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