Cellular Basis of Innate/Adaptive Immunity

Dr SMH Ghaderian

Association professor of Medical Genetics

The Immune System. A schematic representation of the separation of the immune system into its innate and adaptive

components in which key processes are isolated and identified.

The “danger” theory. Diagrammatic representation of the principles of the “danger” theory of innate immune activation

highlighting that the activation of the innate immune response is dependent on the threat presented by a molecule and not on

whether it is exogenously or endogenously derived.

The immune system has four main functional areas. The immune responses are provided via four main functional areas:

barrier functions; immune tissues; immune cells; and immune-functioning proteins and peptides.

Introduction

The phagocytes in the blood and tissues originate in bone marrow from a common

committed stem cell and acquire unique functions as they differentiate.

All of these motile cells are attracted to sites of infection or inflammation by (in

humans) a family of about 40 small proteins called chemokines.

Tissue cells and leukocytes secrete chemokines at sites of infection or inflammation.

Chemokines with many unrelated names (IL-8, RANTES, eotaxin, MCP-1, etc.) have

similar structures and bind to a family of 14 different chemokine receptors expressed

selectively by lymphocytes, monocytes, and granulocytes.

These seven-helix receptors are coupled to trimeric G-proteins that mediate

chemotaxis toward the source of the chemokine.

Hematopoietic stem cells in the bone marrow give rise to all blood cells and to different tissue cells with immune system

functions.

THE MOLECULAR MECHANISM FOR PHAGOCYTOSIS OF A BACTERIUM BY A MACROPHAGE. Macrophage surface receptors are activated by contact with a bacterium; this triggers actin rearrangements that lead to protrusion of the plasma membrane to engulf the bacterium. The actin filaments encasing the newly formed phagosome depolymerize, and membrane traffic to and from the phagosome leads to its maturation. Hydrolytic enzymes are delivered to the mature phagosome through fusion with primary and secondary lysosomes, and the bacterium is degraded.

SPHINGOMYELIN/CERAMIDE SIGNALING PATHWAY. Stimulation of the tumor necrosis factor (TNF) receptor activates a neutral sphingomyelinase, which cleaves choline from sphingomyelin. Ceramide flips across the bilayer and activates a cytoplasmic kinase as well as PKC-ζ and a protein phosphatase.

• In response to infection, lymphocytes of the immune systems of vertebrates produce

two kinds of adaptive responses: humoral (in the body fluids) and cellular.

• B lymphocytes produce the humoral response by secreting antibodies

(immunoglobulins), soluble proteins that diffuse in the blood and tissue fluids.

• Two types of T lymphocytes mediate the cellular arm of the adaptive immune response.

Cytotoxic T lymphocytes (killer T cells) destroy cells infected with viruses, whereas

helper T cells regulate other lymphocytes.

• These responses protect against infection but fail in acquired immunodeficiency

syndrome (AIDS) when the human immunodeficiency virus (HIV) kills helper T cells.

• A blood smear reveals lymphocytes of various sizes and shapes but not their

remarkable heterogeneity at the molecular level.

Cellular Basis of Adaptive Immunity

BLOOD CELLS. A, Light micrograph of a dried blood smear prepared with Wright's stain. B, Family tree of blood cells showing the developmental relationships of the various lineages. Looping-back arrows indicate renewal of the cell type. Forward-oriented arrows indicate differentiation and proliferation. RBC, red blood cell. (A, Courtesy of J.-P. Revel, California Institute of Technology, Pasadena.)

ACTIVATION AND ADAPTATION OF A SEVEN-HELIX RECEPTOR. A, Ligand binding shifts the equilibrium from the resting conformation toward the active conformation. B, The active receptor promotes dissociation of guanosine diphosphate from the α-subunit of multiple trimeric G-proteins, allowing GTP to bind. Typically, this dissociates Gα from Gβγ, each of which activates downstream effectors that produce, for example, the second messengers cAMP and diacylglycerol (DAG). cAMP and DAG activate PKA and PKC, which phosphorylate active receptors on their C-terminus. C, This attracts arrestin, putting the receptor into the inactive adapted state. PKA, protein kinase A; PKC, protein kinase C.

