2DL1, 2DL2 and 2DL3 All Contribute to KIR Phenotype Variability on Human NK Cells

ORIGINAL ARTICLE 2DL1, 2DL2 and 2DL3 all contribute to KIR phenotype variability on human NK cells

SE Dunphy1, KJ Guinan2, C Ní Chorcora1, J Jayaraman3, JA Traherne3,4, J Trowsdale4, D Pende5, D Middleton6 and CM Gardiner1

Natural killer (NK) cells are lymphocytes that function as part of the innate immune system. Their activity is controlled by a range of inhibitory and activating receptors, including the important killer-cell immunoglobulin-like receptors (KIR). The KIR are a multi-gene family of receptors that interact with the human leukocyte antigen (HLA) class I family of molecules and are characterised by extensive allelic polymorphism. Their expression on the cell surface of NK cells is highly variable, but the factors responsible for this variability are not yet clearly understood. In the current study, we investigated KIR expression in a healthy human cohort that we had previously characterised in depth at a genetic level, with KIR allele typing and HLA class I ligand genotypes available for all donors (n = 198). Allelic polymorphism significantly affected the phenotypic expression of all KIR analysed, whereas HLA ligand background influenced the expression levels of 2DL1 and 2DL3. In particular, we found that although 2DL2 may influence 2DL1 expression, this appears to be owing to variation in 2DL1 copy number. Finally, the inhibitory receptor LILRB1 had higher expression levels in individuals with B/B KIR genotypes, suggesting a possible relationship between KIR and non-KIR receptors, which serves to balance NK cell activation potential.

Genes and Immunity (2015) 16, 301–310; doi:10.1038/gene.2015.15; published online 7 May 2015

INTRODUCTION expression of receptors and the difficulty in procuring large Natural killer (NK) cells are large granular lymphocytes, which act genetically well-characterised donor cohorts that are necessary to as part of the innate immune system. They are an important puzzle out the contribution of genetic factors. In this current source of inflammatory cytokines early in the immune response study, phenotypic KIR expression was determined for a large and are also key mediators of cytotoxicity against virally infected cohort of Irish donors (almost 200 donors), who had previously 17 and tumour cells.1,2 NK cell recognition of target cells is mediated been typed to the allele level for KIR genes. Recently available in part by the killer-cell immunoglobulin-like receptors (KIR), a antibodies with defined specificity for individual KIR receptors also multi-gene family of receptors that contains both inhibitory and facilitated characterisation of important receptors which had activating members and whose known ligands belong to the previously been challenging to assess. In this current study, it was human leukocyte antigen (HLA) class I family of molecules.3 found that the KIR phenotype is influenced by both allelic Similar to their HLA ligands, the KIR are genetically complex. polymorphism and the presence of HLA ligands for the KIR in the Individuals vary in terms of the number and type of genes present Irish population. The role of allelic polymorphism is of particular in their KIR gene haplotypes. The highly polymorphic nature of the interest for 2DL1 and 2DL3, as these KIR have not been well KIR genes, for which multiple alleles exist at each locus, further characterised in other studies to date. One comprehensive study contributes to variation.3,4 On the basis of KIR gene content, two carried out in the Japanese population was hindered by the more broad KIR haplotypes exist. While the A haplotype is more restricted KIR allele repertoire in this population, with both 2DL1 and 2DL3 variation dominated by single alleles found at high conserved in terms of gene content and has only one short-tailed 11 activating KIR present, group B haplotypes show greater variation frequencies in the cohort analysed. A recent study in the German population found that allelic polymorphism did influence and can contain a number of activating KIR genes. Although our 18 understanding of KIR genetics has advanced rapidly, phenotypic 2DL1 expression. Allelic diversity in the Irish population studies have lagged behind. However, we know that KIR permitted examination of a number of allotypes of both 2DL1 expression on the surface of NK cells also varies between donors, and 2DL3. In addition, the KIR genotype affected the expression of both in terms of percentages of NK cells expressing individual an important non-KIR receptor, in a manner that suggests a receptors and the density at which these receptors are expressed. potential balancing of activating and inhibitory signalling The factors underlying this variable KIR phenotype are still not potential on NK cells. fully understood, although allelic polymorphism, promoter variation, copy number variation, HLA ligand background and prior infection with human cytomegalovirus (HCMV) have all been RESULTS identified as possible contributing variables.5–16 KIR expression is influenced by allelic polymorphism Phenotyping studies have been hampered by both a lack of To investigate expression patterns of 2DL1, 2DL3/L2, 3DL1, 3DL2, suitable monoclonal antibodies for measurement of cell surface 2DL4 and 2DS4 genes, we phenotyped cell surface expression, by

