunodeficiency 141cytotoxic T-cells 23–4, 90–1, 94

activation 76graft rejection 167, 168fmechanism of action 23f, 90–1, 91fMHC class I recognition 53, 90tumor immunology 179viral infections 122

Daclizumab see anti-IL-2R antibody (Daclizumab/Basiliximab)

decay accelerating factor (DAF) 116defensins

infection 118–19neutrophil granules 3

delayed-type hypersensitivity see hypersensitivityreactions, type IV

dendritic cells (DCs) 71ontogeny 106fTh1/Th2 cell balance 85vaccination, tumor immunotherapy 182see also follicular, interdigitating

dermatitis herpetiformis, HLAassociations 173tdiabetes mellitus, type I see insulin-dependent

diabetes mellitus (IDDM)diapedesis 114–15, 115f

inflammation see inflammationlymphocytes 65fneutrophils 10

DiGeorge syndrome 139, 139fdiphtheria, pooled gammaglobulin 128tdiphtheria–tetanus–pertussis vaccine (DTP) 128f,

132, 132t, 134“direct pathway,” allograft recognition 167discoid lupus erythematosus 160disulfide bonds, antibodies 27, 30diversity (D) genes 28, 44DNAvaccines 131–2Doherty, Peter 54drug reactions, type II hypersensitivity 156

eczema 161Efalizumab (anti-LFA-1 antibody) 203effector cell production 82–96Ehrlich, Paul 189elderly, immune response in 102, 103electrophoresis, countercurrent 53endocrine system 103

immunosuppression 101, 101fendocytosis 57endosomes 57endothelial cells, local inflammation 10enzyme-linked antibodies 62enzyme-linked immunosorbent assays (ELISAs)

53, 53feosinophils 4f

allergic reactions 149–50anaphylatoxin effects 9chemokine specificities 116tdiseases/disorders 150granules 150inflammation 115ontogeny 106fparasite killing 14

eotaxin, inflammation 115epitopes 50, 51fEpstein–Barr virus (EBV) 177, 179

AIDS 143polyclonal lymphocyte activation 193X-linked lymphoproliferative syndrome 141

erythrocytes, ontogeny 106fE-selectin, inflammation 114, 115estrogen, immune system effects 102Etanercept (TNF receptor-IgG1 fusion protein) 207

psoriasis therapy 203rheumatoid arthritis therapy 202

experimental allergic encephalomyelitis (EAE) 200experimental autoimmune thyroiditis 189–90, 189fextracellular antigens 55–7, 56f, 60extravasation see diapedesisextrinsic allergic alveolitis 158f

Fab antibody fragments 27, 28f, 31F(ab’)2 antibody fragments 27, 28ffactor B 7, 12factor D 7factor H 8, 116

deficiency 137factor I 8, 116

deficiency 137familial hemochromatosis 192farmer’s lung 158

INDEX 215

Fas/FasL interaction 98, 99, 99fautoimmune diseases 195–6natural killer cell killing 13, 13ftumor immunology 179

Fc antibody fragments 27, 28fFcaR 35FceRI 36, 149FceRII 36, 149–50Fcg receptors 23, 34, 39, 192FcRn 34, 35ffetus

as allograft 174, 174fhematopoiesis in liver 105

fibrinogen 12FK506 (Tacrolimus) 170

mechanism of action 170ffluorescence activated cell sorting (FACS) 62, 64f

lymphoma diagnosis 185ffluorescence microscopy 64f

cell surface markers 62follicular dendritic cells (FDCs) 71–2

complement receptors 117–18lymph nodes 66memory cell stimulus 94

food allergy 152formoterol 152fungal infections 123, 124, 126furrier’s disease 158

gd TCR 42, 43f, 58gd T-cells 58, 60, 122gc chain, cytokine receptors 82–3

mutations 140gas gangrene, pooled gammaglobulin 128tgene cloning, subunit vaccines 131genes

cytokines 83, 85expression, T-cell activation 82, 83f, 94immunoglobulins see immunoglobulin(s)

germinal centers 66, 67f, 92, 93f, 94spleen 68f

Giardia lamblia 138glomerulonephritis 156f

passive transfer 198fglucocorticoids

immunosuppression 101, 101finflammation regulation 116–17

glycoproteins, tumor antigens 178Goodpasture’s syndrome 156, 198, 208t

HLAassociations 173t, 198GPI (glycosylphosphatidylinositol)- anchor

deficiencies 137graft rejection 24, 164–76

acute 167, 168fantibody-dependent cellular cytotoxicity 168ffirst set 164graft-vs-host reaction 165–6, 166fhistorical research 165–6, 166fhyperacute 167, 171mechanisms 166–8, 168f, 169f, 175

allograft recognition 166–7lymphocytes 167

MHC incompatibility 165–6, 175mixed lymphocyte reactions 165prevention 168–71, 175

immunosuppressive drugs 168, 170, 170fsee also individual drugs

lymphoid population targeting 170–1second set 164type II hypersensitivity reactions 155types 167–8, 168f

graft-vs-host disease (GVHD) 165–6, 166fhematopoietic stem cell transplants 172–3