Chemotaxis of a Dictyostelium amoeba toward cAMP. A, Live cell attracted to cAMP (gold) released from a micropipette. A time series of differential interference micrographs shows the rapid formation of a new pseudopod and reorientation of the direction of movement when the position of the micropipette is moved at the 60-second time point. B, Cells have a uniform distribution of cAMP receptors (yellow and red dots) over their surface. A shallow gradient of cAMP activates these seven-helix receptors (red), which activate a trimeric G-protein and phosphatidylinositol-3 kinase, an enzyme that rapidly converts PIP2 to PIP3. On a slower time scale, the active G-protein activates PTEN, a PIP3 phosphatase, throughout the cell. The combination of these two signals creates a steep gradient of PIP3 across the cell. C, Fluorescence micrograph of a cell exposed to a point source of cAMP (yellow). A GFP-PH domain fusion protein inside the cell binds to PIP3 (green) on the inside of the plasma membrane, revealing the steep gradient of PIP3. (A, Courtesy of Susan Lee and Richard Firtel, University of California, San Diego. B, Redrawn from a sketch by Pablo Iglesias, Johns Hopkins University, Baltimore, Maryland. C, Courtesy of Pablo Iglesias, Johns Hopkins University, Baltimore, Maryland. Reference: Janetopoulos C, Ma L, Devreotes PN, Iglesias PA: Chemoattractant-induced phosphatidylinositol 3,4,5 trisphosphate accumulation is spatially amplified and adapts, independent of the actin cytoskeleton. Proc Natl Acad Sci U S A 101:8951-8956, 2004.)

Neutrophils

Neutrophils, also known as polymorphonuclear leukocytes or "polys," are the main

phagocytes circulating in blood on their way to connective tissues.

They are distinguished by a multilobed nucleus and two types of granules.

The more abundant specific granules contain lysozyme (an enzyme that digests

bacterial cell walls) and alkaline phosphatase.

These granules do not stain with either the basic or acidic dyes used for blood

smears, so these cells are called neutrophils.

Azurophilic granules are true lysosomes containing hydrolytic enzymes bound to

acidic proteoglycans.

Neutrophils have copious glycogen but few mitochondria, so they rely on glycolysis for

ATP synthesis in poorly oxygenated wounds.

They are among the most motile cells in the body.

Human bone marrow produces about 80 g of neutrophils each day.

In response to infection or injury, a circulating factor releases neutrophils from the

bone marrow into the blood.

Neutrophils spend about 10 hours in blood, alternating between a circulating pool and

a socalled marginated pool adherent to endothelial cells, chiefly in the lung.

Exercise and epinephrine release marginated neutrophils into the circulating pool;

smoking increases the marginated pool.

Neutrophils leave the blood by receptor-mediated attachment to endothelial cells and

then crawl between endothelial cells into the connective tissue, where they perish after a

day or two of phagocytosis.

Neutrophils are humans' first line of defense against bacterial infection, as they are

highly specialized for finding and destroying bacteria.

Provided that the concentration of neutrophils is high enough (about 10 million cells

per milliliter), these motile phagocytes can find and destroy bacteria faster than the

invaders can reproduce.

Bacterial products, especially N-formylated peptides, attract neutrophils by binding

plasma membrane receptors and stimulating locomotion, similar to chemotaxis by other

cells.

Neutrophils bind and ingest bacteria by phagocytosis.

Both types of granules fuse with phagosomes, delivering antibacterial proteins and

proteolytic enzymes that kill the ingested bacteria.

Some granules fuse with the plasma membrane, releasing antibacterial proteins

outside the cell.

Phagosome membranes produce millimolar concentrations of superoxide (O2-)

radicals and other reactive oxygen species that help to disperse the granule enzymes

and contribute to killing bacteria.

These toxic oxygen species may also cause collateral damage to the neutrophil.

Genetic defects in the enzymes that produce superoxide cause chronic

granulomatous disease, a serious human disease, because neutrophils cannot kill

ingested bacteria and fungi.

Neutrophils and their role in the innate immune response. Overview of the major morphological features of neutrophils, their

mechanisms of killing, and some of the contents of the three major types of cytoplasmic granules they possess.

SORTING PATHWAYS USED BY MANNOSE 6-PHOSPHATE RECEPTORS AND COAT ASSEMBLY AT THE TRANS-GOLGI NETWORK. A, MPRs carry newly synthesized lysosomal hydrolases containing mannose-6-phosphate (M6P) from the TGN, via endosomes, to lysosomes, after which they return to the TGN. Receptors missorted to the cell surface are recovered by endocytosis and returned to the pathway in endosomes. B, Coordination of coat assembly and cargo recruitment at the TGN. An exchange factor activates the small GTPase Arf to bind GTP, which triggers recruitment of AP1 coat constituents to the TGN membrane. The MPR is concentrated in the emerging coated vesicle through interactions between a tyrosine-based sorting motif in its cytoplasmic domain and the μ-subunit of AP1.

