GENETIC BASIS OF ANTIBODY STRUCTURE AND DIVERSITY. Steven J. Norris, Ph.D

Transcription

1 GENETIC BASIS OF ANTIBODY STRUCTURE AND DIVERSITY Steven J. Norris, Ph.D

2 Topics I. General principles II. 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

3 10 15 to Different Antibodies and T cell Receptors Produced!

4 Antibody Structure and Function Variable Domains Antigen-Binding Activity Constant Domains Functional Activities Coico et al., Fig. 4.3

5 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

6 Isotype Switching - after antigen exposure Coico et al., Fig. 1.1 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

7 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

8 Immunoglobulin heavy chain gene segments Abbrev. Meaning Number Size Function V Variable ~50 ~95 aa D Diversity ~20 ~3-6 aa Form the Variable Domain J Joining ~6 ~13 aa C Constant 9* ~110 aa /Domain Form the Constant Regions *One locus for each isotype

9 Heavy Chain Locus and VDJ Rearrangement V-D joining D-J joining V domain exon

10 Coico et al., Fig. 6.3 Looping Out of DNA During Rearrangement

11 Clinical vignette B cell maturation, from Geha and Notarangelo, Case Studies in Immunology Case 1 X-linked Agammaglobulinemia (XLA) medical student Bill Grignard has normal T cell function, but has almost no B cells or antibodies due to a defect in signaling at the pre-b cell stage. Defect Bruton s tyrosine kinase (btk) mutation B cell development arrested B cells No B Cells! T cells T cells

12 Glacier National Park

13 Expression of Surrogate Light Chains Prior to Light Chain Expression 5 Coico et al., Fig. 7.2

14 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

15 Kappa Gene Rearrangement V-J joining V κ domain exon

16 Result - functional surface IgM! 5

17 Coexpression of IgM and IgD - Alternate Splicing

18 Stages of B Cell Development Coico et al., Fig. 7.1

19 Mesa Verde National Monument

20 Mechanisms of Ag-Binding Diversity Each individual can produce B and T cells with to 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

21 Basis of Diversity Variation within Complementarity Determining Regions V D J Coico et al., Fig. 4.4

22 CDRs (L1, L2, L3, H1, H2, H3) form the Ag-binding pocket (Paratope) and determines Ag-binding specificity Coico et al., Fig. 4.5

23 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

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

25 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 ~ VJ combinations in kappa and lambda loci Affects the diversity of CDR3

26 Chinese Restaurant Example Column A Egg Drop Soup Won Ton Soup Hot and Sour Soup Column B Egg Foo Young Sweet and Sour Pork Mongolian Beef Moo Shoo Pork Shrimp Chow Mein 3 X 5 = 15 different combinations

27 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 x 160 ~2 x 10 6 possibilities

28 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

29 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

30 Puuhonua o Honaunau (Place of Refuge)

31 Isotype Switching Definition change in the heavy chain isotype expressed by a given B cell Example switching from IgM IgG3 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 doesn t change Isotype switching is stimulated by antigen exposure + cytokines produced by T cells

32 Mechanism deletion of DNA between switch regions Coico et al., Fig. 6.5

33 Isotype switching is stimulated by cytokines produced by T helper cells Actor 2 nd edition, Fig. 3-6

34 Clinical Vignette from Geha and Notarangelo, Case Studies in Immunology Case 2 CD40 Ligand Deficiency Dennis Fawcett has recurrent sinus infections due to a defect in CD40L production and isotype switching, which reduces T cell activation and thereby precludes production of immunoglobulins other than IgM. Defect in signalling protein, CD40 ligand X Geha and Notarangelo, Fig. 2.3

35 Membrane vs. Secreted Ig Expression B cells express only membrane-bound 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

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38 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

39 Effect of Enhancers Enhancer Sequence Weak Mucho gusto! +

40 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

41 All members of Ig gene superfamily have domain structure Sequence similarity amino acids per domain Beta-pleated sheet structure Intrachain disulfide bond (Cys-Cys) Example Ig light chain

42 Members of the Ig gene superfamily include:

43 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

44 Summary Antigen binding diversity occurs during VDJ rearrangement, BEFORE antigen exposure. The 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.

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