FHS Option: Infection and ImmunityTheme: Lymphocyte recognition and signallingLecture: Antigen receptors: diversity, structure and functionLecturer: Anton van der Merwe

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Antigen recognition is mediated by specific antigen receptors

• Recognition is a central feature of the immune response, required for detection and elimination of dangerous organisms (pathogens).

• This recognition is mediated by specific antigen receptors which bind to structures (antigens) on, or derived from, pathogens.

• Antigen receptors can be secreted or cell-associated.• Antigenany structure which elicits an immune response

The innate and adaptive immune responses have fundamentally different recognition receptors• Innate immune response

– receptor genes are germ-line encoded (i.e. inherited) and do not change during the life-time of an individual.

– they have evolved slowly over many generations, and tend to recognise invariant structures common to groups of pathogens that have posed a persistent threat.

• Adaptive immune response– Recognition (antigen) receptor genes are newly generated and selected within each individual (not

germ-line encoded). Thus recognition capability is able to adapt quickly within a single individuals life-time.

– In theory this system can recognize any potential pathogen, even if never encountered before by humans.

– Only found in higher multicellular organisms– Has evolved independently at least twice

• Jawed vertebrates ( Immunoglobulin system RAG system), • Jawless vertebrates (Variable Lymphocyte Receptor system)

– This may be because, unlike simpler animals, these organisms are long-lived and have limited reproductive capacity.

Properties of antigens recognised by the innate immune response

• Because pathogens will try to evade recognition, receptors in the innate immune response have evolved to recognize antigen structures which are functionally critical and therefore cannot change or be lost.

• Because a limited number of receptors can be used,

there is a tendency to recognize structures present in large groups of pathogens (e.g. flagellin, lipopolysaccharide).

Examples of receptors of the innate immune reponse

Receptors LigandsSoluble (plasma) Collectins microbial polysaccharides

Mannose binding lectin bacterial and fungal carbohydrates

Complement non-host surfaces

Cell associated

Cell surface Toll-like receptors (TLR) various microbial products

Mannose receptors multiple carbohydrates

Endosomal Toll-like receptors (TLR) microbial DNA

Intracellular NOD-like receptors (NLR) bacterial products

RIG-like receptors (RLR) viral DNA

Mannose-binding lectin

There is spontaneous generation of C3b in plasma which covalently couples to surfaces via a reactive thioester

Host cell-surfaceshave proteins (CD59, DAF, MCP, CD1, H) which quickly inactivate C3b

All other surfaces lack these factors so C3b remains active

Complement activation:• Inflammation• Opsonization • Lysis

Alternative activation pathway of complement An elegant mechanism for pathogen recognition

No effect

Cell-associated innate receptors

From Akirii et al, 2008

Innate response to viruses

From Pichlmair and Reis e Sousa, 2007

Collaboration between innate and adaptive immune responses

• By recognition of microbial products the innate response alerts the adaptive immune response to presence of infection (or danger?). This strongly stimulates, and is probably essential for, the adaptive response. – Does this by activation antigen presenting cells so that they express ‘costimulatory

molecules’ such as B7-1 and B7-2

– Hence immunologists’ “dirty little secret”, that foreign antigens must be mixed with inflammation-causing ‘adjuvants’ to provoke an adaptive immune response.

• Because its receptors have evolved to distinguish particular classes of pathogen, the innate response helps to direct the type of adaptive immune response that is mounted.

• Provides effector mechanisms deployed by the adaptive immune response in the effector phase. These include complement and cells with Fc receptors such as macrophages, granulocytes and NK cells.

Receptors of innate immune response help distinguish between types of pathogen

(chitin, proteases)

• The central features of the adaptive immune response are– that highly-specific responses can be generated against an

enormous variety of foreign antigens.

– that the response is much enhanced upon subsequent exposure (a phenomenon termed memory).

• These properties are explained by the ‘clonal selection hypothesis’, which has now been verified.

Antigen receptors of the adaptive immune response

• B cell antigen receptor (BCR)– antibodies or immunoglobulins

– initially cell-surface, and later secreted

• T cell antigen receptor (TCR)– two types of T cell lineages expressing either

• TCR common one, well-understood

• TCR less common, poorly understood

Structure of an immunoglobulin molecule (1)

Structure of an immunoglobulin molecule (2)

Antigen binding site on antibodies

lysozyme

Small ligands: pockets or grooves; Large ligands: irregular complementary surface

Antibody diversity• Structural variation in the constant regions (different effector

functions)– heavy chain

• (isotype switching)

• membrane or secreted

– light chain• (functional significance not known

• Diversity in variable regions (different antigens recognised)– See later

Antibody isotypes

Important functional differences between isotypes

•Valency (IgM) - important in primary response•Complement activation - IgM, IgG3•Opsonization - IgG1, IgG3•Placental transfer - IgG2, IgG4•Binding to mast cells and basophils - IgE•Secretion onto mucosal surfaces - IgA

Isotype switching (IgM to IgD)

Involves differential RNA processing. Thus..• it is reversible• the same cell can express both IgM and IgD (only two isotypes expressed on the same cell).