FIVE STEPS IN THE MIGRATION OF A NEUTROPHIL FROM THE BLOOD TO THE CONNECTIVE TISSUE. Endothelial cells exposed to inflammatory agents like histamine expose selectins on their surface and snare mucins on neutrophils flowing in the bloodstream (1). As a neutrophil rolls along the surface (2), chemotactic factors activate their integrins (3), causing the neutrophil to bind tightly to Ig-CAMs on the endothelium (4). The neutrophil then migrates between the endothelial cells into the connective tissue (5). (Reference: Springer T: Traffic signals for lymphocyte and leukocyte emigration: The multi-step paradigm. Cell 76:301-314, 1994.)

TOLL-LIKE RECEPTORS. Most TLRs are homodimers or heterodimers, but this figure shows a ribbon diagram of a single receptor molecule assembled from crystal structures of different receptors. Ribbon diagram of the extracellular domain of TLR3, an endosomal TLR consisting of 23 leucine-rich repeats that binds double-stranded RNAs released from viruses. A transmembrane helix connects to the cytoplasmic TIR domain from TLR2, a receptor for bacterial lipoproteins. Ligand binding to receptor dimers initiates a signal that is transmitted through adapter proteins to kinases, which activate cytoplasmic transcription factors including NF-κB. NF-κB moves to the nucleus and stimulates expression of TNF and other inflammatory mediators.

Monocytes and Macrophages

o Monocytes in the blood are precursors of tissue macrophages.

o Monocytes are large cells with an indented nucleus and a small number of azurophilic

granules.

o After circulating in the blood for about three days, monocytes enter tissues and

differentiate into macrophages under the influence of local growth factors, including

lymphokines secreted by lymphocytes.

o They enlarge and amplify their machinery for locomotion, phagocytosis, and killing

microorganisms and tumor cells.

o Macrophages are professional phagocytes, which generally follow neutrophils to

wounds or infections to clean up debris, including cellular debris and foreign material.

o Plasma membrane receptors for antibodies allow macrophages to recognize foreign

matter marked with antibodies and to facilitate its ingestion.

o Primary lysosomes fuse with phagosomes to degrade the contents.

o Eventually, the cytoplasm fills with residual bodies containing the remains of ingested

material.

oThese "professional phagocytes" may divide and survive for months in tissues, where

they ingest foreign material, participate in immune responses, and secrete growth

factors that influence other cells.

o When confronted with large foreign bodies, macrophages can fuse together to form

giant cells. No challenge is too great.

o Giant multinucleated microphages will even try to ingest a Petri dish if it is coated with

antibody.

o Local growth factors in bone stimulate monocytes to fuse and differentiate into

multinucleated osteoclasts that degrade bone matrix during bone remodeling.

o Macrophages participate in the immune response by degrading ingested protein

antigens and presenting fragments on their surface bound to MHC class II proteins.

o This complex activates helper T lymphocytes carrying the appropriate T-cell receptors.

o Activated T cells proliferate and secrete growth factors that stimulate B lymphocytes to

produce antibody.

o Macrophages also secrete a variety of factors involved with host defense and

inflammation.

o Interleukin-1, transforming growth factor-α, transforming growth factor-β, and platelet-

derived growth factor stimulate the proliferation and differentiation of the cells required to

heal wounds.

o Chemokines attract cells of the immune system to sites of inflammation.

Natural killer (NK) cellsNK cells, a class of giant, granular, cytotoxic lymphocyte (but lacking antigen- specific

receptors), have an effector function that is broadly similar to that of cytotoxic T

lymphocytes (CTLs) in the adaptive immune system: to induce apoptosis in virus-

infected cells and other damaged or abnormal body cells, such as tumor cells.

In the former case, recall that extracellular viruses can be coated by complement C3b

and then targeted for destruction by macrophages, but viruses remaining inside cells

are not visible to the complement system.

However, once viruses have been detected inside body cells, NK cells are recruited

to induce the virus-infected cells to undergo apoptosis.

Both intrinsic and extrinsic apoptosis pathways are used by NK cells.

In the former case, NK cells bind to diseased cells, and via exocytosis release the

contents of their secretory granules (perforins and granzymes) into the intercellular

space.

