Topics

I. II. General principles The heavy chain Ig locus and VDJ rearrangement III. Light chain rearrangement. IV. Mechanisms of Ag-binding diversity V. Isotype switching VI. Membrane vs. secreted Ig expression VII. Regulation of Ig expression VIII. The immunoglobulin gene superfamily

1015 to 1018 Different Antibodies and T cell Receptors Produced!

Antibody Structure and Function

Variable Domains Antigen-Binding Activity

Constant Domains Functional Activities

Coico et al., Fig. 4.3

Antibody Structure and Function

Ag-binding diversity Multiple V segments V(D)J joining Heavy, light chain assortment Junctional/insertional diversity Somatic mutation Functional diversity IgM C fixation IgG C fixation, opsonization IgA secreted Ig IgE allergic reactions

Generation of antigen-binding diversity occurs before antigen exposure through V(D)J rearrangement

VDJ rearrangement Surface IgM/IgD expressing B cells Exposure to Ag, helper T cells

Development of plasma cells, secretion of Agspecific antibodies Isotype Switching after antigen exposure

Coico et al., Fig. 1.1

Heavy Chain Ig Locus

Located on chromosome 14 Expresses IgM and IgD in B cells initially
and constant region genes

Other heavy chain isotypes expressed after antigen exposure (IgG1, IgG2, IgG3, and IgG4, IgA1, IgA2, IgE)
Encoded by 1, 2, 3, 4, 1, 2,

Found in germline (unrearranged) configuration in all cells except B cells

Immunoglobulin heavy chain gene segments

Abbrev.

Meaning Variable Diversity Joining

Constant

Number

Size

Function

V D J

~50 ~20 ~6 9*

~95 aa ~3-6 aa ~13 aa

~110 aa /Domain Form the Constant Regions Form the Variable Domain

*One locus for each isotype

Heavy Chain Locus and VDJ Rearrangement

D-J joining V-D joining

V domain exon

Looping Out of DNA During Rearrangement

Coico et al., Fig. 6.3

Expression of Surrogate Light Chains Prior to Light Chain Expression

Coico et al., Fig. 7.2

Light Chain Loci

Two separate loci kappa () and lambda () on two different chromosomes Express kappa and lambda light chains Have V and J gene segments (no D segments) After successful heavy chain rearrangement, kappa gene rearrangement occurs If kappa rearrangement is successful, the resulting immature B cell expresses IgM with kappa light chains If kappa rearrangement is not successful, lambda gene rearrangement occurs Successful lambda rearrangement results in expression of IgM with lambda light chains

Kappa Gene Rearrangement

V-J joining

V domain exon

Result - functional surface IgM!

5

Coexpression of IgM and IgD Alternate Splicing

Stages of B Cell Development

Coico et al., Fig. 7.1

Mechanisms of Ag-Binding Diversity

Each individual can produce B and T cells with 1015 to 1018 different specificities! We make antibodies and T cell receptors that react with almost any compound, including those that are synthetic and have never occurred in nature Most of this diversity is generated during V(D)J rearrangement, which occurs prior to antigen exposure The diverse group of B and T cells produced is called a repertoire Of this repertoire, <1% of B and T cells will respond to any single antigen or infectious agent

CDRs (L1, L2, L3, H1, H2, H3) form the Ag-binding pocket (Paratope) and determines Ag-binding specificity

Coico et al., Fig. 4.5

Five Mechanisms of Antibody Diversity

Availability of multiple V gene segments Combinatorial diversity (different VDJ and VJ combinations) Assortment of heavy and light chains Junctional and insertional diversity Somatic hypermutation

1. Multiple V regions
Heavy chain locus - ~50 V regions Kappa and lambda loci 40 V regions each Encode the CDR1 and CDR2

2. Combinatorial diversity (different VDJ and VJ combinations)

Different V, D, and J combinations are selected randomly during B cell development Number of V genes X D genes X J genes = number of possibilities 50 X 20 X 6 = 6000 heavy chain combos ~160-200 VJ combinations in kappa and lambda loci Affects the diversity of CDR3

3. Ig H and L chain combinations

6000 H chain VDJ combinations 200 kappa chain VJ combinations 160 lambda chain VJ combinations

H and L combinations = 6000 x 200 + 6000 x 160 ~2 x 106 possibilities

4. Junctional and insertional diversity

V(D)J recombination sites not precise, but instead are sloppy Recombination a few base pairs one direction or another will change amino acid sequence N-region addition random insertion of nucleotides to DJ or VD junctions of heavy chain by terminal deoxynucleotide transferase Affects sequence of CDR3

5. Somatic hypermutation
Point mutations occurring in the V regions Mutation rate in heavy and light chain V regions ~10,000 times higher than background following B cell activation Only form of Ab diversity that occurs after antigenic stimulation Results in affinity maturation, i.e. selection of mutants that have a higher affinity for the antigen in secondary responses

 Definition change in the heavy chain isotype expressed by a given B cell

Example switching from IgM IgG3

Isotype Switching

Constant region downstream of V region is expressed Naive B cells can only express IgM and IgD; plasma cells that develop from naive B cells express only IgM V region and thus antigen binding specificity doesnt change Isotype switching is stimulated by antigen exposure + cytokines

Mechanism deletion of DNA between switch regions

Coico et al., Fig. 6.5

Isotype switching is stimulated by cytokines produced by T helper cells

Membrane vs. Secreted Ig Expression

B cells express only membranebound immunoglobulins Plasma cells express only secreted immunoglobulins Membrane-bound Ig has a hydrophobic tail that anchors it to the cytoplasmic membrane; this tail is lacking from secreted Ig Results from alternative splicing of the RNA transcript secreted form transcript is shorter, lacks region encoding the membrane (M) exon

Regulation of Ig Expression
Enhancer element a region of DNA near the J gene segments that increases transcription from the Ig gene promoters V(D)J joining brings the enhancer element close to the promoter increased transcription Differentiation into plasma cells increases Ig expression ~1,000 fold

Effect of Enhancers
Enhancer Sequence

Weak

Mucho gusto!

Ig Gene Superfamily
Immunoglobulins and T cell receptors are found in vertebrates, not in invertebrates Ig and TCR most likely evolved from related proteins involved in cell-cell interactions These proteins are members of the Ig gene superfamily

All members of Ig gene superfamily have domain structure

Sequence similarity 100-110 amino acids per domain Beta-pleated sheet structure Intrachain disulfide bond (Cys-Cys)

Example Ig light chain

Members of the Ig gene superfamily include:

Big Bang Theory

Recombinase proteins RAG1 and RAG2 central to the VDJ rearrangement process RAG1 and RAG2 resemble bacterial recombinases Big bang theory RAG genes were acquired from bacteria or fungi, resulting in the development of Ig and TCR systems in an early vertebrate ancestor

Summary
Antigen binding diversity occurs during VDJ rearrangement, BEFORE antigen exposure. The 1015-1018 different antigen binding site combinations result from five different mechanisms: multiple gene segments, combinatorial diversity, junctional and insertional diversity, expression of different H and L chain pairs, and somatic hypermutation. The adaptive immune system may have developed from a big bang event in which a onetime acquisition of bacterial recombinases changed the function of the Ig superfamily Ig and TCR genes FOREVER.