Gram-negative bacteria, lipopolysaccharide 5Gram-positive bacteria, peptidoglycans 5granulocyte colony-stimulating factor (G-CSF) 84t

FcgRI regulation 34receptors 83

granulocyte-macrophage colony-stimulatingfactor (GM-CSF) 84t

natural killer cell production 14T-helper cell production 85tumor immunotherapy 182

granulomas 117chronic 161

Graves’ disease (thyrotoxicosis) 156, 190f, 197, 208t

CTLA-4 polymorphisms 192diagnosis 204tgender ratio 193tHLAassociations 173tplacental crossing 198ftherapy 205

growth factors, hematopoiesis 85fGuillain–Barré syndrome 193

Haemophilus influenzaevaccine 132, 132tX-linked agammaglobulinemia 138

haplotypesMHC 46T-cell restriction 54

haptens 50, 51fHashimoto’s thyroiditis 156, 188–9, 202–3, 208t,

209diagnosis 204tHLAassociations 173trelative studies 190–1

hay fever 36, 150HBsAg see hepatitis Bheart transplants 172heat-shock proteins 203heavy chain disease 185heavy chains, antibodies 27, 29, 29f

gene(s) 28gene recombination 31f

helminths see parasitic infectionshelper T-cells see T-helper cellshematopoiesis 85f, 105hematopoietic stem cells 105, 112

transplants 172–3see also bone marrow transplants

hemolytic anemia, autoimmune see autoimmunehemolytic anemia

hemolytic disease of the newborn 155, 155fheparin 149hepatitis A

pooled gammaglobulin 128tvaccine 132t

hepatitis Bpooled gammaglobulin 128, 128tvaccine 130, 130f, 131, 132t

hepatitis C, vaccine 132tHER-2/neu oncogene 178herceptin (anti-HER-2/neu) 184herd immunity, vaccination 128hereditary angioedema 137, 137fherpes simplex virus (HSV), AIDS 143heteroconjugate antibodies 184, 184fhighly active antiretroviral therapy (HAART)

145high-walled endothelium of the postcapillary

venules (HEVs) 63–4, 65fhistamine

allergic reactions 149infection 119

HIV infection 142–5, 146–7antiviral antibodies 145CD4+ cell numbers 144–5characteristics 143immune response evasion 144, 145laboratory diagnosis 145natural history 143f, 144–5, 144f

infection of CD4 cells 143–4opportunistic infections 143reverse transcriptase 144T-helper cell depletion 144–5transmission/infection methods 142, 143–4treatment 145viral structure 143f

HLA(human leukocyte antigen)disease associations 173–4, 173t, 175

atopic allergy 149autoimmune diseases 191–2celiac disease 202Goodpasture’s syndrome 198

insulin-dependent diabetes mellitus 173–4,173t, 192, 192f, 200

rheumatoid arthritis 173t, 174, 192, 200polymorphism and inheritance 169f

homing, lymphocyte circulation 63–4, 66fhoming receptors 64humanization, monoclonal antibodies 37, 38fhuman papilloma virus (HPV) 177, 179human T-cell leukemia virus-1 (HTLV-1) 177humoral immunity

age effects 102cell-mediated immunity vs 128cytokine control 88fdefects 141innate immunity 11–13, 15parasites 123see also antibodies; immunoglobulin(s)

hybridomas 37“hygiene hypothesis” 148hyperacute graft rejection 167, 171hyper IgM syndrome, X-linked 138–9, 138fhypermutation, class switching 45hypersensitivity reactions 148–63, 153f

see also individual typeshypersensitivity reactions, type I 148–54, 153f, 162

inflammatory mediators 149see also atopic allergy

hypersensitivity reactions, type II 154–6, 162anti-receptor autoimmune diseases 156autoimmune types 156complement activation 154drug reactions 156mechanisms 154forgan transplant rejection 155transfusion reactions 154–5

hypersensitivity reactions, type III 153f, 157–60,162–3

Arthus reaction 157–8circulating complexes 158–60

immune complex glomerulonephritis 159–60,159f

serum sickness 158–9complement proteins 157inhaled antigens 158, 158fmechanisms 157f

hypersensitivity reactions, type IV 153f, 160–1, 163atopic allergy vs 150graft rejection 167mechanism 160f

hypervariable regions 27, 29f, 30, 31

idiopathic thrombocytopenic purpura (ITP) 197,208t

idiotype network 97–8, 98f, 103idiotypes 30, 31fIg-a (CD79a) 41Ig-b (CD79b) 41immune complexes

autoimmune diseases 199–200, 199fglomerulonephritis 159–60, 159fhypersensitivity reactions see hypersensitivity

reactions, type III immune response evasion

bacterial capsules 117extracellular bacteria 117–19HIV infection 144, 145intracellular bacteria 119–20, 119ftumors see tumor immunology

immune surveillance 179immune tolerance see toleranceimmunity, passive–acquired 127–8, 128t, 133immunization see vaccinationimmunoblasts 92, 93fimmunodeficiency 24, 135–47