The perforins insert into the membrane of the target cell in a way that creates pores in

the membrane to allow the pro-apoptotic granzymes to enter the target.

For the extrinsic pathway, Fas ligands on NK cell membranes activate Fas receptors

on the surface of target cells to initiate apoptosis.

NK cells can induce apoptosis by secreting perforins and granzymes close to the surface of target cells. (A) Cytoplasmic

secretory granules, containing perforin and granzyme molecules, are transported via the microtubule network toward the NK

cell membrane at a point close to the target cell. (B) The secretory granules fuse with the NK cell plasma membrane

(exocytosis) and released perforins form large transmembrane pores in the target cell membrane, enabling the diffusion of

granzymes into the cytosol of the target cell. The granzymes then initiate intrinsic apoptosis pathways by cleaving

procaspases or by cleaving the proapoptotic BID protein to activate the mitochondrial apoptosis pathway

How immune system phagocytes kill microbial pathogens. (A) A single neutrophil (yellow) engulfing rod-shaped anthrax

bacilli (orange). Like other phagocytes, neutrophils engulf microbial pathogens, which are then destroyed within the

phagocyte. (B) Immune system phagocytes, such as macrophages, have cell surface receptors that can recognize certain types

of pattern on microbes (such as components of bacterial cell walls), identifying them as foreign cells.

The process of phagocytosis begins with binding of a bacterium (or other microbe) by cell surface receptors, followed by

internalization of the microbe within a vacuole called a phagosome.

WHITE BLOOD CELLS. Transmission electron micrographs of thin sections of each cell and interpretive drawings with lysosomes shown in brown. A, A neutrophil showing the multilobed nucleus (the connections between lobes are in other sections) and the two classes of granules. B, An eosinophil showing the bilobed nucleus and the large, specific granules containing a darkly stained crystalloid. C, Basophil with large specific granules colored blue. D, Blood monocyte. E, Macrophage grown in tissue culture. (Micrographs courtesy of D. W. Fawcett, Harvard Medical School, Boston, Massachusetts. Drawings modified from T. Lentz, Yale Medical School, New Haven, Connecticut.)

Eosinophils

o Eosinophils are identified in blood smears as cells with a bilobed nucleus and large

specific granules that stain brightly with eosin.

o Specific granules contain a cationic protein, a ribonuclease and peroxidase, in addition

to a crystalloid of a basic protein.

o Like neutrophils, eosinophils pass briefly through the blood on their way to connective

tissue, where they survive for about two weeks.

o Chemotactic factors generated by the complement system, basophils, some tumors,

parasites, and bacteria all attract eosinophils.

o Many of the same factors attract other leukocytes, but particular chemokines are

specialized for eosinophils.

o Eosinophils accumulate in blood and tissues in response to parasitic infections.

o Eosinophils bind parasites and lyse them, like killer lymphocytes (see later

discussion), by secreting a cationic protein that forms pores in their membrane.

o Production of superoxide, hydrogen peroxide, and antimicrobial peptides also

contributes to killing.

o Activated eosinophils contribute to inflammation in some allergic disorders such as

asthma.

Basophils

• Basophils are the least abundant and least understood granulocytes.

• They look much like neutrophils but have a bilobed nucleus and large, basophilic,

specific granules containing heparin, serotonin, and all of the blood histamine.

• Basophils are weak phagocytes.

• Like mast cells, they have cell surface receptors that bind IgE and release the

vasoactive agents stored in their granules when antigens bind to these bound

immunoglobulins.

• Basophils and mast cells have much in common, but they appear to have different

origins.

• Basophils arise from bone marrow stem cells, whereas mast cells are derived from

connective tissue mesenchymal cells.

• Humans have both circulating basophils and tissue mast cells, but this is not universal.

Mice have mast cells but no basophils.

• Turtles have basophils but no mast cells.

THE IMMUNE RESPONSE BY THREE CLASSES OF LYMPHOCYTES THROUGH THREE PARALLEL STEPS. A, Genetic recombination produces populations of cells with a wide variety of antigen specificities provided by cell surface immunoglobulins (Ig) or T-cell receptors (TCR). B, The binding of specific antigens (Ag) to surface immunoglobulins or T-cell receptors selects a subset of the cells. C, Proliferation of clones of the selected cells yields many cells specialized to produce antibody (Ab) to soluble antigens, secretion of growth factors by helper T cells in response to ingested and degraded antigens, or killing of virus-infected cells identifiable by the viral peptides on their surface. The helper and killer T cells use a common set of T-cell receptors and are guided to the appropriate target cells by the CD4 and CD8 accessory molecules.