1School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, 152-160 Pearse Street, Trinity College, Dublin 2, Ireland; 2Current address: BioAtlantis Ltd., Kerry Technology Park, Tralee, Co. Kerry, Ireland; 3Cambridge Institute for Medical Research, University of Cambridge; Division of Immunology, Cambridge, UK; 4Department of Pathology, University of Cambridge, Cambridge, UK; 5Immunology Laboratory, IRCCS AOU San Martino-IST, Largo Rosanna Benzi 10, Genova, Italy and 6Transplant Immunology Laboratory, Royal Liverpool University Hospital, Liverpool, UK. Correspondence: Dr CM Gardiner, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, 152-160 Pearse Street, Trinity College, Dublin 2, Ireland. E-mail: [email protected] Received 2 December 2014; revised 6 March 2015; accepted 31 March 2015; published online 7 May 2015 Impact of allelic polymorphism on KIR expression SE Dunphy et al 302 measuring both the percentage of NK cells expressing a receptor allele content (n = 198).19 The recent development of single-KIR- and the level of expression of receptor on the surface of cells, in a speciﬁc antibodies allowed us to determine the impact of allelic population that we have previously characterised for KIR gene and polymorphism on KIR expression. Given the complication of

Figure 1. Allelic polymorphism influences KIR expression. Donors (n = 198) were typed to the allele level for the KIR genes. PBMCs from these donors were analysed by flow cytometry to determine KIR expression on NK cells. Expression of 2DL1 (a), 2DL2/3 (b), 3DL1 (c) and 3DL2 (d) was evaluated using the single KIR-specific antibodies 143211, 180701, DX9 and Q66, respectively. 3DS1 expression was assessed using a combination of the Z27 and DX9 antibodies. 2DL2 expression was assessed using a combination of the GL183 and 180701 antibodies. The percentage of NK cells expressing each KIR and MFI of expression on these NK cells for individual donors is shown. MFI values were not calculated for donors who showed no surface expression of a KIR. Donors that typed homozygous for particular KIR alleles were stratified according to their underlying allele background. Differences in expression between groups containing more than one donor were analysed using unpaired t-tests or one-way ANOVA followed by Bonferroni’s Multiple Comparison post tests. *Po0.05; **Po0.01; ***Po0.001.

Figure 1. Continued. interpretation when donors are heterozygous for a particular although 2DL3*005 was not recognised by the anti-2DL3 locus, we first identified donors homozygous for KIR alleles. antibodies available and was therefore omitted from analysis.21 2DL2 was associated with the highest percentage expression 2DL/2DS Receptors. Within the donor cohort there were five 2DL1 (mean = 34.6 ± 11.2%, n = 18) (Figure 1b) of the 2DL3 alleles alleles present in a homozygous state—2DL1*001, *002, *003, *004 studied; however, it should be noted that as the antibody used and *007. Only a single suitable donor was identified for each of to detect 2DL2 expression (GL183) also binds 2DS2, and as 2DL2 2DL1*001 and 2DL1*007, and further studies would need to be and 2DS2 are in perfect linkage disequilibrium in this current performed to fully determine the expression patterns of these rare cohort, it was not possible to rule out a possible contribution of alleles. In this current cohort, the 2DL1*001 allele was associated 2DS2 expression. A different antibody specific for 2DL3 only was with the highest percentage of 2DL1-positive NK cells relative to used for the other 2DL3 alleles, allowing for direct comparisons of the other alleles studied (Figure 1a). 2DL1*003 was also found at a expression patterns. The percentage of NK cells expressing 2DL3 high frequency (mean = 18.3 ± 7.8%, n = 34) and 2DL1*002 was in donors homozygous for 2DL3*001 was statistically significantly found on a moderate percentage of NK cells compared with the higher than those seen in donors homozygous for 2DL3*002 other allotypes (mean = 15.3 ± 9.2%, n = 17). Donors homozygous (24.8 ± 10.4%, n = 39 vs 17.2 ± 6.8%, n = 27, Po0.01). Furthermore, for 2DL1*004 or *007 were found to express 2DL1 on a low 2DL3*001 was also expressed at a higher density than 2DL3*002. percentage of their NK cells compared with the other alleles. The Among the 2DS4 alleles assessed (2DS4*00101, *003, *004 and percentage of NK cells expressing 2DL1 was statistically signifi- *006), only 2DS4*00101 was expressed on the surface of NK cells. cantly different between donors homozygous for 2DL1*003 and 2DL4 expression was negligible. Only 2DL4*00102 was found on 2DL1*004 (18.3 ± 7.8%, n = 34 vs 7.4 ± 8.2%, n =4, Po0.05). In the surface of NK cells, and expression was confined to the terms of the level of expression measured by mean fluorescent CD56bright subset (data not shown). intensity (MFI), 2DL1*002 had the highest density of receptor expression on NK cells (mean = 719.2 ± 261.4). This was statistically 3DL Receptors. Nine different 3DL1 alleles (3DL1*00101, *002, significantly different from the allele that showed the lowest *01502, *004, *005, *007, *008, *009 and *019) and 3DS1, which level of receptor expression—2DL1*004 (mean = 371.9 ± 103.7, segregates as an allele at the 3DL1 locus, were identified for Po0.05). 2DL1*001 and *003 had similar moderate MFI values phenotypic analysis. Relative to the other alleles assessed, donors (581.8 and 595.4 ± 242.4, respectively), whereas the donor homo- homozygous for 3DL1*00101 had the highest percentage of 3DL1- zygous for 2DL1*007 had a lower level of cell surface expression. positive NK cells (Figure 1c). This was statistically significant when 2DL2 and 2DL3 segregate as alleles of the same locus.20 compared with 3DL1*002, *01502, *004, *005, *007 and 3DS1 In addition to 2DL2, there were three 2DL3 alleles (2DL3*001, receptors. 3DL1*002, *01502, *007, *008, *009 and 3DS1 were *002 and *005) found in a homozygous state in the donor cohort, expressed on a moderate percentage of donor NK cells, whereas

