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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