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Characterisation of the KIR genes in HLA homozygous cell lines Christian Garcia Anthony Nolan Research Institute Royal Free Hospital School of Medicine.

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Presentation on theme: "Characterisation of the KIR genes in HLA homozygous cell lines Christian Garcia Anthony Nolan Research Institute Royal Free Hospital School of Medicine."— Presentation transcript:

1 Characterisation of the KIR genes in HLA homozygous cell lines Christian Garcia Anthony Nolan Research Institute Royal Free Hospital School of Medicine University College London

2 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.

3 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.

4 DAPCD66SIGLECLAIRILT KIR Fc  R FcGRTNKp46 19q13.4 LRCExtended LRC Introduction

5 Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex. Introduction

6 Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex. Introduction A

7 Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex. Introduction B

8 KIR gene polymorphism is not restricted to a single exon, but distributed all along the gene. Introduction 12 (  )

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-C C2 specificity HLA-B Bw4 specificity HLA-A HLA-C C1 specificity

10 1.75 kb 0.26 kb 0.28 kb 2.2 kb 0.23 kb A B C D E F G Typing strategy 36 bp327 bp300 bp294 bp51 bp 102 bp 53 bp ca 200 bp 34 bp 12 (  ) ABCDEGF *001 *002 *005 *00301 *00302 *004 ABCDEGF ABCDEGF ABCDEGF ABCDEGF ABCDEGF ABCDEGF KIR 2DL1

11 1.75 kb 0.26 kb 0.28 kb 2.2 kb 0.23 kb A B C D E F G KIR2DL1: *002, *003 AT Cell Line Typing strategy 36 bp327 bp300 bp294 bp51 bp 102 bp 53 bp ca 200 bp 34 bp 12 (  )

12 1.75 kb 0.26 kb 0.28 kb 2.2 kb 0.23 kb A B C D E F G KIR2DL1: *002, *003 AT Cell Line Typing strategy 36 bp327 bp300 bp294 bp51 bp 102 bp 53 bp ca 200 bp 34 bp 12 (  )

13 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

14 Results Profiled the KIR repertoire of 107 cell lines. Same cell lines are completely characterised for HLA. KIR2DL1KIR2DL3KIR3DL1KIR3DL2 KIR3DSs 12345

15 Gene/allele combinations consistent with previously published data. PF ,002002/6,001002/3/6/7/8, ,010 2DL12DL33DL13DL2Haplotype 05,20 SCHU 003,-001,-002/3/6/7/8, ,00112,19 2DS HOR 002,-002/6,-NEGATIVE006,00724,35 RML 003,-001,-002/3/6/7/8,-002,00712,33 Gene/allele combinations novel or previously not known. CB6B 004,-NEGATIVE 007,010 2DL12DL33DL13DL2Haplotype NOVEL WT47 004,-NEGATIVE 007,010 2DS NOVEL Results

16 Cell lines homozygous for KIRs. SPO ,-001,-005,-001,- 2DL12DL33DL13DL2 Hp 19,- SA 003,-001,-002/3/6/7/8,-002,- 2DS ,- SPL 003,-001,-005,-010,-20,- EMJ 003,-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

17 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

18 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

19 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 Acknowledgements


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