© 2015 Macmillan Publishers Limited Genes and Immunity (2015) 301 – 310 Impact of allelic polymorphism on KIR expression SE Dunphy et al 304 3DL1*005 expression was relatively low. 3DL1*019 was not significant difference (data not shown). As previously seen for expressed on the cell surface. 3DL1*004, an allele known to 2DL1, 2DL3 was more densely expressed on the NK cells of donors retained intracellularly,22 was not expressed, with the interesting when its ligand was absent, and the MFI of the C2/C2 donor group exception of two donors who were positive for cell surface was statistically significantly higher when compared with the expression (see Supplementary Figure 2). In terms of MFI, combined C1/C1 and C1/C2 donor groups (data not shown). 3DL1*01502 had the highest level of 3DL1 expression on NK Therefore, although it appears that HLA ligand background cells. 3DL1*008 was also found at a high level relative to the other influences the expression of both 2DL1 and 2DL3, the effects on allotypes studied. 3DL1*00101, *002 and *007 showed relatively 2DL1 expression are more pronounced. In general, there are a moderate levels of receptor density, whereas 3DL1*004, *005 and higher percentage of NK cells expressing these KIR in donors with *009 were found at low levels. the genes for their appropriate HLA ligands and higher density of Seven different 3DL2 alleles were present in a homozygous receptor expression in donors lacking the cognate HLA ligands. state. 3DL2*002 was associated with the highest percentage expression (mean = 42% ± 8.6%, n = 8) (Figure 1d). This was 2DL1 expression is influenced by the presence of 2DL2 fi statistically signi cantly higher than the expression in donors A previous study in Caucasian donors found that there was an homozygous for the 3DL2*007 allele (mean = 21.7 ± 9.2%, n = 8). association between 2DL1 expression and HLA background in 3DL2*001, *003 and *005 were found to be relatively moderately donors homozygous for the A KIR haplotype, but this relationship expressed. 3DL2*009 donors and the single donor homozygous for was not seen in donors with the B KIR haplotype.12 However, 3DL2*010 had lower percentages of 3DL2-expressing NK cells in when the B haplotype donors were stratified on the basis of the their peripheral blood (25.8 ± 2.1%, n = 2 and 13.4%, respectively). presence or absence of 2DL2, it was found that in donors lacking In terms of the density of receptor expression, 3DL2*007 had 2DL2 the relationship between the percentage of NK cells fi statistically signi cantly lower levels than 3DL2*001, *002, *003 expressing 2DL1 and HLA ligand background was re-established. or *005. This suggested that 2DL1 expression is influenced by the presence of 2DL2. To see whether this relationship was present in this The expression of 2DL1 and 2DL3 receptors are affected by HLA current cohort, donors carrying the B haplotype were stratified on ligand background the basis of the presence or absence of the 2DL2 gene, and the The genes for the KIR receptors and their HLA ligands are found expression of 2DL1 in donors with different underlying HLA on separate chromosomes, and thus each KIR is inherited backgrounds was assessed (Figure 3). There were no statistically independently from its cognate HLA ligand; however, there have significant associations between the percentage of NK cells been reports that the KIR phenotype may be partly dependent on expressing 2DL1 and the HLA background in either the 2DL2+ HLA ligand background.11,12 or the 2DL2 − groups. However, although the pattern of To determine whether KIR expression is influenced by the expression between the whole cohort and donors lacking 2DL2 presence of cognate HLA ligand in our cohort, donors were was similar (percentage of NK cells expressing 2DL1 increasing stratified according to HLA ligand background, and KIR expression with HLA-C2 copy number, Figure 3a), in donors with 2DL2 it was patterns were assessed. HLA-C ligands were considered in analysis found that C2/C2 donors no longer had the highest percentage of of 2DL1, 2DL3, 2DL2 and 2DS1, whereas 3DL1 expression was 2DL1-positive cells (Figure 3b). In fact, these donors had lower examined in the context of HLA-B ligands. It was found that HLA frequencies of 2DL1-expressing NK cells than either C1/C1 or ligand background significantly affected the expression of C1/C2 donors. The statistical significance of the relationship 2DL1 and 2DL3 (Figures 2a and b), but not 2DS1, 2DL2 or 3DL1 between 2DL1 MFI and HLA background also was reduced in (Figures 2c–e). donors positive for the 2DL2 gene, but this may be owing to lower fi The percentage of NK cells expressing 2DL1 was highest in donor numbers as a result of the strati cation. Overall, it would fl donors homozygous for its cognate HLA-C2 ligand, and this appear that 2DL2 does indeed in uence the cell surface percentage decreased through donors heterozygous for HLA-C2, expression of 2DL1. down to HLA-C1 homozygous donors who lacked the cognate ligand completely (Figure 2a). Although there were no statistically The relationship between 2DL2 and 2DL1 expression depends on significant differences between these groups, a statistically 2DL1 copy number significant linear trend for increased expression of 2DL1 with To better understand these observations, the effect of the 2DL2 increased HLA-C2 copy number was observed. Furthermore, the gene on 2DL1 expression was examined in more detail. 2DL1 underlying HLA background had a statistically significant impact expression was assessed in donors with and without the 2DL2 on the density at which the 2DL1 receptor is found on the surface gene to investigate whether there was a relationship separate of donor NK cells, with expression levels in donors homozygous from any involvement of HLA ligand background. It was found for its non-cognate HLA-C1 ligand being statistically significantly that 2DL1 expression was statistically significantly higher, both in higher than both levels in heterozygous donors and donors terms of percentage expression and MFI, in donors lacking 2DL2 homozygous for HLA-C2. Donors homozygous for HLA-C2 showed compared with those who had this gene (Figure 4a). We the lowest level of 2DL1 expression. postulated that this effect may be owing to the fact that 2DL1 For 2DL3, similar to 2DL1, the highest percentage of receptor- and 2DL2 are generally located in the centromeric KIR region. positive NK cells were found in donors homozygous for its HLA 2DL1 is generally found in the centromeric region of the A KIR ligand HLA-C1 (Figure 2b). This difference was statistically haplotype (CenA), whereas 2DL2 is present in the centromeric significant when compared with HLA-C1/C2 heterozygotes. The region of the B KIR haplotype (CenB). This means that donors who percentage of 2DL3-positive NK cells in donors lacking its cognate do not have 2DL2 (CenA/CenA donors) may have two copies of ligand (C2/C2 donors) was similar to that seen for the 2DL1, whereas donors possessing the 2DL2-containing CenB heterozygous donors, but it did not reach statistical significance region will be more likely to only have a single copy of 2DL1 when compared with the C1/C1 donor group, possibly owing to present. Therefore, the observed differences may be owing to a the lower number of donors in the C2/C2 group. Overall, it gene dose effect for 2DL1, as opposed to a complex relationship appeared that carriage of the C2-encoded epitope was associated between 2DL1 expression and 2DL2. This was assessed by with a reduced percentage of circulating NK cells expressing 2DL3. comparing donors carrying a B haplotype who had two copies Indeed, comparing expression in C1/C1 homozygotes with of the 2DL1 gene, donors carrying a B haplotype with only a single expression in donors where C2 was present yielded a statistically copy of 2DL1 and donors with the A/A genotype with two copies