EXAMPLES OF SIGNALS THAT PROMOTE DIFFERENTIATION OR PROGRAMMED CELL DEATH OF IMMATURE THYMOCYTES IN THE THYMUS AND MATURE T CELLS IN THE PERIPHERY. Thymocytes that make functional T-cell receptors and do not recognize self-antigens mature, provided that they receive survival signals, such as interleukin-7. Thymocytes undergo apoptosis if they produce defective T-cell receptor, recognize self-antigens, suffer DNA damage, or receive a death stimulus (glucocorticoid hormone). More than 95% of immature thymocytes die without leaving the thymus. (Based on Strasser A: The role of BH3-only proteins in the immune system. Nat Rev Immunol 5:189-200, 2005.)

Cellular Basis of Adaptive Immunity

In response to infection, lymphocytes of the immune systems of vertebrates produce

two kinds of adaptive responses: humoral (in the body fluids) and cellular.

B lymphocytes produce the humoral response by secreting antibodies

(immunoglobulins), soluble proteins that diffuse in the blood and tissue fluids.

Two types of T lymphocytes mediate the cellular arm of the adaptive immune

response. Cytotoxic T lymphocytes (killer T cells) destroy cells infected with viruses,

whereas helper T cells regulate other lymphocytes.

These responses protect against infection but fail in acquired immunodeficiency

syndrome (AIDS) when the human immunodeficiency virus (HIV) kills helper T cells.

A blood smear reveals lymphocytes of various sizes and shapes but not their

remarkable heterogeneity at the molecular level.

Antibodies produced by B cells provide a chemical defense against viruses, bacteria,

fungi, and toxins.

Antibodies, or immunoglobulins, are an incredibly diverse family of proteins, each with

a binding site that accommodates one of millions of different ligands termed antigens.

Innate lymphoid cells (ILCs). Schematic diagram showing the relationship between ILCs and T cells, the principle cytokines

they secrete, and their main effector functionality.

Type I Innate Lymphoid Cells

Type I ILCs have many similarities with NK cells.

They are also activated by the action of the cytokines IL-12, -15, and -18, in

response to intracellular infections and tumor cells.

Just as is the case for NK cells, this also causes them to secrete IFNγ, but unlike NK

cells, they are not directly cytotoxic and instead, also secrete TNFα.

This has the net result of stimulating macrophage activation and the production of

oxygen radicals, both of which are important defenses against intracellular infection.

ILC1 cells drive a type 1 immune response and help promote the development and

maturation of the adaptive response.

They are thought to potentially contribute to the pathology of a number of forms of

inflammatory bowel disease.

Type II Innate Lymphoid Cells

Type II ILCs are activated by cytokines that are produced by damaged epithelial cells.

These are commonly released in response to the presence of helminths, by the action

of allergens or as a result of tissue injury.

The cytokines secreted by activated ILC2 cells are classic type 2 cytokines.

They stimulate the process of vasodilation, the production of mucus, and repair to

damaged tissues and the extracellular matrix and can influence the host’s thermal

regulation.

ILC2 cells still activate macrophages, but the pathway differs from that used by ILC1

cells.

ILC2 cells provide protection against fat-induced inflammatory responses and may

also protect against the development of metabolic syndromes, insulin resistance, and

diabetes.

An inability to produce ILC2 cells leads to increased pathogenicity upon infection with

the parasites Nippostrongylus brasiliensis and Schistosoma mansoni.

Type III Innate Lymphoid Cells

Type III ILCs become activated as a result of exposure to IL-1β and IL-23 produced in

response to infection.

This in turn causes the ILC3 cells to secrete IL-17, IL-22, and GM-CSF themselves.

This stimulates phagocytic processes, the secretion of antimicrobial peptides, and

improved epithelial cell survival.

Intestinal ILC3 cells are reliant on the host microbiota to develop properly.

Pathologically, ILC3 cells are important contributors to fat-induced inflammation and

therefore can be considered as working in a manner opposite to ILC2 cells in this

context.

ILC3 cells have also been connected with the immunopathology of inflammatory

bowel disease and colitis.

All ILCs respond quickly to infection or injury.

Their influence in directing the final direction of the immune response along

particular pathways makes them an attractive target for immunomodulation.

