Immunoglobulin Genetics

Problem…the immune system makes over one billion different antibody proteins

• In 1950’s: central dogma stated DNA—to RNA—to protein

• One gene for each protein• Required millions of genes just for the immune system• Does not seem possible, but most scientists thought it

might be• Today we know the human genome is less than 30,000

genes• So, what is really going on???

B lymphocyte development

B lymphocyte development (2)

Immunoglobulin GeneticsHistory

• Same C region could associate with many V regions– IgG Ab with different specificities

• Same V region could associate with many C regions– Same idiotype on IgG and IgM Ab

• V and C regions coded for by separate genes

Genes for immunoglobulin proteins are found on different chromosomes

Light Chain Gene FamiliesGerm line gene organization

J1 C1

E

J2 C2

E

J3 C3

E

J4 C4

EP

L V1

P

L Vn

P

L V2

Lambda light chain genes; n=30

P

L V 1

P

L V n

P

L V 2 J2 J3 J5 C

E

J1 J4

Kappa light chain genes; n=300

Light Chain Gene FamiliesGene rearrangement and expression

L V 1 L V nL V 2 J2 J3 J5 CJ1 J4

DNAP PP E

P P E

L V 1 L J5 C

V 2 J4

DNA

DNA Rearrangement

V CJ

V C

Protein

Transport to ER

Protein

TranslationCL V J

E

L V CJ

RNA

Transcription

Primary transcript

CL V JRNA Processing

RNAmRNA

Heavy Chain Gene FamilyGerm line gene organization

Introns separate exons coding for H chain domains

Heavy chain genes; Vn=1000, Dn=15

P

L V1

P

L Vn

P

L V2 D2 D3D1 Dn

C C C3 CC 2C 1 C 4 C1 C2

J2 J3 J5J1 J4

E

CH1 H CH2 CH3 CH4

Heavy Chain Gene FamilyGene rearrangement and expression

P

L V1

P

L Vn

P

L V2 D2 D3D1 Dn C CJ2 J3 J5J1 J4

E

DJ rearrangement

P

L Vn

P

L V1

P

L V2 D2D1 C CJ5J4

EDNA

VDJ rearrangement

D2 C CJ5J4

EP

L V1

P

LV2

DNA

Transcription

D2 C CJ5J4

E

LV2

RNAPrimary transcript

Heavy Chain Gene FamilyGene rearrangement and expression

EPrimary transcript

E

J4D2V2 C CJ5L C CJ5L D2 J4V2

RNA Processing

An

mRNA for C

CL DJV CL DJV

An

mRNA for C

Translation

C heavy chain

CL DJV CL DJV

C heavy chain

Transport to ER

C heavy chainV C

CDJV CDJV

C heavy chainV C

Mechanism of DNA Rearrangements

• Recombination signal sequences (RSS)– Nonmer

– Heptamer

– 1 or 2 turn signals

• Rag-1 and Rag-2

L V

2

J C

1

Lambda light chains

L V

1

J C

2

Kappa light chains

Heavy chains

L V

2

J C

211

D

Heptamer

CACAGTG

GTGTCAC

Nonamer

ACAAAAACC

TGTTTTTGG

23 bp

23 bp

Two turn RSS

2

Nonamer

GGTTTTTGT

CCAAAAACA

Heptamer

CACTGTG

GTGACAC

12 bp

12 bp

One turn RSS1

Order of Ig Gene Expression

+

-

Heavy chain gene

VDJ recombination

Productive rearrangement ?

Yes

No

Transcribe/translate

1st

2nd

or

Apoptosis

Progenitor B cell

Pre B cell

() Heavy chain

PaternalMaternal

Order of Ig Gene Expression

+

-

Light chain gene

VJ recombination

Productive rearrangement ?

Yes

No

Transcribe/translate

Light chain

4 th

Apoptosis

1st -

2nd -

3rd -

4th -

or

Pre B cell

Mature B cell

+

Order of Ig Gene Expression Explains:

• Why an individual B cell only produces one kind of Ig with one kind of heavy and one kind of light chain

• Why an individual B cell makes only makes antibodies of one specificity

• Why there is allelic exclusion

Origin of Antibody DiversityHistory

• Definition – Diversity (repitoire) is the total of all the Ab specificities that an organism is capable of expressing

• Theories– Germ line theory– Somatic mutation theory

Origin of Antibody DiversityCurrent concepts

• Multiple V genes

• V-J and V-D-J joining

• Junctional diversity

• N region insertions– Amino acid sequences not encoded in the germ line

D region

T GGC CC

Pro Trp

C C C C C C

V gene

T GG

D region

C C C C C C

V gene

T GG

C GGC CC

Pro Arg

C C C C C C

V gene

T GG

D region

C GCC CC

Pro Pro

-GTCAATG

-GTCAATG- +

Break DNA

TdT adds nucleotides

Ligate

N region insertion Intron

JD

J

J

J

D

D

D

Generation of N region insertions

Origin of Antibody DiversityCurrent concepts

• Multiple V genes

• V-J and V-D-J joining• Junctional diversity• N region insertions• Somatic mutation• Combinatorial association• Multispecificity

Generation of DiversityB cell receptor

(Immunoglobulin)

Heavy Light

V gene segments 1000 300

D gene segments 15 -

J gene segments 4 4

N region insertion ++ -

Junctional diversity +++ +

Somatic mutation + +

V x D x J

1000 x 15 x 4 V x J

300 x 4 Total 6 x 104 1.2 x 103

Combinatorial association 7.2 x 107

• Ag independent process • Clonal selection

Copyright © 2007 by W. H. Freeman and Company

ANTIBODY GENE REARRANGEMENTS

CLASS SWITCHING:

Selection of CH Genes

Mechanism Unclear, but InvolvesSwitch RegionSwitch RecombinaseIsotype Specific Cytokine Signals

Class Switching among Constant-Region GenesClass Switching among Constant-Region Genes