B-lymphoproliferative disorders 141–2corticosteroids 141cytotoxic drugs 141innate immune response 135–7, 146primary 135–41, 146recognition of 141, 146secondary 141–2, 146

lymphoproliferative disorder 185see also individual diseases/disorders

INDEX216

immunogens 50immunoglobulin(s)

class switching see class switching colostrum 127deficiencies 138–9, 142t, 146

see also individual diseases/disordersgene rearrangement 109, 110f, 111

k light chains 30fnonproductive 109

genes 28–9, 38–9, 44TCR vs Igs 42see also individual genes/regions

milk 127neonate levels 111fstructure see antibodies, structure see also antibodies; humoral immunity

immunoglobulin A(IgA) 29, 33t, 34–5, 39deficiency 138in neonate 111fsecretory 35, 36f, 119, 119f, 127

immunoglobulin D (IgD) 29, 33t, 35cell surface expression 41in neonate 111f

immunoglobulin E (IgE) 29, 33t, 35–6atopic allergy 148–9, 149fin neonate 111fparasitic worm infections 124secretory system 119

immunoglobulin G (IgG) 29, 33–4, 33tin neonate 111fplacental transfer 111subclasses 36, 37ttransplacental passage 34, 35f

immunoglobulin M (IgM) 29, 33t, 35cell surface expression 41membrane associated 42fin neonate 111fswitch from membrane to secreted form 42f

immunoglobulin superfamily (IgSF) receptors 83immunopathology 24, 25immunoreceptor tyrosine-based activation motifs

(ITAMs) 76–7, 78f, 79immunosenescence 102, 102f, 102t, 103immunosuppression 101, 101f

fungal infections 124tumor immunology 180

immunosuppressive drugsautoimmune disease therapy 205graft rejection prevention 168, 170, 170fsee also individual drugs

immunotherapy see tumor immunotherapyimmunotoxins 183–4“indirect pathway,” allograft recognition 167inducible nitric oxide synthase (iNOS) 7infections 114–26

autoimmune diseases 193defensins 118–19histamine 119mast cells 119type IV hypersensitivity reactions 161

inflammation 114–17, 125allergy therapy 153cell types 115chemokines 114, 115cytokines 115ICAM-1 114integrins 114interleukins 115, 116leukocyte migration/diapedesis 114–15, 115f

age effects 102cytokines 11macrophages 10–11, 12regulation 91, 94

local 9, 10Mac-1 114mediators 114

type I hypersensitivity 149RANTES 115–16regulation/resolution 116–17selectins 114, 115

inflammation, acute 125classical complement pathway 18complement 10–11, 11f, 15

immunoglobulin E 36initiation 114–15

inflammation, chronic 88–90, 117early events 88macrophage activation 90, 121f

Infliximab (anti-tumor necrosis factor antibody)202, 207

influenza vaccine 132tinnate immunity 1–16

acquired immunity vs 25fcell types 4f

see also individual typescomplement see complement cytokine networks 85fexternal barriers 1, 3, 14–15humoral mechanisms 11–13, 15

see also individual mechanismsimmunodeficiency 135–7, 146interferons 85finterleukins 85fTh1/Th2 cell balance 86TNF 85ftumors see tumor immunology viral infections 120–1see also individual systems

insect bites 161insulin-dependent diabetes mellitus (IDDM) 200,

208–9CTLA-4 polymorphism 196diagnosis 204tHLAassociations 173–4, 173t, 192, 200therapy 205twin studies 192

integrins 64inflammation 114lymphocyte circulation 65

intercellular adhesion molecule-1 (ICAM-1)antigen presentation to T-cells 75inflammation 10, 114

intercellular adhesion molecules (ICAMs)65–6

interdigitating dendritic cells (IDCs) 71migration/maturation 72fthymus 106

interferon(s) 84finnate immunity 11, 85flymphocyte development 85freceptors 83see also individual types

interferon a (IFNa) 84tbiological functions 120–1innate immunity 11tumor immunotherapy 181

