Characterisation of the KIR genes in HLA homozygous cell lines.

Report
Anthony Nolan Research Institute
Royal Free Hospital School of Medicine
University College London
Characterisation of the KIR genes
in HLA homozygous cell lines
Christian Garcia
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.
Introduction
19q13.4
Extended LRC
DAP
CD66
LRC
SIGLEC
FcGRT
ILT
LAIR
ILT
KIR
FcR NKp46
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.
A
Introduction
• Killer cell Immunoglobulin-like receptors are encoded by a
polygenic, polymorphic, and polyhaplotypic gene complex.
B
Introduction
• KIR gene polymorphism is not restricted to a single exon, but
distributed all along the gene.
1
2
() 3
4
5
6
7
8
9
Typing strategy
•PCR-SSP approach for the subtyping of inhibitory KIRs:
2DL1, 2DL3, 3DL1, 3DL2
HLA-C
C2 specificity
HLA-C
C1 specificity
HLA-B
Bw4 specificity
•As well as for the typing of the following activating KIRs:
2DS1, 2DS2, 2DS3, 2DS4 & 2DS5.
HLA-A
Typing strategy
1
2
() 3
4
5
6
7
8
9
34 bp
36 bp
327 bp
300 bp
294 bp
51 bp
102 bp
53 bp
ca 200 bp
A
B
C
KIR 2DL1
1.75 kb
0.26 kb
0.28 kb
D
2.2 kb
E
2.2 kb
F
0.23 kb
G
0.23 kb
A
B
C
D
E
F
G
*001
A
B
C
D
E
F
G
*002
A
B
C
D
E
F
G
*005
A
B
C
D
E
F
G
*00301
A
B
C
D
E
F
G
*00302
A
B
C
D
E
F
G
*004
A
B
C
D
E
F
G
Typing strategy
1
2
() 3
4
5
6
7
8
9
34 bp
36 bp
327 bp
300 bp
294 bp
51 bp
102 bp
53 bp
ca 200 bp
A
B
C
1.75 kb
AT Cell Line
0.26 kb
0.28 kb
D
2.2 kb
E
2.2 kb
F
0.23 kb
G
0.23 kb
KIR2DL1: *002, *003
Typing strategy
1
2
() 3
4
5
6
7
8
9
34 bp
36 bp
327 bp
300 bp
294 bp
51 bp
102 bp
53 bp
ca 200 bp
A
B
C
1.75 kb
AT Cell Line
0.26 kb
0.28 kb
D
2.2 kb
E
2.2 kb
F
0.23 kb
G
0.23 kb
KIR2DL1: *002, *003
Strategy
Our current SSP strategy allows us to detect 36 different
KIR alleles and permits us to call unequivocally 26 of them.
KIR2DL1
*001
*002
*00301
*00302
*004
*005
KIR2DL3
*001
*002
*003
*004
*005
*006
KIR3DL1
*00101
*00102
*002
*003
*00401
*00402
*005
*006
*007
*008
KIR3DL2
*001
*002
*003
*004
*005
*006
*007
*008
*009
*010
*011
*012
Results
•Profiled the KIR repertoire of 107 cell lines.
•Same cell lines are completely characterised for HLA.
KIR2DL1
KIR2DL3
KIR3DL1
KIR3DL2
1
KIR3DSs
2 3
4
5
Results
Gene/allele combinations consistent with previously published data.
2DL1
2DL3
3DL1
3DL2
2DS
1 2 3 4 5
Haplotype
PF97387
003,002
002/6,001
002/3/6/7/8, 005
002,010
05,20
SCHU
003,-
001,-
002/3/6/7/8, 005
002,001
12,19
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.
2DL1
2DL3
3DL1
3DL2
2DS
1 2 3 4 5
Haplotype
CB6B
004,-
NEGATIVE
NEGATIVE
007,010
NOVEL
WT47
004,-
NEGATIVE
NEGATIVE
007,010
NOVEL
Results
Cell lines homozygous for KIRs.
2DL1
2DL3
3DL1
3DL2
2DS
1 2 3 4 5
Hp
SPO010
003,-
001,-
005,-
001,-
19,-
SA
003,-
001,-
002/3/6/7/8,-
002,-
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,-
Conclusions
• 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.
Acknowledgements
Anthony Nolan Research Institute
HLA Informatics Group
Steven G.E. Marsh
James Robinson
J. Alejandro Madrigal
Stanford University, California US
Department of Structural Biology
Peter Parham
Heather Schilling
Libby Guethlein

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