online May 1, 2013 originally publisheddoi:10.1182/blood-2012-10-461442

2013 121: 4703-4707  

John Trowsdale and Karl-Johan MalmbergVivien Béziat, James A. Traherne, Lisa L. Liu, Jyothi Jayaraman, Monika Enqvist, Stella Larsson, Influence of KIR gene copy number on natural killer cell education 

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Brief Report

IMMUNOBIOLOGY

Influence of KIR gene copy number on natural killer cell educationVivien Beziat,1 James A. Traherne,2 Lisa L. Liu,1 Jyothi Jayaraman,2 Monika Enqvist,1 Stella Larsson,3 John Trowsdale,2

and Karl-Johan Malmberg1,4,5

1Karolinska Institutet, Stockholm, Sweden; 2Cambridge Institute for Medical Research, Cambridge, United Kingdom; 3Department of Clinical Immunology and

Transfusion Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; 4Institute for Cancer Research, Oslo University

Hospital Radiumhospitalet; and 5Institute of Clinical Medicine, University of Oslo, Norway

Key Points

• KIR gene copy numbervariation influences NKcell education at therepertoire level due to alinear effect on KIRexpression.

• No effect of KIR gene doseon NK cell education atthe single cell level.

Natural killer (NK) cells are functionally tuned by education via killer cell immunoglobulin

receptors (KIRs) interacting with HLA class I molecules. We examined the effect of KIR

gene copy number variation on the education of human NK cells. The frequency of NK

cells expressing a given KIR correlated with the copy number of that gene. However,

coexpression of multiple copies from a single locus, or duplicated loci, was infrequent,

which is in line with independent transcriptional regulation of each allele or copy.

Intriguingly, coexpression of 2 KIR alleles, resulting in higher surface expression, did not

lead to enhanced functional responses in vitro or to selective advantages during in vivo

responses to cytomegalovirus infection, suggesting that receptor density does not

influence NK education at the single cell level. However, individuals with multiple KIR

gene copies had higher frequencies of responding cells, consistent with heightened

overall responsiveness. (Blood. 2013;121(23):4703-4707)

Introduction

Killer cell immunoglobulin-like receptors (KIRs) interact with HLAmolecules and shape the functionality of natural killer (NK) cells. Atthe genetic level, there is evidence that epistatic interactions betweenKIR and HLA influence outcomes in several clinical conditions,including infections, autoimmunity, and cancer (reviewed in Khakooand Carrington1). More recently, the KIR gene dose was associatedwith simian immunodeficiency virus and HIV-1 viral load in rhesusmacaque and humans, respectively, suggesting that KIR gene copynumber variation (CNV) influences antiviral immunity.2,3 Onesuggested mechanism for how theKIR gene CNV affects theNK cellresponse to viral infection is by promoting education.3 During NKcell education, interactions between inhibitory KIRs and theircognate HLA class I ligands set the threshold for NK cell activationupon stimulation with target cells lacking the corresponding HLAclass I ligands.4 Hence, the expression of multiple copies of aninhibitory KIR could potentially lead to enhanced NK cell education,thereby strengthening antiviral immunity.

KIR expression on NK cells is largely random and is deter-mined by the KIR gene content, polymorphism, and stochasticepigenetic regulation at the promoter level.5,6 In addition, extensiveCNV contributes to diversity of KIR genes.7 At the protein level,KIR2DL3 (2DL3) and 3DS1 expression frequencies were shown tobe higher in individuals with 2 copies of the respective KIR genecompared with those with only 1 copy, suggesting a gene doseeffect on KIR expression.3,5 However, the overarching impact ofKIR CNV on human NK cell repertoires and functionality remainsunknown.

Using a recently developed high throughput methodology for KIRgene typing, we investigated the effect of KIR CNV on the expressionof 7major inhibitory and activating KIRs and determined its influenceon NK cell education at the single cell and repertoire level.

Study design

Healthy donors

This study was approved by the regional ethics committee in Stockholm,Sweden, with 204 healthy adults (mean age, 42.2 years; range, 18 to 69 years).This study was conducted in accordance with the Declaration of Helsinki.