interferon b (IFNb) 84tbiological functions 120–1tumor immunotherapy 181

interferon g (IFNg) 22–3, 84tdeficiency 136FcgRI regulation 34innate immunity 11intracellular bacteria 120macrophage activation 90, 121fnatural killer cell production 14NK-T-cell secretion 58T-helper cell production 85viral infection control 123

interleukin(s) 84freceptors 82–3see also individual types

interleukin-1 (IL-1) 12, 84f, 91inflammation 115natural killer cell production 14

interleukin-1 (IL-1) receptor antagonists 207interleukin-2 (IL-2) 84f

activation-induced cell death 99defects 139T-cell activation 77T-cell expression 82, 85T-cell proliferation 86–7, 87ftumor immunotherapy 181, 182

interleukin-2 receptor (IL-2R) 170, 171, 175interleukin-3 (IL-3) 84f

T-helper cell production 85

interleukin-4 (IL-4) 84fFcgRI regulation 34fetal production 174NK-T-cell secretion 58parasitic worm infections 124T-helper cell production 85, 86

interleukin-5 (IL-5) 84fin allergic reactions 150T-helper cell production 85

interleukin-6 (IL-6) 12, 84fsecretion by Th2 cells 85

interleukin-8 (IL-8) 84tinflammation 115

interleukin-10 (IL-10) 84tas anti-inflammatory cytokine 91fetal production 174immunosuppressive effect 100inflammation 116Th2 cell secretion 85

interleukin-12 (IL-12) 84tdeficiency 136Th1 development 86

interleukin-13 (IL-13)FcgRI regulation 34parasitic worm infections 124Th2 cell secretion 85

intestinal intraepithelial lymphocytes 70intestinal lymphocytes 70intracellular antigens 54–5, 56f, 60intracellular pathogens 22

bacteria 119–20, 119f, 120f, 125interferon-g 120macrophage killing 23f

invariant chain 55, 56f, 57isografts 164isohemagglutinins 155isotypes 29–30, 31fITAM (immunoreceptor tyrosine-based activation

motifs) 76–7, 78f, 79

Janus kinase (JAK)-STAT signaling pathway83

JAK-3 mutations 140J chain 34–5Jenner, Edward 22, 129Jerne, Niels 97–8joining (J) genes, immunoglobulins 28

Kaposi’s sarcoma, AIDS 143k light chains 28, 29–30

gene recombination 30fkidney transplants 171–2, 171fkilled vaccines 129, 133Köhler, Georges 37Kupffer cells 3

lactoferrinneutrophil granules 3phagocytosis 7

l light chains 28, 29–30Lambert–Eton syndrome 208tLangerhans’ cells 71latex allergy 151–2Lck tyrosine kinase 76–7, 78fLeishmania 22

cell-mediated immunity 123Th1/Th2 cell balance 86

leprosy 161leukemia 185f

acute myelogenous 178leukemia inhibitory factor (LIF) 84tleukocyte adhesion deficiency (LAD) 136leukocytes

chemokine specificities 116tmigration/diapedesis 114–15, 115f, 116

leukotrienesallergies 149antagonists 152

light chains, antibodies 27, 28, 29, 29flinkage disequilibrium 173lipocortin-1 116–17lipopolysaccharide (LPS) 5

CD14 binding 117

INDEX 217

macrophage binding 12Toll-like receptor binding 5, 5f, 117

Listeria 119live attenuated vaccines 129–30, 130f, 133liver transplants 172LMP2 55, 56fLMP7 55, 56flocal inflammation see inflammationL-selectin 114lung surfactant proteins 11–12lung transplants 172lymph nodes 66–8, 73

B-cell areas 66, 67fmemory B-cell development 67fplasma cell development 67fstructure 67fT-cell areas 67f, 68

lymphoblasts 20flymphocyte(s) 20f

activation 75–81cytokine networks 85f

chemokine specificities 116tcirculation 63–6, 65f, 66f, 73

homing 63–4, 64, 66fintegrins 65intercellular adhesion molecules 65–6lymphocyte function-associated antigen-1 65mucosa-associated lymphoid tissue 71f

development 85fgraft rejection mechanisms 167intestinal 70see also B-cell(s); T-cell(s)

lymphocyte function-associated antigen-1 (LFA-1)antigen presentation to T-cells 75inflammation 114lymphocyte circulation 65

lymphocyte function-associated antigen-3 (LFA-3)75

lymphoid neoplasia 187cellular phenotype 179fdiagnosis 184

lymphoid tissue 62–74cell populations 63functional organization 64frequirement for 62–3see also individual types

lymphoma diagnosis 185flymphopenia, autoantibodies 208tlymphoproliferative disorders, secondary

