mechanisms of immunoglobulin diversity

6th ERIC Educational Workshop on IG gene analysis in CLL

Uppsala, 22-23 september 2016

Mechanisms of Immunoglobulin diversity Frédéric Davi

Hopital Pitié-Salpêtrière

Université Pierre & Marie Curie

Paris, France

Ig genes are a different set of genes !

Non-Ig genes

• Structure

– 1 gene / several exons

• Variations

– SNP

– pathogenic mutations

– inherited or acquired

Ig genes

•Struture

– Combination of 2 or 3 genes

(« rearranged » Ig gene)

– Modified (loss of nucleotides)

– Insertion of non-templated nucleotides

•Variations

– SNP

– physiologic mutations

– acquired (affinity maturation)

The B-cell receptor

V region

C region

Ig

CD79a/b

H chain

L chain

Signalling subunits

Antigen binding site

Ig structure

V J C

IGL genes V D J C

IGH genes

VH

CH1

CH2

CH3

VL

CL

V

J

V

J

D1

2

3

VH

CH1

CH2

CH3

VL

CL

V

J

V

J

D1

2

3

Framework Regions (FR)

Complementarity

Determining Regions (CDR)

3D structure of V regions

IMGT®, http://imgt.cines.fr

Generation of diversity

• Combinatorial diversity (1)

– V region coded by 2 or 3 genes

– Reservoir of multiple IG V, D, J genes

– Random assembly

• Junctional diversity

– Imprecise joining at CDR3

• Combinatorial diversity (2)

– Pairing of H and L chain

• Maturation diversity

– Somatic hypermutations

Central (bone marrow)

Peripheral (secondary lymphoid organs)

VDJ recombination

IGHV IGHD IGHJ IGHM

CH1 CH2 CH3 CH4 L

5’ 3’ germline DNA

partially rearranged DNA

rearranged DNA

precursor mRNA

mature mRNA

polypeptide chain

Mu heavy chain

D-J rearrangement

V-D rearrangement

Transcription

Splicing

Translation

Elimination of the signal peptide

Recombination signals (1)


Recombination signals (2)


Types of rearrangements

5’ 3’

V J

Deletion

+

Inversion

7

7 9 9

99

7 7

7 7

9

7

9

coding joint

signal joint

7 9 7 9

7 9

7 9

7 79 9

coding joint signal joint

5’

5’

3’

3’

5’

5’

V J

5’ 3’

V J

Deletion

+

Inversion

7

7 9 9

99

7 7

7 7

9

7

9

coding joint

signal joint

7 9 7 9

7 9

7 9

7 79 9

coding joint signal joint

5’

5’

3’

3’

5’

5’

V J

Mechanisms of VDJ recombination

1. Binding of RAG1/2 and DNA

nicking OH

V J

RS

RAG1/RAG2

2. Synapsis

OH OH

3. Hairpin formation and

cleavage

Coding ends

Signal ends

4. Hairpin opening

5. Processing of DNA ends

6. Ligation

NNNNN

Coding joint Signal

joint

DNA-

PK Artemis

Ku70-

Ku80

TdT

endonucleases

exonucleases

polymerases

XRCC4, DNA ligase IV, Cernunnos

Junctional diversity (P)

G-C-A-T-C-C

C-G-T-A-G-G

T-T-G-G-G-C

A-A-C-C-C-G

G-C-A-T-C-C

C-G-T-A-G-G T-T-G-G-G-C

A-A-C-C-C-G

G-C-A-T-C-C-G

C-G-T-A-G

G-G-G-C

T-T-A-A-C-C-C-G

G-C-A

C-G-T

G-G-G-C

T-T-A-A-C-C-C-G

G-C-A-G-G-C-T-C

C-G-T

G-G-G-C

T-T-A-A-C-C-C-G

G-C-A-G-G-C-T-C-

C-G-T-C-C-G-A-G-

A-A-T-T-G-G-G-C

T-T-A-A-C-C-C-G

N P

RAG cleavage

Hairpin opening

Deletion

N nucleotide addition

Ligation

Terminal

deoxynucleotide

Transferase

Exonuclease

Ligase

IGH V-D-J region


IGHD genes reading frames


Semantics : rearranged IG genes

• Productive

– the coding region has an open reading frame,

– with no stop codon

– and no defect described in the initiation codon, splicing sites and/or regulatory elements

– and an in-frame junction

• Unproductive

– an out-of-frame junction

– and/or the presence of stop codon(s)

– and/or frameshift mutation(s)

– and/or a defect described in the splicing sites

– and/or the regulatory element(s)

– and/or unusual features (translocation, gene fusion...)