Disruption or alteration of ILC function could be used to alter or modify the immune

response that arises in response to chronic inflammation, vaccination, or as a

general means of controlling the immune response.

Locations of principal lymphoid tissues within the human body.

ASSEMBLY OF IMMUNOGLOBULIN GENES BY REARRANGEMENT OF GENE SEGMENTS. A, Region of germ line DNA with multiple, tandem V, D, J, and C segments for assembling an immunoglobulin heavy chain, B, Same region after rearrangement of D and J segments in a Pro-B cell. C, After rearrangement of V in a Pre-B cell. D, B-cell. E, Immunoglobulin mRNA produced by a B-cell. (Based on a drawing in Chiorazzi N, Rai KR, Ferrarini M: Chronic lymphocytic leukemia. New Engl J Med 352:804-815, 2005.)

Antigens include proteins, polysaccharides, nucleic acids, lipids, and small organic

molecules produced biologically or chemically.

Antibody binding can mark an antigen for phagocytosis or neutralize its toxicity.

The huge repertoire of antigen-binding sites present in the collection of antibodies that

circulate in a single individual arises through rearrangement and somatic mutations of

immunoglobulin genes.

This remarkable process was exploited during evolution specifically for the use of the

immune system.

Each mammalian immunoglobulin is composed of four polypeptide chains-two identical

heavy chains and two identical light chains-each encoded by different genes.

Light chains and heavy chains both contribute to the antigen-binding site. In vertebrate

genomes, immunoglobulin genes exist in segments aligned along a chromosome.

Several of these gene segments must be combined in the proper order to make a full-

length functional antibody gene.

Some of these gene segments encode the framework of the antibody protein, which is

essentially identical within each class of antibodies.

Other gene segments, present in many variations, encode the part of the polypeptide

chain that forms the antigen-binding site.

MODULAR PROTEINS CONSTRUCTED FROM EVOLUTIONARILY HOMOLOGOUS, INDEPENDENTLY FOLDED DOMAINS. A, Examples of protein domains used in many proteins: fibronectin 1 (FN I), fibronectin 2 (FN II), fibronectin 3 (FN III), immunoglobulin (Ig), Src homology 2 (SH2), Src homology 3 (SH3), kinase.B, Immunoglobulin G (IgG), a protein composed of 12 Ig domains on four polypeptide chains. Two identical heavy chains (H) consist of four Ig

domains, and two identical light chains (L) consist of two Ig domains. The sequences of these six Ig domains differ, but all of the domains are folded similarly. The two antigen-binding sites are located at the ends of the two arms of the Y-shaped molecule composed of highly variable loops contributed by domains H1 and L1.C, Examples of proteins constructed from the domains shown in A: fibronectin, CD4, PDGF-receptor, Grb2, Src, and twitchin. Each of the 31 FN3 domains in twitchin has a different sequence. F1 is FI, F2 is FII, and F3 is FIII.

During maturation of a particular B cell, recombination enzymes (RAG1 and RAG2)

assemble immunoglobulin gene segments into one unique full-length gene for a heavy

chain and one for a light chain.

As a result of random gene arrangements, each B cell assembles and expresses

novel immunoglobulin genes.

The process is precise in that the right number of segments is always chosen to make

a heavy chain or a light chain, but it is also random in that any one of the variable

segments may be chosen.

The resulting antibody contains two identical but unique antigen-binding sites.

The gene segments can be assembled in many different combinations, and most

heavy chains can assemble with most light chains.

The diversity arising from the combinatorial process is expanded further in two ways.

First, the recombination process inserts a variable number of nucleotides between the

gene segments.

Second, pre-B cells use enzymes to mutate codons for amino acids in the antigen-

binding site, creating variations in the antigen-binding specificity in different cells.

In principle, about 3000 different light chains and 60,000 heavy chains can combine to

produce about 100 million different antibodies even without taking mutations into

account.

Accordingly, it is possible experimentally to induce a mouse to make an antibody that

is specific for almost any naturally occurring or synthetic chemical.

Infection by a pathogen results in the production of antibodies that bind to the

pathogen but not to any of the individual's own molecules.

This response comes from activation and proliferation of preexisting B cells that have

the capacity to make antibodies to molecules of the pathogen.

Activation requires a chance encounter of particular B cells with the pathogen and

stimulates the cell to mature into a factory for secreting antibodies.