Figure 2. KIR expression is influenced by HLA background. Donors (n = 198) were genotyped for the KIR genes and the HLA class I ligands for KIR genes. PBMCs from these donors were analysed by flow cytometry to determine KIR expression on NK cells. The percentage of NK cells expressing each KIR and MFI of expression on these NK cells for individual donors is shown for 2DL1 (a), 2DL3 (b) and 3DL1 (c). The percentage of NK cells expressing 2DS1 and 2DL2/S2 are shown in d and e, respectively. Donors were stratified according to their underlying HLA class I ligand background. Differences in expression between the groups was analysed using one-way ANOVA followed by a post test for a linear trend (dotted line) and Bonferroni's Multiple Comparison post tests (solid line). *Po0.05; ***Po0.001.

Figure 3. The influence of 2DL2 on the relationship between 2DL1 expression and HLA background. Donors (n = 198) were genotyped for the 2DL1 and 2DL2 genes and the HLA class I ligands (C1 and C2) for KIR genes. PBMCs were stained with an anti-2DL1-specific antibody, and the expression of 2DL1 on NK cells was measured by flow cytometry. The percentage of 2DL1-positive NK cells and MFI of expression on these NK cells for individual donors is shown. Donors were stratified on the basis of the absence (a) or presence (b) of 2DL2 and also according to the underlying HLA class I ligand background. Differences in expression between the HLA ligand groups were analysed using one-way ANOVA followed by Bonferroni's Multiple Comparison post tests. **Po0.01; ***Po0.001.

of the 2DL1 gene. Donors carrying either the A/A genotype or a B haplotypes. These subgroups were designated as A/A, A/B and B/B haplotype who had two copies of the 2DL1 gene had similar based on the combination of KIR haplotypes making up the donor percentages of 2DL1-expressing NK cells, whereas donors with a B genotype. Owing to the limited presence of activating KIR genes haplotype with only one copy of 2DL1 had statistically significantly on the A haplotype and the more abundant occurrence of these lower percentages than either of the other two groups (Figure 4b). genes on B haplotypes, donors in the A/A haplotype group have Furthermore, 2DL1 expression in donors with two copies of 2DL1 less activation potential through their KIR gene repertoire was not statistically significantly different in the absence or compared with donors who have a B haplotype present. Thus, it presence of 2DL2 (Figure 4c). This suggests that the relationship can be examined whether other NK cell receptors show altered observed between 2DL2 and 2DL1 expression is owing to expression profiles in donors who have KIR genotypes with differences in 2DL1 copy number. differing activation potentials. The non-KIR receptors assessed were the inhibitory LILRB1 and NKG2A (as they recognise HLA The expression of non-KIR receptors is influenced by the KIR molecules and use similar signalling to KIR) and the activating genotype NKG2D receptor (different ligands and signalling) to allow for a In addition to KIR, NK cells have other important inhibitory and comparison of the effect of KIR genotype on receptor systems with activating receptors on their cell surface. Several of these non-KIR differing levels of potential cross talk and redundancy. receptors also recognise class I HLA molecules, and intracellular It was found that donors possessing the B/B KIR genotype had signalling of KIR and some non-KIR receptors use similar statistically significantly higher percentages of NK cells expressing mechanisms and molecules.23–27 Therefore, there is the potential inhibitory LILRB1 than either A/A or A/B group donors (Figure 5a). for redundancy and cross talk between KIR and non-KIR receptors The B/B group also showed a higher density of LILRB1 expression which may have an impact upon NK cell activation, and it is than the other two donor groups, but this was not statistically feasible that the expression of non-KIR receptors may be significant. Although no statistically significant differences were influenced by the KIR in order to maximise NK cell responsiveness. found in the expression of NKG2A or NKG2D between donors with To investigate whether there is a potential relationship between different underlying KIR haplotypes, expression (both percentage the expression of these receptors and KIR, the donor cohort was positive and MFI) of the inhibitory NKG2A receptor was highest stratified into three subgroups on the basis of their underlying KIR among the B/B genotype donors when compared with both A/A

Figure 4. The presence of 2LD2 gene affects 2DL1 expression. Donors (n = 198) were genotyped for the 2DL1 and 2DL2 genes. PBMCs from these donors were stained with an anti-2DL1-specific antibody, and the expression of 2DL1 on NK cells was measured by flow cytometry. The percentage of 2DL1-positive NK cells and MFI of expression on these NK cells for individual donors is shown. The whole cohort of donors was stratified based on (a) the absence or presence of 2DL2, or (b) 2DL1 copy number. (c) Donors with two copies of the 2DL1 gene were stratified based on the presence or absence of the 2DL2 gene. Differences in expression between the groups were analysed using one-way ANOVA followed by Bonferroni's Multiple Comparison post tests or unpaired t-tests. **Po0.01; ***Po0.001. and A/B donors. Overall expression of non-KIR inhibitory receptors was identified in this current cohort). A recent study in the was increased in donors with more activating KIR genotypes. This German population also found lower levels of expression suggests a potential relationship between KIR and non-KIR associated with 2DL1*004.18 Both 2DL1*004 and *007 belong to receptors whereby various NK cell receptors serve to balance a group of 2DL1 allotypes that have a cysteine residue at position the activating potential determined by an individual’s KIR 245 and which have been reported to possess less inhibitory genotype. capacity than other 2DL1 allotypes.28 From this current study, it appears that this reduced inhibitory potential might simply be owing to reduced expression of Cys245 2DL1 alleles on the cell DISCUSSION surface. The KIR are a complex family of receptors and the factors For many years, the role of HLA genotype in the expression of controlling their expression are not fully understood. In support of KIR proved controversial. Although some studies claimed that HLA other studies, the current study confirms that allelic polymorphism genotype played no part,29 others found a clear association significantly affects cell surface expression for all KIR examined. between KIR expression and the presence of HLA ligands.11,12 These data are of particular importance for 2DL1 and 2DL3, as the Recent work investigating the effect of HCMV on KIR expression effect of allelic polymorphism on these KIR had not been found that NK cells expressing 2DL1 and 2DL3 were expanded in previously well characterised. For 2DL1, it was found that donors seropositive for this common virus and that this expansion 2DL1*004 and *007 exhibited lower levels of expression relative was dependent on the presence of the appropriate HLA to other 2DL1 allotypes (although only a single 2DL1*007 donor ligands for these KIR.16,30 Authors postulated that the previous

