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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)
IMGT®, http://imgt.cines.fr
Recombination signals (2)
IMGT®, http://imgt.cines.fr
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
IMGT®, http://imgt.cines.fr
IGHD genes reading frames
IMGT®, http://imgt.cines.fr
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
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
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
Secondary IGV rearrangements (2)
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
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
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
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
IMGT®, http://imgt.cines.fr
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