Alternate splicing of messenger RNA (mRNA) selects domains required to direct the

same antibody to either the plasma membrane or the secretory pathway.

ALTERNATIVE SPLICING CAN GENERATE MULTIPLE DIFFERENT PROTEINS FROM A SINGLE GENE. Here are some of the possible mRNA and protein products of a gene whose pre-mRNA is subject to alternative splicing. Left, Examples show the other effects of skipping one or more internal exons, which produces a set of related proteins with different combinations of"modules." Right, Examples show the effects of alternative splice sites. In the case shown, the use of alternative 3' splice sites redefines the 5' end of the downstream exon. This can lead to the inclusion of additional amino acids in the protein product. Use of an alternative splice site can also cause the exon to be read in a different reading frame (green asterisk), changing the amino acid sequence. If the alternative reading frame contains a translation stop codon (red asterisk), a truncated protein will be produced, and the mRNA will generally be targeted for rapid degradation by the NMD pathway.

Cells that are activated by antigen binding to a surface immunoglobulin divide to

increase their numbers.

This process, called clonal expansion, amplifies the production of antibody specific

for the antigen.

The mature cellular product of the B-cell response is a plasma cell, which is highly

specialized to secrete one specific antibody.

Other B cells that are activated by the antigen become memory cells.

These long-lived cells display the specific antibody on their surface and stand poised

to mount an amplified response on subsequent exposure to the same antigen.

This immunologic memory explains why exposure to a particular pathogen or

vaccination against a pathogen results in protection, in the form of antibodies, for many

years.

Specialized B lymphocytes and plasma cells secrete different antibody isoforms or

isotypes.

Formation of immunoglobulins with the various isotypes requires further

recombination events to join the variable region with the antigen-binding site to the

isotype constant domain.

IgG isotypes, produced in lymph nodes and spleen, circulate in blood and tissue

fluids.

IgA isotypes, produced by lymphoid nodules in the respiratory and gastrointestinal

tracts and by mammary glands, are first taken up and then secreted by epithelial cells of

these organs (transcytosis).

IgE isotypes bind to receptors on the surface of mast cells and basophils (see earlier

discussion).

T lymphocytes provide cellular responses to pathogens. Cytotoxic T cells execute

tumor cells and virus-infected cells.

Helper T cells stimulate antibody production by B cells.

The specificity of these responses is provided by variable cell surface receptors called

T-cell receptors.

A set of segmented genes analogous to immunoglobulin genes encode T-cell

receptors.

In contrast to antibodies, T-cell receptors do not bind free antigens but rather

recognize peptide antigens displayed on the surface of target cells complexed to

proteins called major histocompatibility complex (MHC) antigens.

These highly variable MHC proteins are responsible for the rejection of tissue grafts

from nonidentical individuals.

A, Electron micrograph of a thin section of a muscle capillary showing caveolae ("little caves"), which are abundant in endothelial cells that mediate transcytosis. Arrows show "cave" openings. B, Electron micrograph of the inside surface of a fibroblast prepared by quick-freezing, deep-etching, and rotary shadowing. The whorl-like coat on the caveolae formed by

self-assembly of caveolin (white arrows). Caveolae are typically smaller than clathrin-coated pits shown in the upper left and right. (A, Courtesy of D. Fawcett, Harvard Medical School, Boston, Massachusetts. B, Courtesy of John Heuser, Washington

University, St. Louis, Missouri.)

T-LYMPHOCYTE ACTIVATION. A, Resting T cell with inactive nonreceptor tyrosine kinase Lck and the T-cell receptor complex (TCR) with unphosphorylated cytoplasmic phosphorylation sites (ITAMs). B, An encounter with an antigen-presenting cell with an MHC-antigenic peptide complex complementary to the particular TCR initiates signaling. Active Lck phosphorylates various ITAMs. C, The nonreceptor tyrosine kinase ZAP-70 is activated by binding via its two SH2 domains to phosphorylated ITAMs on the zeta chains. D, Ribbon diagrams of MHC II (green) with bound peptide from moth cytochrome c (orange). The main model is reduced in size and tilted 90 degrees forward in the view in the upper right corner, the same orientation as in the panels B, C, E, and H. E, Active ZAP-70 phosphorylates various targets, including the transmembrane protein LAT and the adapter protein SLP76, which then propagate the signal. Phospholipase Cγ binds a LAT phosphotyrosine and produces IP3 and DAG. IP3 releases Ca2+ from vesicular stores. Ca2+ activates calcineurin (protein phosphatase 2B), which activates the latent transcription factor NF-AT. Vav, the nucleotide exchange factor of the small GTPase Rac, is activated by binding to SLP76. Grb2-SOS binds another phosphorylated ITAM and initiates the MAP kinase cascade. F, Micrographs of the time course of the interaction of a T cell with an artificial membrane mimicking a specific antigen-presenting cell. Each image comprises a superimposition interference reflection micrograph, showing the closeness of contact as shades of gray (with white being closest apposition), and a fluorescence micrograph, showing TCRs (green) and ICAM1 (red). The stable arrangement of ICAM1 around concentrated TCRs is called an immunologic synapse. G-H, Immunologic synapse with a central zone of TCRs boun