immunodeficiency 185lymphotoxin 84t

Th1 cell secretion 85lysozyme

innate immunity 11neutrophil granules 3phagocytosis 7

Mac-1, inflammation 114macrophage(s) 4f

activation 90antigen presentation 71asthma 150complement receptors 117–18inflammation 10–11, 12, 121fintracellular pathogen killing 23f, 120, 120fphagocytosis 3in spleen 69tissue types 3tumor immunology 179–80

macrophage colony-stimulating factor (M-CSF)84t

MAGE gene 178major basic protein 150malaria 123malnutrition 103mannose-binding lectin (MBL) 12, 117Mantoux reactions 161maple bark stripper’s disease 158marginal zone, spleen 68fMASP proteins 8–9, 117

deficiencies 136mast cells 4f, 10f

allergy see atopic allergy

mast cells (cont.)anaphylatoxin production 9asthma 150FceRI 36infection 119inflammation 115ontogeny 106fstabilization 152

maternally acquired antibody, passive–acquiredimmunity 127

M-cells 70, 71f, 72measles

pooled gammaglobulin 128ttype IV hypersensitivity reactions 161

measles–mumps–rubella (MMR) vaccine 132, 132tadults 133

MECL-1 55, 56fMedawar, Peter 165medullary cords 67fmedullary sinuses 67fmembrane attack complex (MAC) 9, 9f, 18memory 19–21, 24, 93, 94memory B-cells 94

development 66, 67fgerminal centers 92, 93f

memory T-cells 88, 94Metchnikoff, Elie 2methotrexate 170f, 205MHC (major histocompatibility complex) 22, 45–8,

49functions 48gene map 46, 48fgenetic polymorphism 46, 48graft rejection 165–6, 175haplotype 46tissue distribution 48

MHC class Iantigen presentation 54–5, 56f, 57f, 58, 60cytotoxic T-cell recognition 90expression 13MHC class II vs 46placental lack 174structure 45–6, 47ftumor immunology 178, 180

MHC class IIantigen presentation 55, 56f, 57, 57f, 58, 60invariant chain 55, 56f, 57MHC class I vs 46placental lack 174structure 46, 47fT-helper cell recognition 53tissue expression 105–6

MHC class II enriched compartment (MIIC),antigen processing 56f

MHC class III 46MHC restriction 54, 57, 76fmicroarrays, antigen detection 54b2-microglobulin 46

antigen processing 56fCD1 association 57–8

milk, immunoglobulins 127Milstein, César 37mitochondrial pathway, apoptosis 98, 99, 99fmitogen-activated protein kinase (MAPK)

pathway 77, 78fmixed lymphocyte reactions (MLR) 165mizorbine 170fmolecular mimicry, autoimmune diseases 193

atherosclerosis 203Hashimoto’s thyroiditis 202multiple sclerosis 200organisms 193

monoclonal antibodies 27, 37, 38f, 39monocytes 4f

allergic reactions 149–50chemokine specificities 116tontogeny 106f

monomeric immunoglobulin M (IgM) 35mononuclear phagocyte system see macrophage(s)mucosa-associated lymphoid tissue (MALT)

69–70, 69f, 73lymphocyte circulation 71fM-cells 72

multiple myeloma 184–5paraproteins 186f

multiple sclerosis (MS) 200, 209CTLA-4 polymorphisms 192HLAassociations 173tmolecular mimicry 200

myasthenia gravis 156, 197–8, 208tdiagnosis 204tgender ratio 193tHLAassociations 173t

Mycobacterium 22Mycobacterium tuberculosis

attenuated strains 130Th1/Th2 cell balance 86

mycophenolic acid 170fmyeloma, multiple see multiple myelomamyeloma proteins 27myeloperoxidase 3myxedema, primary see primary myxedema

NADPH oxidase 6–7narcolepsy, HLAassociations 173t“natural” antibodies 35natural killer (NK) cells 13–14, 15

activation 121age effects 102antibody-dependent cellular cytotoxicity 23,

23f, 46fantigen recognition 45, 46fapoptosis induction 12, 13–14, 13fchemokine specificities 116tcytokine production 13cytotoxic T-cells vs 91Fcg receptors 23graft rejection 168finflammation 115ontogeny 106f, 111perforin 12tumor immunology 180

negative selection, T-cell development 108–9, 108f,112

Neisseria infections, complement deficiency 137neonatal immune responses 111, 111f, 112neonatal lupus erythematosus 198nephelometry 53, 54neutral proteases 149neutrophils 3f, 4f

anaphylatoxin effects 9chemokine specificities 116tchronic granulomatous disease 136fcomplement receptors 117–18defects 135extravasation 10granules 3inflammation 10, 115ontogeny 106fphagocytosis 3

NFAT 77NFkB 77NFkB transcription pathway 4, 5fnickel 161nitric oxide, phagocytosis 7NK-T-cells 58non-Hodgkin’s lymphoma, diagnosis 184nonproductive immunoglobulin gene

rearrangement 109nucleotide insertion (N-region diversity) 42, 44“nurse” cells 105

