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