![Page 1: Characterisation of the KIR genes in HLA homozygous cell lines Christian Garcia Anthony Nolan Research Institute Royal Free Hospital School of Medicine](https://reader030.vdocument.in/reader030/viewer/2022032722/56649cda5503460f949a47bb/html5/thumbnails/1.jpg)
Characterisation of the KIR genes in HLA homozygous cell lines
Christian Garcia
Anthony Nolan Research InstituteRoyal Free Hospital School of Medicine
University College London
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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.
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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.
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DAP CD66 SIGLEC LAIRILT ILT KIR FcRFcGRT NKp46
19q13.4
LRCExtended LRC
Introduction
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• Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex.
Introduction
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• Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex.
Introduction
A
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• Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex.
Introduction
B
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• 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
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•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
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B
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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
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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
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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
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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
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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
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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
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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
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• 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
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• 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
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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