OKT-3 (anti-CD3 monoclonal antibody) 170–1oncofetal antigens 177oncogenic DNAviruses 177oncostatin M 84tontogeny 105–13, 106f

see also individual cellsopportunistic infections, AIDS 143opsonization 117–18, 118f

complement-mediated 9, 117–18immunoglobulin G 34mannose-binding lectin 117

oral immunization 130organ transplants see transplantsovarian cancers, gene mutations 178

INDEX218

p53 gene, mutations 178pancreatic carcinomas 178papain, antibody digestion 27, 28fparaproteins, multiple myeloma 186fparasitic infections 123–4, 126

eosinophils 14helminths 124, 125, 125f

paratopes 50, 51fparoxysmal nocturnal hemoglobinuria 137passive–acquired immunity 127–8, 128t, 133pathogen-associated molecular patterns (PAMPs)

4pattern recognition receptors (PRRs) 4

dendritic cells 71NFkB transcription pathway 4, 5f

pemphigus vulgaris 199diagnosis 204t

pepsin, antibody digestion 27, 28fpeptidoglycans 5perforin 12periarteriolar lymphoid sheath 68fperipheral tolerance, T-cell development 109pernicious anemia 199, 208t

diagnosis 204ttherapy 205

pertussis vaccine, subunit acellular 131Peyer’s patches 69f, 70, 72

M-cells 70, 71fstructure 64

phagocytosis 3–7defects 135–6, 142t, 146

see also individual diseases/disordersgranules 7history 2macrophages see macrophage(s) mechanism of action 5–6, 6fpathogen recognition 4reactive oxygen intermediates 6–7, 7f

phagosomes 5–6phosphatidylinositol pathway 77, 78fphosphodiesterase (PDE) inhibitors, allergy

therapy 152–3phospholipase C 77, 78fpigeon-fancier’s disease 158placental transfer, immunoglobulin G 34, 35f,

111“plantibodies” 38plasmablast development 66, 69plasma cells 20f

development 19, 66germinal centers 92, 93fin lymph nodes 67fspleen 69

dyscrasias 184–5plasmapheresis, autoimmune disease therapy

205–6plasmodia 3f, 124platelet activating factor (PAF) 149platelets 106fPneumocystis carinii

AIDS 143severe combined immunodeficiency 140X-linked hyper IgM syndrome 139

poison ivy 161poliomyelitis vaccine 129, 132tpolymorphonuclear neutrophil see neutrophilspolymyositis, gender ratio 193tpooled gammaglobulin 127–8, 128tpositive selection, T-cell development 107–8, 108f,

112Prankulast 152precipitation reactions, antibody detection 53primary biliary cirrhosis 189

diagnosis 204tprimary follicles, lymph nodes 66primary immunodeficiency 135–41, 142t, 146primary myxedema 173t, 208t

diagnosis 204ttherapy 205

primary response 20, 22f, 94fprivileged sites 70–1

autoimmune diseases 194transplants 170

proinflammatory cytokines, autoimmune disease196

properdin 8prophylaxis 127–34

passive–acquired 127–8, 128t, 133vaccination see vaccination

prostaglandin D2 149prostaglandin E2 116prostate-specific antigen (PSA) 178proteases, neutral 149proteasomes 54–5, 55fprotein kinase C 77, 78fP-selectin 10“pseudo light chain” expression 109psoriasis 203–4, 209–10purine nucleoside phosphorylase (PNP) deficiency

140pyogenic bacterial infections, X-linked

agammaglobulinemia 138

rabiespooled gammaglobulin 128, 128tvaccine 131, 132t

radioimmunoconjugates 183–4RAG genes

mutations 140receptor editing 44VDJ recombination 29

RANTESin allergic reactions 150inflammation 115–16

rapamycin (Sirolimus) 170, 170fras gene mutations 178Ras–MAP kinase signaling pathway 83reactive oxygen intermediates (ROIs) 6–7, 7freceptor editing, RAG genes 44recombinant DNA 130recombination signal sequences (RSS) 29red pulp, spleen 68–9, 68fregulatory T-cells 100

in autoimmune disease 196Reiter’s disease 173treproductive immunology 174, 175respiratory syncytial virus (RSV) 128treverse transcriptase 144Rhesus (Rh) blood groups 155rheumatic fever 193, 198–9rheumatoid arthritis (RA) 200, 201f, 202, 209