– (and/or changes of conserved amino acids demonstrated as leading to uncorrect folding) ?

Temporal regulation of IG gene rearrangements

Jung, Annu Rev. Immunol. 2006

Allelic exclusion / inclusion

V-D-J (+)

D-J

V-D-J (+)

V-D-J (-)

V-D-J (+)

V-D-J (+)

V-D-J (+) V-D-J (+)

33% 2% 65%

Secondary IGV rearrangements (1) V1 V2 V3 V4 J1 J2 J3

V1 V2 V3 J2 J3

V1 V2 J3

Light chain edition

IGVH replacement

Initial V-J rearrangement

Secondary V-J rearrangement

J1 J2 J3

D1 D2 D3 D4

J2 J3

D4

V4V3V2V1

V3V2V1

J2 J3

D4

V2V1

Initial V-D-J rearrangement

Secondary V-V rearrangement

V1 V2 V3 V4 J1 J2 J3

V1 V2 V3 J2 J3

V1 V2 J3

Light chain edition

IGVH replacement



J1 J2 J3

D1 D2 D3 D4

J2 J3

D4

V4V3V2V1

V3V2V1

J2 J3

D4

V2V1



J1 J2 J3

D1 D2 D3 D4

J2 J3

D4

V4V3V2V1

V3V2V1

J2 J3

D4

V2V1



Secondary IGV rearrangements (2)

V1 V2 V3 V4 J1 J2 J3

V1 V2 V3 J2 J3

V1 V2 J3

Light chain edition

IGVH replacement



J1 J2 J3

D1 D2 D3 D4

J2 J3

D4

V4V3V2V1

V3V2V1

J2 J3

D4

V2V1



V1 V2 V3 V4 J1 J2 J3

V1 V2 V3 J2 J3

V1 V2 J3

Light chain edition

IGVH replacement



J1 J2 J3

D1 D2 D3 D4

J2 J3

D4

V4V3V2V1

V3V2V1

J2 J3

D4

V2V1



J1 J2 J3

D1 D2 D3 D4

J2 J3

D4

V4V3V2V1

V3V2V1

J2 J3

D4

V2V1



Peripheral maturation

B-cell peripheral maturation

Kuppers, Nat Rev Cancer, 2005

Somatic hypermutations

• Mostly point mutations

• Mutation rate = 10-3 to 10-4 per base cell per cell generation

• Localized : start near the 5’ end of the V-D-J-EXON and extend for 1-2 kb

• Preferred targets motifs (hotspots)

– (A/T)A [WA] G(C/T)(A/T) [GYW]

– Complementary T(A/T) [TW] (A/T)(A/G)C [WRC]

• Transitions (e.g. C>T, G>A) are more frequent than transversions (e.g. C>A or G, G>C or T).

• Silent (S) or aminoacid replacement (R) or stop codon, in FR and CDR

• Antigen selection: R/S ratio high in CDR and low FR

Evolution of SHM during immune response

X 1

x 10

x 100

affinity

Germline IG genes : semantics

• Functional gene

the coding region has an open reading frame without stop codon, and if there is no described defect

in the splicing sites, recombination signals and/or regulatory elements

• Open Reading Frame (ORF)

the coding region has an open reading frame, but :

– alterations in the splicing sites, recombination signals and/or regulatory elements

– and/or changes of conserved amino acids that can lead to uncorrect folding

– and/or the entity is an ORPHON

• Orphons

Genes on chromosomal localizations outside the main loci, and do not contribute to the synthesis of

Ig, even if they have an ORF

• Pseudogene

the coding region has stop codon(s) and/or frameshift mutation(s) or there is a mutation in the

initiating ATG codon (L-PART1)

Germline organization of the IGH locus

5’

3’

