October 12, 2004

IMMUNITY

ADAPTIVE INNATE

CELL MEDIATED HUMORALANTIBODIES

EFFECTOR SYSTEMSFc ReceptorsComplement

RECEPTORSEFFECTORS

CellsMolecules

ANTIGENS

Innate immunity

Adaptive immunity

Immunologic memory

First response to infection

Triggered by persisting infection

Protects against subsequent infection

Functions of innate immunity

Initial response to infection

Frequently is sufficient to eliminate the infection

Effector mechanisms of innate immunity are often used to eliminate pathogens in adaptive immune

response

Innate immunity stimulates adaptive immune response and influences the nature of the adaptive response

Receptors recognize pathogen associated molecular patterns (PAMP)

Nucleic acids unique to microbes (e.g. double stranded RNA or unmethylated CpG DNA)

Features of proteins found in microbes (e.g. N-formylmethionine)

Complex lipids and carbohydrates synthesized by microbes but not mammalian cellsLPS in gram-negative bacteria

Teichoic acids in gram-positive bacteriaMannose-rich oligosaccharides found in microbial

glycoproteinsHas evolved to recognize microbial proteins often essential

for their survival

Receptors of the Innate immune responseSpecificity inherited in genome

Expressed by all cells of a particular type- not clonally distributed

Trigger immediate response

Recognize a broad class of pathogens

c

Function

Recognize pathogen

Attract effector cells

Induce effector molecules

Contribute to innate immunity

Influence the nature of the subsequent adaptive immune response

Trigger inflammation

Overview of the events during inflammation

The recruitment of leukocytes and extravasation of several plasma proteins to a site of infection with activation of the leukocytes and proteins eliminating the infectious agents

Epithelium

Barriers to infectionMechanical

Chemical Cells

Infection

Epithelial barriers to infection

Mechanical: Tight junction of epithelial cells form a physical barrier

EpitheliumInfection

Epithelial barriers to infectionChemical

Antibacterial peptides (defensins)

Enzymes (e.g. lysozyme, pepsin)

Low pH

EpitheliumInfection

Epithelial barriers to infection

CellsMast cells

Intraepithelial T lymphocyes

B-1 B cells

EpitheliumInfection

CD5 B cell binds capsular polysaccharide

CD 5 cell secretes IgM anti-

polysaccharide antibody

IgM

Phagocytes

Neutrophils

Produced and lost in large number every day

Abundant in bloodNot present in healthy tissue but recruited to

site of infection

Short-lived (6 hours) and pus contains dead and dying neutrophils

Contain granules with anti-bacterial proteins and peptides

Can eliminate pathogens by phagocytosis

Phagocytes

Macrophages

Circulating precursors are called monocytes

Longer lived than neutrophils

Can divide at the site of infection

PhagocytesWhen pathogens cross the epithelial barrierthey are recognized by phagocytes in the

subepithelial connective tissues

Trapping,engulfment and destruction by phagocytosis

Phagocytes

When pathogens cross the epithelial barrierthey are recognized by phagocytes in the

subepithelial connective tissues

Trapping,engulfment and destruction by phagocytosis

Induction of co-stimulatory molecules

Cytokine secretion by phagocyte

Antigen uptake, processing and presentation

Neutrophils and monocytes are recruited from blood to sites of infection

Resident tissue macrophages that recognize microbes secrete cytokines and chemokines that act on endothelial cells to produce adhesion molecules and attract circulating neutrophils and macrophages

Neutrophils and macrophages have receptors that recognize microbes and stimulate their phagocytosis and killing

Receptors that directly bind microbes

Mannose receptors

Scavenger receptors

Integrins

Pathogen Associated Molecular Patterns

Not clonal

Neutrophils and macrophages have receptors that recognize microbes and stimulate their phagocytosis and killing

Receptors for opsonins

FcRs

CR1, 3 and 4(recognize cleavage products of C3)

Triggering these receptors both stimulates phagocytosis and activates the phagocytes

Neutrophils and macrophages have receptors Toll-like receptors

TLR-5:flagellin

Activation through these receptors triggers cytokine production and expression of co-stimulatory

molecules

TLR-2: zymosan from yeast,bacterial lipoproteins andlipteichoic acid and peptidoglycan on Gram-positive bacteriaTLR-4: LPS on Gram-negative bacteria; viral proteins