CTLA-4 polymorphisms 192diagnosis 204tgender ratio 193tHLAassociations 173t, 174, 192, 200pathogenesis 200, 201ftherapy 202, 205

rheumatoid factor 200, 202rolling, lymphocytes 65f

saliva, innate immunity 1Salk polio vaccine 129salmeterol 152Salmonella, use in vaccines 130schistosomiasis 24, 126, 163scleroderma

diagnosis 204tgender ratio 193t

sebaceous secretions, innate immunity 1secondary antibody responses 20–1, 22f, 94fsecondary follicles, lymph nodes 66secondary immunodeficiency 141–2, 146second set graft rejection 164secretory component of IgA 35, 36fselectin(s) 10, 64, 114, 115self/nonself discrimination 21–2

see also autoimmune diseases; toleranceserum amyloid P 12serum sickness 127

type III hypersensitivity reactions 158–9severe combined immunodeficiency (SCID) 42,

138f, 140–1, 140f, 142t, 146sex hormones, immune system regulation 102signaling pathways

cytokine receptors 83T-cell activation see T-cell(s), activation

Sirolimus (rapamycin) 170, 170fSjögren’s syndrome

diagnosis 204tgender ratio 193tHLAassociations 173t

skin, innate immunity 1skin tests, atopic allergy 149smallpox

eradication through vaccination 129type IV hypersensitivity reactions 161

sodium cromoglycate 152somatic hypermutation 44–5

germinal centers 92spleen 68–9, 68f, 73Src family kinases 79staphylococcal enterotoxins 58Staphylococcus aureus

chronic granulomatous disease 135superantigens 58X-linked agammaglobulinemia 138

steel locus factor (SLF) 84tStreptococcus

molecular mimicry 193X-linked agammaglobulinemia 138

stress, immunosuppression 101subunit vaccines see vaccinessuperantigens 58–9, 60

TCR interactions 58, 59fsuppressor T-cells 100f, 101sweat, innate immunity 1Syk kinase, B-cell activation 79systemic lupus erythematosus (SLE) 160, 189,

199–200CTLA-4 polymorphisms 192diagnosis 204tFcgR polymorphisms 192gender ratio 193timmune complexes 159–60, 199ftherapy 205twin studies 192–3

Tacrolimus see FK506 (Tacrolimus)T-cell(s) 22–4, 24–5

activation 75–9, 76f, 78f, 81CD40/CD40L interaction 75–6cell surface markers 75, 78fgene expression 77, 82, 83f, 94regulation 77, 79signal transduction 76–7, 77f, 78f, 81TCR–CD3 complex 75, 78f

age effects 102anergy 75, 109, 180antigen presentation to 71, 75, 80

see also T-cell(s), activation antigen recognition 54, 60autoimmune diseases 196–7B-cells vs 75, 76t, 80CD3 complex 75cytokine production 82cytotoxic see cytotoxic T-cellsdefects/deficiency 101, 139, 142t, 146development 22, 24, 63, 105–9, 108f, 112

double-negative cells 107, 108fdouble-positive cells 107–8, 108fnegative selection 108–9, 108f, 112peripheral tolerance 109positive selection 107–8, 108f, 112self-reactive apoptosis 109single-positive cells 107, 108fsurface marker changes 106–7TCR rearrangement 107, 112tolerance development 109, 112

double-negative T-cells 107, 108fdouble-positive T-cells 107–8, 108fenumeration 141gd 58, 60, 122graft rejection 167helper see Th1 cells; Th2 cells; T-helper cellsimmunoglobulin class switching stimulus

92–3leukemias/lymphomas 178, 179flymph nodes 67f, 68membrane receptors see TCRs

INDEX 219

MHC restriction 54, 76fnatural killer 58ontogeny 106fproliferation 86–7, 87f, 94purging, bone marrow transplants 172, 184regulation 98–100, 103

activation-induced cell death 98–9CTLA-4 expression 99–100

single-positive 107, 108fspleen 68f, 69vaccination, autoimmune disease therapy 206see also specific types

T-cell receptors (TCRs) 22, 41–3, 48ab 42, 43fCD3 complex 42–3, 44f

T-cell activation 75, 78fCDR3 region 46diversity calculations 45tgd 42, 43f, 58gene structure 42, 43fMHC-peptide recognition 53nucleotide insertion/deletion 42rearrangement 107, 112structure 41–2, 44fsuperantigen interactions 58, 59fsee also antigen membrane receptors

tears, innate immunity 1terminal deoxynucleotidyl transferase (TdT) 42,

106–7tetanus

pooled gammaglobulin 128tvaccine 132t, 133

Th1 cells 85, 94development 86production 87f

Th1/Th2 cell balance 85–6autoimmune diseases 196dendritic cells 85innate immunity 86parasitic infections 123–4

Th2 cells 85, 94atopic allergy 148–9development 86parasitic worm infections 124production 87f