L IGHV IGHD IGHJ E IGHM IGHD IGHG3 IGHG1

IGHEP1 IGHA1 IGHGP IGHG2 IGHG4 IGHE IGHA2 E

Germline organization of the IGK locus

L IGKV (distal) L IGKV (proximal) IGKJ E IGKC E KDE

3’ 5’

Germline organization of the IGL locus

L IGLV IGLJ1 IGLC1 J2 IGLC2 J3 IGLC3 J4 IGLC4 J5 IGLC5 J6 IGLC6 J7 IGLC7 E

5’ 3’

Polymorphism of the IGHV genes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

E V Q L V E S G G G L V Q P G G S L R

IGHV3-13*01 GAG GTG CAG CTG GTG GAG TCT GGG GGA ... GGC TTG GTA CAG CCT GGG GGG TCC CTG AGA

H A

IGHV3-13*02 --- --- --T --- --- --- --- --- --- ... --- --- --- --- --- --- --- G-- --- ---

IGHV3-13*03 --- --- --- --- --- --- --- --- --- ... --- --- --- --- --- --- --- --- --- ---

___________________CDR1-IMGT____________________

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

L S C A A S G F T F S S Y D M H

IGHV3-13*01 CTC TCC TGT GCA GCC TCT GGA TTC ACC TTC AGT AGC TAC GAC ... ... ... ... ATG CAC

N

IGHV3-13*02 --- --- --- --- --- --- --- --- --- --- --- -A- --- --- ... ... ... ... --- ---

C

IGHV3-13*03 --- --- --- --- --- -G- --- --- --- --- --- --- --- --- ... ... ... ... --- ---

_______________CDR2-

41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

W V R Q A T G K G L E W V S A I G T A G

IGHV3-13*01 TGG GTC CGC CAA GCT ACA GGA AAA GGT CTG GAG TGG GTC TCA GCT ATT GGT ACT GCT GGT

N

IGHV3-13*02 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --C -A- --- --- --- ---

IGHV3-13*03 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

IMGT________________

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

D T Y Y P G S V K G R F T I S R

IGHV3-13*01 GAC ACA ... ... ... TAC TAT CCA GGC TCC GTG AAG ... GGC CGA TTC ACC ATC TCC AGA

IGHV3-13*02 --- --- ... ... ... --- --- --- --- --- --- --- ... --G --- --- --- --- --- ---

Q

IGHV3-13*03 --- --- ... ... ... --- --- --- --- --- --- --- ... --- -A- --- --- --- --- ---

81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

E N A K N S L Y L Q M N S L R A G D T A

IGHV3-13*01 GAA AAT GCC AAG AAC TCC TTG TAT CTT CAA ATG AAC AGC CTG AGA GCC GGG GAC ACG GCT

IGHV3-13*02 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

IGHV3-13*03 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---

_CDR3-IMGT__

101 102 103 104 105 106

V Y Y C A R

IGHV3-13*01 GTG TAT TAC TGT GCA AGA GA

IGHV3-13*02 --- --- --- --- --- --- --

IGHV3-13*03 --- --- --- --- --- ---

Polymorphism of the IGLC genes


Combinatorial diversity

55 IGHV 23 IGHD 6 IGHJ 7590 combinations

41 IGKV 5 IGKJ 205 combinations

33 IGLV 5 IGLJ 165 combinations

IGH-IGK 1.6 x 106 combinations

Total 2.8 x 106 combinations

IGH-IGL 1.2 x 106 combinations

IGH

IGK/L

Combinatorial + junctional diversity

55 IGHV 23 IGHD 6 IGHJ 7590 combinations

41 IGKV 5 IGKJ 205 combinations

33 IGLV 5 IGLJ 165 combinations

IGH

IGK/L

2,8 x 106 109 – 1012 combinations

Repertoire limitation and shaping

• Selection at the pre-BCR level

– Pairing with pseudo-light chain

• Selection at the immature BCR level

– Pairing with light chain

– Autoreactivity

• Selection at the mature BCR level

– Affinity maturation

109 – 1012 combinations 106 – 107 BCR types

Normal IGHV repertoire

Wu, Blood 2010

CLL IGHV repertoire

Agathangelidis - Blood 2012

mechanisms of immunoglobulin diversity

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