TLR-9: unmethylated CpG

Neutrophils and macrophages have receptors Seven-transmembrane -helical or G

protein-coupled receptors

Peptides containing N-formylmethionyl residues

Activation induces migration of cells from bloodthrough endothelium and production of microbicidal

substances

Receptors of this class recognize

Chemokines such as IL-8

C5a

Neutrophils and macrophages have receptors for cytokines such as IFN-, the major macrophage-activating cytokine

Macrophage Possessses Many Receptors

LPS receptor (CD14)

Mannose receptor

Scavenger receptors

Fc receptors

CD11b/CD18

Engulfment

Cytokine Secretion

Activation

TLRTLR

Antigen presentation

Deliver additional effectors molecules and cells to site of infectionProvide a physical barrier to prevent the spread of infectionPromote the repair of injured tissues

Role of inflammation in combating infection

Combined local effects increase inflmmatory response

1. Increase in vascular diameter lead to increased local blood volume--

heat and redness from reduced velocity of blood flow

2. Decreased blood flow allows leukocytes to better interact with the vascular endothelium

Extravasation:

Selectins recognize certain leukocyte glycoproteins causing

lymphocytes to roll. ICAM-1 on endothelium interacts with

LFA-1 (a.k.a. CD11a;Cd18) and CR3 (Mac-1) so that leukocytes

attach firmly to the endothelium, to cross the vascular endothelial wall and enter site of infection

3. Increase in vascular permebility leads to local accumulation of fluid-

swelling and pain--

accumulation of Igs, C’ and other blood protein in the tissue.

Combined local effects increase inflmmatory response

4. Mediators induce expression of adhesion molecules on the endothelium neutrophils and monocytes are recruited to the site5. Migration of leukocytes through tissues under the influence of chemoattractant molecules. Direct migration along a gradient of the chemokine that increases as get nearer the site of infection. Chemokines appear to bind to proteoglycan molecules so that they can remain cell associated to create the gradient.

CC chemokines promote the migration of monocytes: MCP-1, MIP-1β, RANTES

CXC chemokines promote migration of : neutrophils -8IL

An increase in vascular permeability leads to local accumulation of fluid

Swelling (edema)

Pain

Accumulation of Igs, complement and other blood proteins in tissue

Entry of fluid into blood at site of infection is prevented

TNF-

Local clots in small vessels are produced.

Fluid in tissue carries pathogen, either directly or within a phagocytic cell, via lymph to regional lymph nodes where an adaptive immune response is elicited

Sepsis

TNF-s released by macrophages

Infection spread to the blood stream

Septic shock requires signaling through TLR-4 (recognizes LPS) and mice (or humans) defective in TLR-4 do not experience septic shock

Vasodilation occurs with increased vascular permeability leading to shock

Mice defective in TLR-4 are highly sensitive to LPS containing pathogens.

TNF-, IL-1 and IL-6 are endogenous pyrogens which elicit acute-phase proteins

virus

IFN-, -IFNβ

Inhibit protein synthesis and DNA - replication in virus infected cells

Increase MHC class I expression and antigen presentation in all cells

- Activate NK cells to kill virus infected cells

Natural Killer Cells

Function against intracellular pathogens such as viruses

Tumor immunity

Activated by IFN-, IFN-β or IL-12

Must be able to distinguish infected from uninfected cells

Summary - Features of Innate Immunity

Triggered by germline encoded receptors of limited diversity

No lasting immunity or memory

Elicit cytokine release by phagocytes

Induced production of acute-phase proteinsElevate body temperature

Induce inflammation

NK cells are able to recognize infected or altered cells

B-1 B cells provide pathogen specific Abs of limited diversity in the absence of T-cell help

Properties of substances that elicit an adaptive immune response

Immunogen: Substance which is capable of eliciting a humoral or cell mediated immune response

B cells + antigen --> effector B cells (plasma cells) + memory B cells; Abs produced

T cells + antigen --> effector T cells (e.g. CTLs) + memory T cells

Antigen: a substance which reacts with an antibody or T-cell receptor

All immunogens are antigens but not all antigens are immunogens

Haptens: small molecules that are antigens (that is can react with Ab or TcR) but which cannot by themselves elicit an immune response

Hapten

(Dinitrophenol) Carrier

(BSA)

Haptens must be linked to a carrier to elicit an immune response. Abs are formed to both the hapten and carrier.