T-helper cells 22–3autoimmune diseases 194–5B-cell activation 79, 80fcytokine secretion 22, 85cytotoxic T-cell activation 90, 90fdepletion, HIV infection 144–5graft rejection 167memory 94MHC II recognition 53viral infection control 122see also Th1 cells; Th2 cells

theophylline 152thrombocytopenia, autoimmune see autoimmune

thrombocytopeniathrombocytopenic purpura, drug-related 156thymus-dependent antigens 79, 80f, 92fthymus gland 22, 112

interdigitating dendritic cells 106MHC class II expression 105–6“nurse” cells 105structure 105–6, 107f

thymus-independent antigens 79, 79f, 92tissue matching, bone marrow grafts 168tissue-specific differentiation antigens, tumor

antigens 178TNF see tumor necrosis factortolerance 21

T-cell development 109, 112tolerogens 206–7Toll-like receptors (TLRs)

dendritic cells 71lipopolysaccharide binding 5, 5f, 117pathogen-associated molecular pattern

recognition 5signal transduction 5f

tonsils 69toxin neutralization 117toxoids, subunit vaccines 131, 131f

Toxoplasma gondiiAIDS 143cell-mediated immunity 123

transforming growth factor-b (TGF-b) 84tfetal production 174immunosuppressive effect 100inflammation regulation 116natural killer cell production 14privileged sites 70–1

transfusion reactions 154–5transgenic plants 131transmigration, lymphocytes 65–6transplant patients, vaccination 133, 134transplants 171–3

heart 172hematopoietic stem cells 172–3kidney 171–2, 171fliver 172lung 172privileged sites 170rejection see graft rejection types 164, 171

transporters associated with antigen processing(TAP) 55, 56f

mutations 141Trichinella spiralis 123Trypanosoma

cell-mediated immunity 123molecular mimicry 193

tryptase 149tuberculosis 161tumor immunology 177–87

acquired immunity 179cell-surface changes 177–8, 178f, 181t, 186cytotoxic T-cells 179, 179fFas/FasL apoptosis 179immune response evasion 180, 180f, 186immune surveillance 179immunotherapy see tumor immunotherapy innate immunity 179–80

tumor immunotherapy 180–2, 186–7B-cell lymphomas 182cell-mediated 181–2

cytokines 180–1dendritic cell vaccination 182, 183fmonoclonal antibody therapy 183–4subunit vaccines 182tumor immunogenicity 182, 183fviral antigen immunization 181whole tumor cell immunization 181–2

tumor necrosis factor (TNF) 84freceptors 83

tumor necrosis factor (TNF) 12, 84tinflammation 115innate immune response 85fnatural killer cell production 14receptor–IgG fusion protein see Etanercept tumor immunotherapy 181viral infection control 122–3

twin studies, autoimmune diseases 192–3type I diabetes mellitus see insulin-dependent

diabetes mellitus (IDDM)typhoid vaccine 129, 132t

umbilical cord blood, hematopoietic stem celltransplants 172

urine, innate immunity 1

vaccination 22, 128–33in adults 133, 134background 22fcell-mediated vs humoral immunity 128history 22immune response sites 128, 129fstrategic considerations 128–9transplant patients 133, 134tumor immunotherapy 181–2

vaccinesadjuvants 132, 134current 132–3, 132t, 134killed 129, 133live attenuated 129–30, 130f, 133subunit 131–2, 133–4

modified toxoids 131, 131ftumor immunotherapy 182

van der Waal forces 50, 52

INDEX220

variable regions, antibodies 27, 28, 29f, 30–1, 32f,45t

varicella zosterpooled gammaglobulin 128tvaccine 132t

vascular addressins 64VCAM-1, leukocyte migration 116VDJ recombination 29, 43venoms, pooled gammaglobulin 128tviral infections 90, 120–3, 122f, 125–6

antibodies 121, 122fcell-mediated immunity 121–3, 122fcytokines 122–3

interferons 120–1immunodeficiency 141innate immune response 120–1tumor antigens 177

VLA-4 (very late antigen-4) 116

Waldenström’s macroglobulinemia 185Wegener’s granulomatosis 197, 208t

diagnosis 204twheal and flare reactions 152white pulp, spleen 68fWiskott–Aldrich syndrome 141, 179

loci 138f

xenografts 164, 171, 175X-linked agammaglobulinemia (XLA) 138, 138fX-linked hyper IgM syndrome 138–9, 138fX-linked immunodeficiency diseases 138fX-linked lymphoproliferative syndrome 138f, 141X-linked severe combined immunodeficiency (X-

SCID) 140X-rays, immunodeficiency 141

yellow fever vaccine 132tYersinia enterocolitica, molecular mimicry 193yolk sac, hematopoiesis 105

ZAP-70 77, 78fdefects 139

Zinkernagel, Rolf 53

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Imunologie medicala