Figure 5. Non-KIR receptor expression is influenced by KIR genotype. Donors (n = 198) were genotyped for the KIR and their underlying KIR haplotypes were determined. PBMCs from these donors were analysed by flow cytometry to determine the expression of non-KIR receptors on NK cells. The percentage of NK cells expressing each receptor and MFI of expression on these NK cells for individual donors is shown. Donors were stratified according to their underlying KIR haplotypes looking at the three possible heterozygous and homozygous haplotype combinations. Differences in expression between the haplotype groups was analysed using one-way ANOVA followed by Bonferroni’s Multiple Comparison posts tests. *Po0.05.

discrepancies in the literature may be because of differences in A previous study carried out in Caucasians found that the the HCMV status of the cohorts investigated.30 In the current relationship between 2DL1 and HLA ligand was found in B study, a distinct correlation between the presence of HLA ligands haplotype donors who lacked 2DL2, and the authors suggested and the expression of 2DL3 and 2DL1 was found. For 2DL3, a that this was owing to the fact that 2DL2 also recognised HLA-C2 higher percentage of NK cells expressed this receptor in donors and, as it was expressed before 2DL1 in the course of KIR homozygous for its cognate ligand HLA-C1. This pattern was also acquisition, its presence would render the expression of 2DL1 seen for 2DL1, but it did not reach statistical signiﬁcance. The unnecessary.12 In this current work, there is evidence that the HCMV status of this current cohort is unknown, and as a impact of 2DL2 on 2DL1 expression may be because of increased retrospective study it is impossible to determine. We are therefore 2DL1 copy number in donors lacking 2DL2 rather than an unable to assess the impact of HCMV as an additional variable adjustment of KIR expression owing to an overlap of ligand contributing to HLA associations with KIR expression that we have recognition. However, co-expression of 2DL2 and 2DL1 on NK cells observed in this Irish cohort. was not assessed in this current study, and thus the possibility that