DOMAIN ARCHITECTURE OF ABC TRANSPORTERS. A, Bacterial transporters. B, Eukaryotic transporters. Each transporter has two ATP-binding domains in the cytoplasm (purple circles) and two transmembrane domains, each consisting of 6 to 10 α-helices (blue or pink squares). CFTR has an additional regulatory (R) domain in the cytoplasm. The four domains required for activity may be four separate polypeptides or may be incorporated in several ways into polypeptides with two or four domains.

The two types of MHC proteins-class I and class II-acquire their antigenic peptides differently.

All somatic cells produce class I MHC proteins.

In cells that have been infected by a virus, cytoplasmic immunoproteasomes degrade some

viral proteins to peptides, which ABC transporters (TAP1, 2) move from the cytoplasm into the

endoplasmic reticulum.

In the lumen of the ER, peptides insert into the binding site of compatible class I molecules

and the complex moves to the plasma membrane.

In contrast, macrophages and other antigen-presenting cells, such as dendritic cells, ingest

foreign matter and degrade it in endosomes and lysosomes.

Peptide fragments bind to class II proteins in endosomes and thence move to the cell surface

of these antigen-presenting cells.

T lymphocytes patrol the body, inspecting the surfaces of other cells.

A chance encounter with a cell displaying a peptide-MHC complex complementary to

its T-cell receptor stimulates the T cell.

The response is proliferation and expansion of a clone of identical T cells.

Accessory membrane proteins CD4 and CD8 on the T-cell surface cooperate with T-

cell receptors to direct the two types of T cells to target cells with the appropriate MHC

proteins.

T-cell receptors provide antigen specificity. Immature T-cells express both CD4 and

CD8 but lose one of them as they mature into cytotoxic (CD8+) or helper (CD4+) T cells.

CD8-positive cytotoxic T cells are specialized to kill cells infected with viruses.

The presence of virus inside is revealed by MHC class I proteins displaying vital

peptides on the surface of the infected cell.

CD8 binds to a constant region of MHC class I proteins carrying viral peptides.

During its intimate encounters with the target cell, a cytotoxic T cell uses three

weapons to kill the target:

First, T cells carry a ligand for the Fas receptor on the target, which stimulates

apoptosis of the target cell.

Second, activated T cells secrete perforin, a protein that inserts into the plasma

membrane of the target cell, forming large (10 nm) pores that leak cytoplasmic contents

and ultimately lyse the cell.

Third, T cells secrete toxic enzymes that enter target cells through the plasma

membrane pores.

CD4 binds a constant part of the MHC class II protein and targets helper T cells to

cells presenting ingested antigens.

The progeny of stimulated helper T cells secrete growth factors (lymphokines or

interleukins) in the vicinity of B cells with the foreign antigen bound to immunoglobulins

on their surface.

Helper T cells are required for B cells to make antibodies against most antigens.

This explains how HIV causes AIDS.

The virus uses CD4 as a receptor to infect and eventually kill helper T cells.

Loss of helper T cells severely limits the capacity of B cells and cytotoxic T cells

(which also require T-cell help) to mount antibody and cellular responses to

microorganisms.

Infections that the immune system normally dispatches with ease then become life-

threatening.

Genetic defects cause a wide variety of immunodeficiency diseases.

For example, defects in Bruton tyro-sine kinase result in failure to produce B cells.

Remarkably, humans who lack function of the enzyme adenosine deaminase have no

B cells or T cells but are otherwise normal.

Deficiencies of many specialized lymphocyte proteins (cytokine receptors, interleukin

receptors, Lck tyrosine kinase, ZAP-70 tyrosine kinase, RAG1 or RAG2, TAP1 or TAP2)

also lead to immunodeficiencies.