Flow cytometry

Peripheral bloodmononuclear cells were separated from buffy coats by densitygravity centrifugation (Ficoll-Hypaque; GE Healthcare, Uppsala, Sweden).KIR repertoire staining and analysis were performed as described in detailelsewhere.8

Functional assays

Peripheral blood mononuclear cells (106 cells) were mixed with K562 cells ata ratio of 10:1 in U-bottomed 96-well plates, centrifuged at 300 rpm for3minutes, and incubated for 2 or 6 hours at 37°C and 5%CO2. CD107a APC-Cy7 or CD107a PE (H4A3) were added prior the start of the assay. Afterincubation, cells were harvested by centrifugation, surface stained, fixed,permeabilized (Fixation & Permeabilization Buffers; Ebioscience) forpolyfunctional assays, stained with intracellular interferon (IFN)-g-AF700

Submitted October 9, 2012; accepted April 22, 2013. Prepublished online as

Blood First Edition paper, May 1, 2013; DOI 10.1182/blood-2012-10-461442.

The online version of this article contains a data supplement.

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(B27) and tumor necrosis factor-a-eF450 (MAb11), and analyzed by flowcytometry.

KIR and HLA typing

Genomic DNAs were isolated from whole blood using a commercial kit(Qiagen) and stored at 220°C. KIR ligands were determined using the KIRHLA ligand kit (Olerup-SSP) for detecting the HLA-Bw4, HLA-C1, andHLA-C2 motifs. KIR genotyping was performed using a recently describedhigh-throughput technology called qKAT as described in detail in Jiang et al.7

The 2DL3*05 genotype was determined using 2 polymerase chainreaction amplifications with the following pairs of sequence-specific primers(polymerase chain reaction-SSP)1: forward 59-GTCCACAGAAAACCTTCCCTCAG-39 and reverse 59-GGTGCAAAGTGTCCTTAAACTTCCTT-392DL3*004/*005/*010-specific and2 forward 59-GTGTCTCCTCTTCTTCCAGGTAATC-39, reverse 59-GCAGGCTCTTGGTCCATTACTA-39 and prod-uct sequencing primer 59-GGGACCATCCTGTCTGTGAG-39 to distinguish2DL3*005 from 2DL3*004 and 2DL3*010.

Statistics

For comparisons of independent groups, Mann-Whitney U tests wereperformed. For comparisons of matched groups, paired Student t test orWilcoxon matched test were performed. The above statistical analyses wereperformed using GraphPad software. Subsets with high KIR expressionrepresenting statistical outliers were identified using Chauvenet’s criterion(see Beziat et al8 for details).

Results and discussion

Previous studies have suggested a gene dose effect on KIRexpression at the messenger RNA level for 2DL1, 2DL2/3, and3DL1/S1,9 and at the protein level for 2DL2/3 and 3DS1.3,5,10 Todetermine whether such gene dose effects apply to a broader set of

Figure 1. The KIR expression frequency correlates linearly with KIR gene copy number. Frequency of CD56dim NK cells expressing the indicated inhibitory (A) and

activating (B) KIR stratified based on KIR gene copy number. (C) Representative examples of expression of the indicated KIR in donors with distinct copy numbers. (D)

Frequency of 2DL11 (top) and 2DL31 (bottom) NK cells within the indicated subsets and stratified based on KIR gene copy number. Red dots represent the donors with

statistical outliers identified by the Chauvenet algorithm. **P , .01; ***P , .001.