Genes and Immunity (2015) 301 – 310 © 2015 Macmillan Publishers Limited Impact of allelic polymorphism on KIR expression SE Dunphy et al 309 2DL2+ NK cells express lower levels of 2DL1, as proposed by 181703), 2DS4-APC (clone 179315), NKG2C-AF488 (R&D Systems, Abing- Schonberg et al.,12 cannot be ruled out. don, UK), CD3-Pacific Blue (Biolegend, London, UK), 2DL3/L2/S2-PE (clone As NK cells express other receptors capable of transmitting GL183), 3DL1/S1-PE (clone Z27), NKG2A-PE, LILRB1-PE (Beckman Coulter, inhibitory and activating signals, there is the potential that the Clare, Ireland) and 2DL1/S1/S3/S5-PerCP/Cy5.5 (clone HP-MA4) fi expression of these receptors may be related to KIR expression, (eBioscience, Hat eld, UK). 3DL2 phenotype was determined using the either to reduce redundancy in the system or to act as buffers unconjugated Q66 antibody in combination with a PE-conjugated anti- against overly inhibitory or activating KIR genotypes. In this study, mouse secondary antibody (Beckman Coulter). Details of the antibodies used in this study are provided in Supplementary Table 1. Expression it was found that inhibitory LILRB1 was more highly expressed in patterns of 2DL1, 2DL3, 2DL4, 2DS4, 3DL1 and 3DL2 were evaluated using individuals with KIR genotypes containing more activating the single KIR-specific antibodies appropriate for these receptors. We receptors. A similar but nonsignificant pattern was found for focused on donors who typed as homozygous for a particular KIR allele to NKG2A. There was also a slight but significant increase in NKG2C avoid confounding phenotype signals from other alleles. Where single KIR- expression in donors with the more inhibitory-biased A/A KIR specific antibodies were unavailable, KIR expression was evaluated using genotype, although these data are difficult to interpret given the antibody combinations (anti-3DL1 and anti-3DL1/S1, anti-2DL1 and anti- unknown HCMV status of this current cohort. On the whole, this 2DL1/S1/S3/S5, anti-2DL3 and anti-2DL3/L2/S2) and KIR allele genotype to suggests that other members of the NK cell receptor repertoire discriminate between the different receptors of interest. Cells were might serve to balance the activating or inhibitory potential acquired on a FACSCanto (BD Biosciences) and analysed using the FlowJo determined by an individual’s KIR genotype. Interestingly, no software (Treestar, Ashland, OR, USA). The following gating strategy was relationship was found between the KIR genotype and NKG2D used and is illustrated in Supplementary Figure 1. Lymphocytes were selected on the basis of their size and granularity. Live/Dead Aqua was expression. NKG2D is the only non-KIR receptor examined in this used to select living cells, and CD45 was used to confirm the lymphoid study that does not signal through the same ITIMs and ITAMs that identity of cells. NK cells were selected as CD56+CD3- lymphocytes. are also used by the KIR and also has notably different ligands than the other receptors discussed, recognising markers of stressed cells as opposed to HLA expression. Therefore, NKG2D Statistical analysis may represent a receptor that enables NK cells to recognise a Statistical analysis was performed using the Prism 5 software (GraphPad, La Jolla, CA, USA). Data were analysed using unpaired t-tests or ANOVA distinct type of target cell, and as such its signalling is uncoupled ’ fi from the KIR, allowing it to override KIR signalling. In addition, it is followed by Bonferroni s Multiple Comparison post tests. Signi cant values were as follows: *Po0.05, **Po0.01, ***Po0.001. noteworthy that the strongest relationship between KIR and non- KIR was seen for LILRB1. This is interesting because, of all the receptors assessed, only LILBR1 is found within close proximity to CONFLICT OF INTEREST the KIR genes in the genome, lying just upstream of the KIR gene The authors declare no conflict of interest. complex in the leukocyte receptor cluster on chromosome 19. Similar to the KIR, LILRB1 is polymorphic and different allotypes have been found to display different expression patterns.31 It is REFERENCES possible that the phenotypic results found in this current study 1 O'Connor GM, Hart OM, Gardiner CM. Putting the natural killer cell in its place. could be explained by the existence of genetic linkage between Immunology 2006; 117:1–10. highly expressed LILRB1 alleles and KIR genotypes with greater 2 Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat Rev Immunol activating potential. 