VDJ Recombination - Diversity · 2011. 1. 27. · VDJ Recombination - Diversity Lecture 4 Finish...
Transcript of VDJ Recombination - Diversity · 2011. 1. 27. · VDJ Recombination - Diversity Lecture 4 Finish...
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VDJ Recombination - Diversity
Lecture 4 Finish antibody subclasses
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Review - Immunoglobin structure
Heavy & Light Chains Disulfide bonds Inter-chain Intra-chain Variable & Constant Regions
– VL & CL – VH & CH
Hinge Region
CH1
VL CL
VH CH2 CH3
Hinge Region
Carbohydrate
Disulfide bond
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Multiple Functions
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Human Immunoglobin Classes
Another way to view the subtypes
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Immunoglobin Classes
• IgG - Gamma (γ) heavy chains • IgM - Mu (µ) heavy chains • IgA - Alpha (α) heavy chains • IgD - Delta (δ) heavy chains • IgE - Epsilon (ε) heavy chains
• Designated by differences in the heavy chain
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Immunoglobin Subclasses
• 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
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Three Dimensional Structure
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Response to a Pathogen
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Humoral Immune Response
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Antigenic Determinants
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Learning Objectives
• Describe the organization of immunoglobulin gene families
• Describe the expression of immunoglobulin genes
• Explain mechanisms of antibody diversity
• Explain mechanisms of class switching
• Describe overview of clonal selection and expansion
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Diversity: Multiple Germline Genes • 123 VH genes on chromosome 14 • 40 functional VH genes with products identified • 79 pseudo VH genes • 4 functional VH genes - with no products identified • 24 non-functional, orphan VH sequences on
chromosomes 15 & 16
VH Locus:
JH Locus: • 9 JH genes • 6 functional JH genes with products identified • 3 pseudo JH genes
DH Locus: • 27 DH genes • 23 functional DH genes with products identified • 4 pseudo DH genes • Additional non-functional DH sequences on the
chromosome 15 orphan locus • reading DH regions in 3 frames functionally increases
number of DH regions
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Heavy Chain Gene Families (Germline)
Introns separate exons coding for H chain domains
Heavy chain genes; Vn=1000, Dn=15 V1 Vn V2 D2 D3 D1 Dn
Cµ Cδ Cγ3 Cε C γ2 C γ1 Cγ4 Cα1 Cα2
J2 J3 J5 J1 J4
E
CH1 H CH2 CH3 CH4
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Heavy Chain Gene Rearrangement and Expression
V1 Vn V2 D2 D3 D1 Dn Cµ Cδ J2 J3 J5 J1 J4
E
Transcription
D2 Cµ Cδ J5 J4
E
L V2
RNA Primary transcript
DJ rearrangement
Vn V1 V2 D2 D1 Cµ Cδ J5 J4
EDNA
Cµ Cδ
VDJ rearrangement
D2 J4 V2 J5
E
V1 L
DNA
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Heavy Chain Gene Rearrangement and Expression
EPrimary transcript
E
J4 D2 V2 Cµ Cδ J5 L Cµ Cδ J5 L D2 J4 V2
RNA Processing
An mRNA for Cµ
Cµ L V Cδ L V
An mRNA for Cδ
Translation
Cµ heavy chain
Cµ L V Cδ L V
Cδ heavy chain
Transport to ER
Cµ heavy chain V C
Cµ V Cδ V
Cδ heavy chain V C
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Diversity: Multiple Germline Genes
• 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
Vκ & Jκ 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
Vλ & Jλ Loci:
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Light Chain Gene Families (Germline)
Jλ1 Cλ1
E
Jλ2 Cλ2
E
Jλ3 Cλ3
E
Jλ4 Cλ4
E
Vλ1 Vλn Vλ2
Lambda light chain genes; n=30
P
L V κ 1
P
L V κ n
P
L V κ 2 Jκ2 Jκ3 Jκ5 Cκ
E
Jκ1 Jκ4
Kappa light chain genes; n=300
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Light Chain Gene Families
Vκ 1 Vκ n Vκ 2 Jκ2 Jκ3 Jκ5 Cκ Jκ1 J4 DNA E
E
Vκ 1 Jκ5 Cκ Vκ 2 Jκ4
DNA
DNA Rearrangement
V J
V C
Protein
Transport to ER
Protein
Translation CV J
E
V C J
RNA
Transcription
Primary transcript
CV JRNA Processing
RNA mRNA
Gene rearrangments and expression
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Final Products!
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Reading D segments in 3 frames
GGGACAGGGGGC GlyThrGlyGly
GGGACAGGGGGC GlyGlnGly
GGGACAGGGGGC AspArgGly
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
Frame 1
Frame 2
Frame 3
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Estimates of Combinatorial Diversity
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 H2L2 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
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Antibody Switches
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Antibody Isotype (Class) Switching
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.
J regions Cα2 Cε Cγ4 Cγ2 Cα1 Cγ1 Cγ3 Cδ Cµ
Organization of the functional human heavy chain C region genes
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Switch Regions
Cα2 Cε Cγ4 Cγ2 Cα1 Cγ1 Cγ3 Cδ Cµ
• 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.
Sγ3 Sγ1 Sα1 Sγ2 Sγ4 Sε Sα2 Sµ
• Upstream of C regions are repetitive regions of DNA called switch regions. (The exception is the Cδ region that has no switch region).
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Switching Recombination
Cα2 Cε Cγ4 Cγ2 Cα1 Cγ1 Cγ3 Cδ Cµ
Cµ
Cδ
Cγ3 VDJ
Sγ3
Cµ
Cδ
Cγ3
VDJ
Cγ1 Sγ1
Cα1
Cγ3
VDJ Cα1
Cγ3 VDJ
IgG3 produced. Switch from IgM
VDJ Cα1
IgA1 produced. Switch from IgG3
VDJ Cα1
IgA1 produced. Switch from IgM
At each recombination constant regions are deleted from the genome An IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1
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Antibody Class Switching
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Maturation and Selection
• Positive Selection preservation of useful specificity
• Negative Selection Apoptosis Receptor Editing
• Central tolerance
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Clonal Selection and Expansion
Independent of exogenous antigen exposure: repertoire of clones are generated (predetermined shape and specificity) greater than 10,000,000 clones