Reactivity with

Antiserum

against

Aminobenzene

o -aminobenzoic acid

m -aminobenzoic acid

p -aminobenzoic acid

Aminobenzene

(aniline)

o - aminobenzoic

acid

m -aminobenzoic

acid

p -aminobenzoic

acid

Small ligands often bind in a deep Ab pocket. There is a tight fit so Ab binding can distinguish structurally related but different haptens.

Epitope or antigenic

determinant is the part

of the immunogen that binds

Ab or the T C R

Abs are designed to interact with the surface of soluble antigens.

Essentially the whole surface of a globular protein can antigenic

B-cell epitopes

May be amino acids located next to each other in sequence (sequential determinant)

Or may be amino acids that are not next to each other in linear sequence but fold into proximity (non-sequential or conformational)

inject

antiserum

antibodies of differentspecificityaffinityisotype

{heterogeneity

anti-

anti-anti-anti-

TCR

MHC

Antigen presenting cell

T cell

EpitopeAgretope

T-Cell EpitopesT cells do not recognize soluble native Ag but instead recognize Ag that has been processed and is presented in association with MHC molecules

Cross reactivity

1. shared epitopes e.g.DNP BSA and DNP- globulin

2. structurally similar epitopes . . e g BSA and HSA

Reaction with other than immunizing Ag

Antigenic determinants on Abs

1. isotypic

constant region determinants that distinguish each

H and L chain class and subclass

2. allotypic

structurally different alleles of the same gene

3. idiotypic

structures unique to a variable region; may be

associated with Ag binding site

NEVER!!

genesgenes

genes

leu

leu

val

val

ANTIBODIES

aa191

or

offspring

For Abs there is allelic exclusion: the products of only one allele are present within one antibody

Isotype : separate constant region

gene. Everyone (who is normal)

has all of the genes for the

different isotypes

Allotype : multiple alleles exist in

the population for a particular

gene.

ForeignnessMust be recognized as "non-self" by the immune system

In general the greater the degree of foreignness, the stronger the response

What determines if something is effective in eliciting an immune response?

SizeBest immunogens usually at least 100,000 daltons

Most (but not all) substances smaller than 5000-10000 Da are poor immunogens

What determines if something is effective in eliciting an immune response?

Composition and Heterogeneity

Are processed and presented

Both the humoral and the cell-mediated immune response are aided by interaction with T cells. Antigen presenting cells present processed Ag in the context of MHC molecules to activate T cells

Molecules that cannot be degraded (e.g. D-amino acids) are poor immunogens

Treatment to increase uptake by antigen presenting cells improves immunogenicity

cross-linking

aggregation

attachment to insoluble matrices

What determines if something is effective in eliciting an immune response?

GenotypeMHC molecules which function in Ag presentation

B-cell and T-cell receptor genes

Genes that encode proteins involved in immune regulation

Route of injection and doseToo little or too much can induce "tolerance"

Boosters further expand B and T cells (memory)

Administration route determines which immune organs and cell populations will respond

Intravenous--> spleen

Subcutaneous--> lymph nodes

AdjuvantsEnhance immunogenicity of Ag

Prolong Ag persistence

Enhance co-stimulatory signalInduce granuloma formation

Nonspecifically stimulate lymphocyte proliferation

Granuloma: Nodule of inflammatory tissue composed of clusters of Activated macrophages and T lymphocytes often with associatedNecrosis and fibrosis.

AlumAluminum potassium sulfate

Causes precipitation

Results in Ag persistance

Increased size improves phagocytosis

Local chronic inflammatory response (granuloma -macrophage rich mass of cells)

Freund's AdjuvantIncomplete: Ag in aqueous solution, mineral oil, and emulsifying agent

Ag is slowly released

Complete: also contains heat-killed Mycobacteria

a muramyl dipeptide of the mycobacterial cell wall activates MΦ

Both result in granuloma formation