2001; 1:41–49. In conclusion, this current study has found that both allelic 3 Vilches C, Parham P. KIR: diverse, rapidly evolving receptors of innate and polymorphism and HLA genotype influence the expression of KIRs adaptive immunity. Annu Rev Immunol 2002; 20: 217–251. and that KIR genes themselves might affect the expression of 4 Robinson J, Waller MJ, Stoehr P, Marsh SG. IPD--the immuno polymorphism database. Nucleic Acids Res 2005; 33 Database issue D523–D526. other important NK cell receptors. 5 Gardiner CM, Guethlein LA, Shilling HG, Pando M, Carr WH, Rajalingam R et al. Different NK cell surface phenotypes defined by the DX9 antibody are due to KIR3DL1 gene polymorphism. J Immunol 2001; 166: 2992–3001. MATERIALS AND METHODS 6 Gagne K, Willem C, Legrand N, Djaoud Z, David G, Rettman P et al. Both the nature Donor cohort of KIR3DL1 alleles and the KIR3DL1/S1 allele combination affect the KIR3DL1 Blood was obtained from healthy Irish donors (n = 198) recruited through NK-cell repertoire in the French population. Eur J Immunol 2013; 43: 1085–1098. the Blood Transfusion Services of Belfast City Hospital, Belfast, Northern 7 Thomas R, Yamada E, Alter G, Martin MP, Bashirova AA, Norman PJ et al. Ireland and St. James’s Hospital, Dublin, Republic of Ireland. Ethics approval Novel KIR3DL1 alleles and their expression levels on NK cells: convergent and patient consent were received in each of the centres according to local evolution of KIR3DL1 phenotype variation? J Immunol 2008; 180: 6743–6750. guidelines. Mononuclear cells were isolated using standard Ficoll gradient 8 Goodridge JP, Lathbury LJ, Steiner NK, Shulse CN, Pullikotil P, Seidah NG et al. centrifugation, and cells were cryopreserved. Genotyping for the KIR genes Three common alleles of KIR2DL4 (CD158d) encode constitutively expressed, and the HLA class I ligands for the KIR was performed using previously inducible and secreted receptors in NK cells. Eur J Immunol 2007; 37:199–211. described PCR-SSOP methods.17 KIR genotypes were assigned as follows: 9 Goodridge JP, Witt CS, Christiansen FT, Warren HS. KIR2DL4 (CD158d) donors genotyped as having only the framework KIR genes (3DL3, 3DP1, genotype influences expression and function in NK cells. J Immunol 2003; 171: 2DL4, 3DL2) and the A haplotype KIR genes (2DL1, 2DL3, 3DL1, 2DS4) were 1768–1774. deemed to be of the A/A KIR genotype. Donors who had all the A 10 Middleton D, Gonzalez A, Gilmore PM. Studies on the expression of the deleted haplotype KIR genes with at least one additional B haplotype (2DL2, 2DS2, KIR2DS4*003 gene product and distribution of KIR2DS4 deleted and nondeleted 3DS1, 2DL5, 2DS3, 2DS1) were placed in the A/B genotype group, whereas versions in different populations. Hum Immunol 2007; 68:128–134. donors lacking at least one of the A haplotype KIR genes were assigned to 11 Yawata M, Yawata N, Draghi M, Little AM, Partheniou F, Parham P. Roles for HLA the B/B genotype group. 2DL1 copy number was determined as previously and KIR polymorphisms in natural killer cell repertoire selection and modulation described.32 The HCMV status of the donors is unknown and impossible to of effector function. J Exp Med 2006; 203:633–645. accurately identify from peripheral blood mononuclear cells. 12 Schonberg K, Sribar M, Enczmann J, Fischer JC, Uhrberg M. Analyses of HLA-C- specific KIR repertoires in donors with group. A and B haplotypes suggest a ligand-instructed model of NK cell receptor acquisition. Blood 2011; 117:98–107. Flow cytometry 13 Beziat V, Traherne JA, Liu LL, Jayaraman J, Enqvist M, Larsson S et al. Influence of The viability of cells was confirmed using Live/Dead Aqua (Life KIR gene copy number on natural killer cell education. Blood 2013; 121: Technologies, Dublin, Ireland). Receptor expression of natural killer cells 4703–4707. was examined using the following monoclonal antibodies: CD56-PE/Cy7, 14 Wright PW, Li H, Huehn A, O'Connor GM, Cooley S, Miller JS et al. Characterization CD45-APC/Cy7, 3DL1-PE (clone DX9), NKG2D-PE (BD Biosciences, Oxford, of a weakly expressed KIR2DL1 variant reveals a novel upstream promoter that UK), 2DL1-FITC (clone 143211), 2DL3-FITC (clone 180701), 2DL4-FITC (clone controls KIR expression. Genes Immun 2014; 15:440–448.

