characterisation of the kir genes in hla homozygous cell lines christian garcia anthony nolan...
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Characterisation of the KIR genes in HLA homozygous cell lines
Christian Garcia
Anthony Nolan Research InstituteRoyal Free Hospital School of Medicine
University College London
Introduction
• The availability of Homozygous Typing Cells for the HLA region has been of huge benefit in the study of the human MHC.
• No such panel of well characterised material exists for the KIR genes.
• In an attempt to provide such material we have analysed the KIR genes in the original 10th IHW cell line panel comprising 107 cell lines.
Aims of this study
• Characterise the KIR gene/allele profile of HLA homozygous cell lines.
• Determine the KIR haplotypes present in HLA homozygous cell lines.
• Define a set of cell lines which are homozygous for the KIR gene region, from those cells which are both homozygous for the HLA region and consanguineous.
DAP CD66 SIGLEC LAIRILT ILT KIR FcRFcGRT NKp46
19q13.4
LRCExtended LRC
Introduction
• Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex.
Introduction
• Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex.
Introduction
A
• Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex.
Introduction
B
• KIR gene polymorphism is not restricted to a single exon, but distributed all along the gene.
Introduction
1 2 () 3 4 5 6 7 8 9
•PCR-SSP approach for the subtyping of inhibitory KIRs:
2DL1, 2DL3, 3DL1, 3DL2
•As well as for the typing of the following activating KIRs:
2DS1, 2DS2, 2DS3, 2DS4 & 2DS5.
Typing strategy
HLA-CC2 specificity
HLA-BBw4 specificity
HLA-AHLA-C
C1 specificity
1.75 kb
0.26 kb
0.28 kb
2.2 kb
2.2 kb
0.23 kb
0.23 kb
A
B
C
D
E
F
G
Typing strategy
36 bp 327 bp 300 bp 294 bp 51 bp 102 bp 53 bp ca 200 bp34 bp
1 2 () 3 4 5 6 7 8 9
A B C D E GF
*001
*002
*005
*00301
*00302
*004
A B C D E GF
A B C D E GF
A B C D E GF
A B C D E GF
A B C D E GF
A B C D E GF
KIR 2DL1
1.75 kb
0.26 kb
0.28 kb
2.2 kb
2.2 kb
0.23 kb
0.23 kb
A
B
C
D
E
F
GKIR2DL1: *002, *003
AT Cell Line
Typing strategy
36 bp 327 bp 300 bp 294 bp 51 bp 102 bp 53 bp ca 200 bp34 bp
1 2 () 3 4 5 6 7 8 9
1.75 kb
0.26 kb
0.28 kb
2.2 kb
2.2 kb
0.23 kb
0.23 kb
A
B
C
D
E
F
GKIR2DL1: *002, *003
AT Cell Line
Typing strategy
36 bp 327 bp 300 bp 294 bp 51 bp 102 bp 53 bp ca 200 bp34 bp
1 2 () 3 4 5 6 7 8 9
Our current SSP strategy allows us to detect 36 different KIR alleles and permits us to call unequivocally 26 of them.
KIR2DL1 KIR2DL3 KIR3DL1 KIR3DL2
*001*002*00301*00302*004*005
*001*002*003*004*005*006
*00101*00102*002*003*00401*00402
*001*002*003*004*005*006
*007*008*009*010*011*012
*005*006*007*008
Strategy
Results
•Profiled the KIR repertoire of 107 cell lines.
•Same cell lines are completely characterised for HLA.
KIR2DL1 KIR2DL3 KIR3DL1 KIR3DL2
KIR3DSs
1 2 3 4 5
Gene/allele combinations consistent with previously published data.
PF97387 003,002 002/6,001 002/3/6/7/8, 005
002,010
2DL1 2DL3 3DL1 3DL2 Haplotype
05,20
SCHU 003,- 001,- 002/3/6/7/8, 005
002,001 12,19
2DS1 2 3 4 5
HOR 002,- 002/6,- NEGATIVE 006,007 24,35
RML 003,- 001,- 002/3/6/7/8,- 002,007 12,33
Gene/allele combinations novel or previously not known.
CB6B 004,- NEGATIVE NEGATIVE 007,010
2DL1 2DL3 3DL1 3DL2 Haplotype
NOVEL
WT47 004,- NEGATIVE NEGATIVE 007,010
2DS1 2 3 4 5
NOVEL
Results
Cell lines homozygous for KIRs.
SPO010 003,- 001,- 005,- 001,-
2DL1 2DL3 3DL1 3DL2 Hp
19,-
SA 003,- 001,- 002/3/6/7/8,- 002,-
2DS1 2 3 4 5
12,-
SPL 003,- 001,- 005,- 010,- 20,-
EMJ 003,- 001,- 001,- 001,- 10,-
DUCAF 003,- 001,- 005,- 001,- 9,-
KT17 003,- 001,- 005,- 006,- -
TUBO 002,- 002/6,- 005,- 001,- 9,-
HID 003,- 001,- 002/3/6/7/8,- 002,- 12,-
Results
• The PCR-SSP technique is suitable for high resolution routine typing.
• This study constitutes the most comprehensive characterisation of the KIR gene repertoire in cell lines that have previously been thoroughly characterised for their HLA profile.
Conclusions
• Most cell lines in this panel have a Caucasoid ethnicity with representatives of Oriental, Amerindian, Hispanic and Black origin also present. However,
• Studies on non- Caucasoid populations should allow us to further characterise the complexity of the KIR gene polymorphism and haplotypes.
Conclusions
Anthony Nolan Research InstituteHLA Informatics Group
Steven G.E. MarshJames Robinson
J. Alejandro Madrigal
Stanford University, California USDepartment of Structural Biology
Peter ParhamHeather SchillingLibby Guethlein
Acknowledgements