Isotype switching (IgM/IgD to IgG/E/A)

Switch involves somaticrecombination-Irreversible-Usually accompanies affinity maturation

Expression of transmembrane and secreted of immunoglobulin

Involves differential RNA processing. Therefore is it reversible.

T cell antigen recognition

• Antibodies (BCR) recognize extracellular antigen in its native form. Essentially any structure can be recognized.

• In contrast T cells (TCR) recognize antigens derived from both intracellular and extracellular sources in processed form. Only certain structures can be recognised - those which can be presented by major histocompatability complex (MHC) molecules (peptides and certain lipids).

• Understanding T cell antigen recognition therefore requires an understanding of how antigen is presented by MHC molecules

MHCclass I

MHCclass II

MHC molecules are most polymorphic in the peptide binding groove

Peptide binding to MHC class I -8 to 10 amino acids long -importance of N and C term -two or more anchor residues

Peptide binding to MHC class II -up to 20 amino acids long -importance of backbone contacts -two or more anchor residues

Peptide recognition by MHC molecules

TCR/pep-MHC I TCR/pep-MHC II

Generation of diversity in antigen recognition sites of BCR (antibody) and TCR

• Diversity of the primary repertoire of receptors on each new (naïve) B and T cell generated by same mechanism.

• B cells can further refine their receptors to improve their binding properties (affinity maturation) by a process of somatic hyper mutation

Diversity in the antigen recognition sites (Variable domains) is generated by two types of mechanism

• Combinatorial diversity– combining two different V

domains to generate Ag binding site

– combining different gene segments to form V domains

• Junctional diversity– imperfect joining of these

segments

Rearrangements generating BCR genes

• Steps involved in the rearrangement of gene segments

• The key enzymes are encoded by Recombination Activating Genes and are called RAG 1 and 2

• These enzymes suddenly appear during evolution of vertebrates around 500 m.y.a.

• Genes similar to transposons

• Essential step in evolution of long-lived animals?

Generation of junctional diversity by P and N nucleotide addition

The 3rd hypervariable loop in V domains has by far the greatest diversity

L1

L3

L2H3

H2

H1

peptide antigen

VH

VLGene segments V CJD

Protein domains CH1

• H1 and H2 coded by V segments• H3 coded by D & J segments and junctions

VH3H1 H2

TCR gene loci

Rearrangements generating TCR genes

The CDR3 loops that interact with peptide have by far the greatest diversity.CDR1 and CDR2 bind to MHC and have much less diversity

Gene segments V CJD

Protein domains C

• CDR1 and CDR2 coded by V segments• CDR3 coded by D & J segments and junctions

V31 2

Diversity in TCR and BCR

Somatic hypermutation• Combinatorial and Junctional diversity generate the primary Ab repertoire.

– This repertoire is sufficient to recognise all possible structures

– However most structures will be recognised with a low affinity (why?)

– Therefore recognition needs to be highly multivalent.• Cell surface Ab binding cells or aggregated Ag

• Soluble IgM binding cells or aggregated Ag

• Somatic hypermutation is the process by which low affinity antibodies are converted to high affinity antibodies. – Strictly speaking this does not ‘increase the size of the repertoire’

– high affinity antibodies are useful because • they can function monovalently e.g. can inactivate soluble bacterial toxins.

• will be able to bind very low concentrations of antigen. Good for eliminating infection and for early response to re-infection.

Somatic hypermutation

• Takes place in germinal centres• Mechanism not well understood• Requires T cell help

To ensure specificity cannot change (how does this work?)

• Associated with class-switching• Can destroy receptor - in which case secondary rearrangment may rescue the B cell.

Day 1

Day 21

Activation-induced cytodine deaminase(AID) plays a central role in somatic hypermutation and class switching

Binding properties of antigen receptors illustrate effects of somatic hypermutation

Receptor Kd (M) Half-life

T cell receptor 10 seconds

Primary antibodies 10 seconds

Secondary antibodies 0.01 minutes to hours

Comparison of TCR and BCR (antibody)

BCR TCR

Ligand Any structure peptide & lipidBind native ligand Yes NoAg processing No YesMHC restriction No YesSomatic mutation Yes NoAffinity for ligand Low to very high LowCo-receptors No Yes