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Figure 2. Influence of KIR gene copy number on NK cell education. (A) Gating strategy to identify single and bi-allelic expression of 2DL3 in donors lacking 2DS1, 2DS2,

and 2DL2 genes. First, gates were set on NKG2C2NKG2A2 NK cells to avoid differences in functionality due to NKG2C expression and/or education by NKG2A. Thereafter,

2DL312DL12 cells were gated out for further stratification into 2DL3*005 single positive cells (1807012EB61), KIR2DL3*xxx single positive cells (1807011EB62) and

2DL3*005/2DL3*xxx double positive cells (1807011EB61). KIR2DL3*xxx represents all 2DL3 alleles recognized by mAb 180701. All donors were confirmed to be 2DL3*005

at the genetic level (B) Representative histogram and (C) bar chart (n 5 9), showing the mean fluorescence intensity (MFI) (1/2 standard deviation) of single and bi-allelic

expression of 2DL3 (GL1831). (D) Expression levels (MFI) of 2DL1, 2DL3, and 3DL1 as a function of CNV. (E) Function (CD107a, IFN-g and tumor necrosis factor-a) of

single KIR-positive NK cells in donors stratified based on KIR gene CNV and the presence or absence of the corresponding KIR ligand. (F) One representative example

and (G) recapitulative bar chart (mean of 3 donors) showing the allelic distribution in the expanded (NKG2C1) and the nonexpanded (NKG2C-) subset after gating on

GL18312DL12NKG2A2 CD56dim NK cells. All donors had the 2DL3*005/2DL3*xxx genotype. (H) Average contribution of the indicated KIR expressing subset to the CD107a

response in donors with 1 or 2 copies of the KIR gene and with the cognate HLA-class I ligand. (I) Overall effect of KIR gene CNV on degranulation in the CD56dim

NKG2A2NKG2C2 NK cell compartment after exclusion of 2DL2/S21 donors. *P , .05; **P , .01; ***P , .001. ns, not significant.

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inhibitory and activating KIRs, we combined a recently developedmethodology for assessing KIR gene CNV with a high-resolutionphenotypic analysis of KIR repertoires. Strikingly, we observeda linear relationship between KIR gene copy number and theexpression of all tested inhibitory and activating KIRs, including2DL1, 2DL2/S2, 2DL3, 3DL1, 3DL2, 2DS1, and 2DS4 (Figure 1A-C).Hence, in line with the effect of multiple copies of 2DL3, previouslyreported by Li et al,5 the results suggest that independent regulation ofdistinct gene copies is common to all KIRs.

Next we plotted KIR expression in distinct NK cell subsets,representing discrete stages of NK cell differentiation. This revealeda number of donors with very high frequencies of specific KIRs,particularly in the NKG2C1NKG2A2 NK cell subset (Figure 1D).These outliers correspond to the recently described clonal-likeexpansions of educated NKG2C1NK cells (expressing self-specificKIRs) in CMV seropositive individuals.8 Importantly, KIR genecopy number stratification revealed that such outliers were foundindependently of the KIR gene copy number (Figure 1D).

Because NK cell education is dependent on the strength of theinteractions between inhibitory KIRs and their cognate ligands, wespeculated that CNV could influence NK cell education at the singlecell level. It is not possible to discriminate cells that express 1 ormorecopies of the same allele. Instead, to test this hypothesis, we tookadvantage of recently described cross-reactivity of the anti-2DL1/S1mAb (EB6) with the 2DL3*005 allele.11 By combining distinctanti-2DL1/S1 and anti-2DL2/3/S2 clones, it is possible to discrim-inate 2DL3*005 single positive, 2DL3*xxx single positive, and2DL3*005/2DL3*xxx double positive NK cells in individualslacking 2DL2/S2 and 2DS1 (Figure 2A). We identified 9 donorswith this particular configuration of KIR genes, out of which 5 hadthe educating ligand HLA-C1. In these sets of unique donors,a number of observations were made. First, NK cells expressing 2alleles of 2DL3 had higher expression of total 2DL3 (Figure 2B-C).Second, the regulation of the 2 distinct alleles was independent andbi-allelic expression frequencies were near or slightly higher thanthose predicted by the product rule. Thus, despite an increase inoverall frequencies of NK cells expressing a given KIR inindividuals with multiple gene copies (Figure 1), most NK cellsonly transcribed from 1 of the available loci. Supporting thisinterpretation, the mean level of KIR expression on CD56dim NKcells did not differ in individuals with multiple KIR gene copies,as exemplified for 2DL1, 2DL3, and 3DL1 (Figure 2D). Third,simultaneous expression of 2 distinct alleles of 2DL3 did notinfluence the education at the single cell level as determined bypolyfunctional responses against K562 targets (Figure 2E andsupplementary Figure 1). Substantiating the latter finding, in vivoexpansion of educated NK cells in CMV seropositive individualsoccurred independently of the KIR gene copy number, becausethere was no preferential expansion of NK cells expressing 2alleles among the expanded NKG2C1 NK cells (Figure 2F-G).The lack of effect of KIR density on NK cell education is in linewith the observation in mice that the quality rather than thequantity of the receptor-ligand interactions determine NK celleducation,12 as well as recent findings in humans suggesting thatthe dose of HLA-C does not influence education.13

