GENERATION OF ANTIBODY DIVERSITY*

*Special Thanks to Steven J. Norris, Ph.D

Jeffrey K. Actor, Ph.D. Jeffrey K. Actor, Ph.D. Pathology and Laboratory MedicinePathology and Laboratory Medicine

The University of TexasThe University of Texas--Houston Medical SchoolHouston Medical School

~1015 to 1018 Different Antibodies

and T cell Receptors Produced!

Antibody Structure and Function

Variable DomainsAntigen-BindingActivity

Constant DomainsFunctional Activities

Generation of antigen-binding diversity occurs before antigen exposure through VDJ rearrangement

VDJ rearrangement

Surface IgM/IgD expressing B cells

Exposure to Ag,helper T cells

Development of plasma cells, secretion of Ag-specific antibodies

Isotype Switching -after antigen exposure

Recombination Events Begin with the Heavy Chain Ig Locus

• B cells rearrange their DNA sequences.• 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 other cell types.

Abbrev. Meaning Number Size Function

V Variable ~50 ~95 aa

D Diversity ~20 ~3-6 aa Form part of Variable Domain

J Joining ~6 ~13 aa

C Constant 9* ~110 aa /Domain

Form the Constant Regions

Immunoglobulin heavy chain gene segments

*One locus for each isotype

Germline DNA

L V1 L V2 L VN D1-26 J1-6 C C C3 C4 CC1 C2C2C1

Heavy Chain Locus and VDJ Rearrangement

D-J joiningV-D joining

Looping Out of DNA During Rearrangement

V(D)J recombinase = RAG1 and RAG2

C

C

Expression of Surrogate Light Chains Prior to Light Chain Expression

After successful rearrangement, the

chain is transported to the surface of the cell along with the surrogate light chain proteins, VpreB and 5. The presence of this complex on pre-B cells triggers the initiation of light chain rearrangement.

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Genetic organization and recombination events.

Germline DNA

Rearranged DNA

Primary RNATranscript

mRNA

Somatic Recombination(D-J joining)

Somatic Recombination(V-D-J Joining)

Transcription,RNA Splicing

Translation, ProcessingVH

CJH

DH

Similarly ProcessedLight Chain

Mature Heavy Chain (IgM)

D2L V1 L V2 L VN D1 J4 C C C3 C4 CC1 C2C2C1D3 J5J6

L V1 L V2 J4 C C C3 C4 CC1C2C1D3 J5J6 C2

L V1 L V2 L VN D1-26 J1-6 C C C3 C4 CC1 C2C2C1

V2 J4D3L C

AAAAAC

Light Chain Loci• Two separate loci – kappa () and lambda () on two

different chromosomes• Code for 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

Light Chain Loci

VDJ

4005

3004

Kappa Gene Rearrangement

D-J joining

Result - functional IgM!

5

Coexpression of IgM and IgD - Alternate Splicing

Coexpression IgM and IgD: Alternate Splicing

Stages of B Cell Development

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

Basis of Diversity – Variation within Complementarity Determining Regions

V D J

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 CDRs

3. Assortment of different H and L chains

• Egg Drop Soup• Won Ton Soup• Hot and Sour Soup

• Kung Pao Chicken • Egg Foo Young• Sweet and Sour

Pork• Mongolian Beef• Moo Shoo Pork• Shrimp Chow Mein

3 X 6 = 18 different combinations

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 CDRs– At VJ and VDJ junctions

Junctional and insertional diversity

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, ie. selection of mutants that have a higher affinity for the antigen in secondary responses

Isotype Switching• Definition – change in the heavy chain isotype

expressed by a given B cell.• 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 first IgM, then switch to other isotypes.

• V region and thus antigen binding specificity doesn’t change during isotype switching.

• Isotype switching is stimulated by Ag exposure + cytokines produced by T cells.

Mechanism – deletion of DNA between switch regions

Isotype Class Switching.

Rearranged DNAin B cell

Deletion of InterveningConstant Region

Sequences

Translation,Processing

VH JHDHL C

AAA

Mature Heavy Chain (IgG1)

L VH JH C C C3 C4 CC1C2C1DH C2

Mature Heavy Chain (IgE)

Mature Heavy Chain (IgA2)

VH JHDHL C1

AAAVH JHDHL C2

AAA

IFN- IL-4IL-13 IL-5

Influence of T cell Cytokines

mRNA

L VH JH C4 CC1C2C1DH C2 L VH JH C C1DH C2 L VH JHDH C2

Transcription

Membrane vs. Secreted Ig Expression

• B cells express characterized by membrane- bound immunoglobulins

• Plasma cells characterized by 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 differential termination of transcription – secreted form transcript is shorter, lacks region encoding the membrane (M) exon

Membrane vs. Secreted Ig Expression

Summary• Antigen binding diversity occurs during VDJ

rearrangement, BEFORE antigen exposure.

• The ~1015 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.

A six-year old black male received scheduled immunizations against Diphtheria, tetanus and pertussis throughout the first two years of his life. Upon boosting, he demonstrated increased antibody affinity to binding diphtheria antigens. The mechanism that affects immunoglobulin antigen binding affinity which occurs after formation of a mature B cell is:A. Isotype switching B. Combinatorial diversity C. Junctional diversity D. Somatic hypermutationE. V gene segment diversityOption D (Somatic hypermutation) is correct. Generation of antigen-binding diversity results from recombination of the variable (V), diverse (D), and joining (J) gene segments during B cell development, with somatic mutation occurring after antigenic stimulation to allow increases to occur in affinity. There are five sources of antibody diversity: 1) presence of multiple V gene segments; 2) Combinatorial diversity, resulting from random recombination of V, D, and J segment combinations; 3) junctional and insertional diversity, resulting in changes in the V-D and D-J junctions; 4) coexpression of different H and L chain pairs; and 5) somatic hypermutation.

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!

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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

Stages of B Cell Development

Kuby, Immunology

Clinical Vignette – from Rosen and Geha, “Case Studies in Immunology”

Case: Hyper IgM Immunodeficiency – Dennis Fawcett has recurrent sinus infections due to a defect in CD40 production and isotype switching, which precludes production of immunoglobulins other than IgM.

Rosen and Geha, Fig. 3.3

Defect in signalling protein, CD40

X

Antibody Structure and FunctionHow is it Formed?

Ag-binding diversity• Multiple V segments• V(D)J joining• Heavy, light chain assortment• Junctional/insertional diversity• Somatic mutation

Functional diversity• Biological activity related to Ig isotype• Isotype switching occurs after antigen exposure