1/26/11. VDJ Recombination - Diversity. Review - Immunoglobin structure. Multiple Functions. Lecture 4 Finish antibody subclasses

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1 Recombination - Diversity Lecture 4 Finish antibody subclasses Review - Immunoglobin structure 2 Heavy & Light Chains Disulfide bonds Inter-chain Intra-chain Variable & Constant Regions V L & C L V H & C H Hinge Region V L Disulfide bond Carbohydrate C L C C H2 C H3 H1 Hinge Region V H Multiple Functions 3 1

2 Human Immunoglobin Classes 4 Another way to view the subtypes Immunoglobin Classes 5 IgG - Gamma (γ) heavy chains IgM - Mu (µ) heavy chains IgA - Alpha (α) heavy chains IgD - Delta (δ) heavy chains Ig - psilon (ε) heavy chains Designated by differences in the heavy chain Immunoglobin Subclasses 6 Can be further divided based on small differences in amino acid sequence in constant region of H chain IgG Subclasses IgG1 - Gamma 1 (γ1) heavy chains IgG2 - Gamma 2 (γ2) heavy chains IgG3 - Gamma 3 (γ3) heavy chains IgG4 - Gamma 4 (γ4) heavy chains IgA subclasses IgA1 - Alpha 1 (α1) heavy chains IgA2 - Alpha 2 (α2) heavy chains 2

3 Three Dimensional Structure 7 Response to a Pathogen 8 Humoral Immune Response 9 3

4 Antigenic Determinants 10 Learning Objectives 11 Describe the organization of immunoglobulin gene families Describe the expression of immunoglobulin genes xplain mechanisms of antibody diversity xplain mechanisms of class switching Describe overview of clonal selection and expansion Diversity: Multiple Germline Genes 12 V H Locus: J H Locus: D H Locus: 123 V H genes on chromosome functional V H genes with products identified 79 pseudo V H genes 4 functional V H genes - with no products identified 24 non-functional, orphan V H sequences on chromosomes 15 & 16 9 J H genes 6 functional J H genes with products identified 3 pseudo J H genes 27 D H genes 23 functional D H genes with products identified 4 pseudo D H genes Additional non-functional D H sequences on the chromosome 15 orphan locus reading D H regions in 3 frames functionally increases number of D H regions 4

5 13 Heavy Chain Gene Families (Germline) Heavy chain genes; Vn=1000, Dn=15 V1 V2 Vn D1 D2 D3 Dn J1 J2 J3 J4 J5 C µ C δ C γ3 C γ1 C γ2 C γ4 C ε C α1 C α2 C H 1 H C H 2 C H 3 C H 4 Introns separate exons coding for H chain domains Heavy Chain Gene Rearrangement and xpression 14 V1 V2 Vn D1 D2 D3 Dn J1 J2 J3 J4 J5 C µ C δ DJ rearrangement V1 V2 Vn D1 D2 J4 J5 C µ C δ DNA rearrangement V1 L V2 D2 J4 J5 C µ C δ DNA Transcription L V2 D2 J4 J5 C µ C δ Primary transcript RNA Heavy Chain Gene Rearrangement and xpression 15 L V2 D2 J4 J5 C µ C δ L V2 D2 J4 J5 C µ C δ Primary transcript RNA Processing L V C µ L V C δ A n A n mrna for C µ mrna for C δ Translation L V C µ L V C δ C µ heavy chain C δ heavy chain V C µ Transport to R V C δ V C C µ heavy chain V C C δ heavy chain 5

6 Diversity: Multiple Germline Genes 16 V κ & J κ Loci: 132 Vκ genes on the short arm of chromosome 2 29 functional Vκ genes with products identified 87 pseudo Vκ genes 15 functional Vκ genes - with no products identified 25 orphans Vκ genes on the long arm of chromosome 2 5 Jκ regions V λ & J λ Loci: 105 Vλ genes on the short arm of chromosome 2 30 functional genes with products identified 56 pseudogenes 6 functional genes - with no products identified 13 relics (<200bp Vλ of sequence) 25 orphans on the long arm of chromosome 2 4 Jλ regions Light Chain Gene Families (Germline) 17 Lambda light chain genes; n=30 V λ 1 V λ 2 V λ n J λ 1 C λ 1 J λ 2 C λ 2 J λ 3 C λ 3 J λ 4 C λ 4 Kappa light chain genes; n=300 L V κ 1 L V κ 2 L V κ n J κ 1 J κ 2 J κ 3 J κ 4 J κ 5 C κ P P P Light Chain Gene Families Gene rearrangments and expression V κ 1 V κ 2 V κ n J κ 1 J κ 2 J κ 3 J J κ 5 C κ apple4 DNA 18 DNA Rearrangement V 2 J 4 V κ 1 κ κ J κ 5 C κ DNA Transcription V J C Primary transcript RNA RNA Processing V J C mrna RNA Translation V J C Protein Transport to R V J Protein V C 6

7 Final Products! 19 Reading D segments in 3 frames 20 Analysis of D (diversity) regions from different antibodies One D region can be used in any of three frames Different protein sequences lead to antibody diversity GGGACAGGGGGC GlyThrGlyGly GGGACAGGGGGC GlyGlnGly GGGACAGGGGGC AspArgGly Frame 1 Frame 2 Frame 3 stimates of Combinatorial Diversity 21 Using functional V D and J genes: 40 VH x 27 DH x 6JH = 6,480 combinations D can be read in 3 frames: 6,480 x 3 = 19,440 combinations 29 Vκ x 5 Jκ = 145 combinations 30 Vλ x 4 Jλ = 120 combinations = 265 different light chains If H and L chains pair randomly as H 2 L 2 i.e. 19,440 x 265 = 5,151,600 possibilities Due only to COMBINATORIAL diversity In practice, some H + L combinations are unstable. Certain V and J genes are also used more frequently than others. Other mechanisms add diversity at the junctions between genes JUNCTIONAL diversity 7

8 Antibody Switches 22 Antibody Isotype (Class) Switching 23 Throughout an immune response the specificity of an antibody will remain the same (notwithstanding affinity maturation) The effector function of antibodies throughout a response needs to change drastically as the response progresses. Antibodies are able to retain variable regions while exchanging constant regions that contain the structures that interact with cells. Organization of the functional human heavy chain C region genes J regions Cµ Cδ Cγ1 Cγ2 Cγ4 Cε Cα2 Switch Regions 24 Cµ Sµ Cδ Cγ1 Cγ2 Cγ4 Cε Cα2 Sγ3 Sγ1 Sα1 Sγ2 Sγ4 Sε Sα2 Upstream of C regions are repetitive regions of DNA called switch regions. (The exception is the Cδ region that has no switch region). The Sµ consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar in may ways to V(D)J recombination. Isotype switching does not take place in the bone marrow, however, and it will only occur after B cell activation by antigen and interactions with T cells. 8

9 Switching Recombination 25 Cµ Cδ Cγ1 Cγ2 Cγ4 Cε Cα2 Cδ Cδ Sγ3 Cµ Cµ Cγ1 Sγ1 IgG3 produced. Switch from IgM IgA1 produced. Switch from IgG3 IgA1 produced. Switch from IgM At each recombination constant regions are deleted from the genome An Ig - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1 Antibody Class Switching 26 Maturation and Selection 27 Positive Selection preservation of useful specificity Negative Selection Apoptosis Receptor diting Central tolerance 9

10 Clonal Selection and xpansion 28 Independent of exogenous antigen exposure: repertoire of clones are generated (predetermined shape and specificity) greater than 10,000,000 clones 10