Together, the analyses made possible in these unique donorsrevealed that NK cells rarely express more than 1 copy, and whenthey do, it does not influence their overall functional responsive-ness. Corroborating these 2 observations, an aggregated analysis ofNK cells in the whole cohort revealed that education of singleKIR1 NK cells, harboring 1 or more gene copies, was independentof the KIR gene CNV (supplementary Figure 2).

However, it is important to consider that differences in thresholdsbetween distinct readouts may influence the interpretation of theresults.4 Thus, in mice, the gene dose of H-2Dd did not influence thepercentage of degranulating NK cells expressing the cognate Ly49Areceptor but was associated with higher mean fluorescence intensityof CD107a, stronger IFN-g responses, and more efficient in vivorejection of major histocompatibility complex class I negativetargets.14 Furthermore, education by NKG2A and KIRs seem toprovide differential effects on IFN-g vs degranulation responses byNK cells after allogeneic stem cell transplantation.15 Despite thispotential limitation of current assays to monitor NK cell education,the CD107a assay is robust enough to consistently detect subtledifferences in NK cell functionality caused by education via differentKIR alleles.6 Indeed, in the present study the effects of combinationsof 2DL3*xxx and 2DL3*005 alleles on NK education wereconsistent, irrespective of the readout used (Figure 2E andsupplementary Figure 1).

Finally, even if CNV had no impact on education at the singlecell level, it could still influence NK cell education at the level ofNK cell repertoires. Indeed, high copy number of an educating KIR(ligand present) led to a relatively larger contribution of NK cellsexpressing this particular KIR to the responding (CD107a1)population (Figure 2H). To further isolate the effect of KIR genecopy number, the analysis was restricted to donors with a fixednumber of KIR ligands (ie, HLA-C1, HLA-C2, and HLA-Bw4).Strikingly, donors with 5 or more copies of 2DL1, 2DL3, and3DL1 responded better to stimulation with K562 cells than thosewith fewer copies (Figure 2I).

In summary, our results reveal a linear effect of KIR gene CNVon the frequency of cells expressing both activating and inhibitoryKIRs and on the overall functional responsiveness of the NK cellrepertoire. The results may have implications for NK cell-mediated alloreactivity in settings of allogeneic stem cell trans-plantation, where education in the donor has a major influence onoutcome.16

Acknowledgments

This work was supported by grants from the Swedish ResearchCouncil, the Swedish Children’s Cancer Society, the SwedishCancer Society, the Royal Swedish Academy of Sciences, theTobias Foundation, the Karolinska Institutet, the Wenner-GrenFoundation, Oslo University Hospital, the MRC and WellcomeTrust with partial funding from the Cambridge BRC-NIHR.

Authorship

Contribution: V.B., L.L.L., J.A.T., J.T., and K.-J.M. designedresearch; S.L. organized sampling of healthy blood donors; V.B., J.J.,L.L.L., M.E., and J.A.T. performed research and analyzed data; andV.B., J.A.T., J.T., and K.-J.M. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Vivien Beziat, Karolinska Institutet, 14186,Stockholm, Sweden; e-mail: vivien.beziat@ki.se; and Karl-JohanMalmberg, Center for Infectious Medicine, F59, KarolinskaUniversity Hospital Huddinge, Stockholm, 14186 Sweden; e-mail:kalle.malmberg@ki.se or k.j.malmberg@medisin.uio.no.

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