© 2015 Macmillan Publishers Limited Genes and Immunity (2015) 301 – 310 Impact of allelic polymorphism on KIR expression SE Dunphy et al 310 15 Li H, Pascal V, Martin MP, Carrington M, Anderson SK. Genetic control of 23 Lanier LL. NK cell recognition. Annu Rev Immunol 2005; 23:225–274. variegated KIR gene expression: polymorphisms of the bi-directional KIR3DL1 24 Borrego F, Masilamani M, Marusina AI, Tang X, Coligan JE. The CD94/NKG2 family promoter are associated with distinct frequencies of gene expression. PLoS Genet of receptors: from molecules and cells to clinical relevance. Immunol Res 2006; 35: 2008; 4: e1000254. 263–278. 16 Charoudeh HN, Terszowski G, Czaja K, Gonzalez A, Schmitter K, Stern M. 25 Pegram HJ, Andrews DM, Smyth MJ, Darcy PK, Kershaw MH. Activating and Modulation of the natural killer cell KIR repertoire by cytomegalovirus infection. inhibitory receptors of natural killer cells. Immunol Cell Biol 2011; 89:216–224. – Eur J Immunol 2013; 43: 480 487. 26 Middleton D, Curran M, Maxwell L. Natural killer cells and their receptors. Transpl 17 Guinan KJ, Cunningham RT, Meenagh A, Gonzalez A, Dring MM, McGuinness BW Immunol 2002; 10:147–164. et al. Signatures of natural selection and coevolution between killer cell 27 Brown D, Trowsdale J, Allen R. The LILR family: modulators of innate and immunoglobulin-like receptors (KIR) and HLA class I genes. Genes Immun 2010; adaptive immune pathways in health and disease. Tissue Antigens 2004; 64: 11: 467–478. 215–225. 18 Babor F, Manser AR, Fischer JC, Scherenschlich N, Enczmann J, Chazara O et al. 28 Bari R, Bell T, Leung WH, Vong QP, Chan WK, Das Gupta N et al. KIR ligand C2 is associated with increased susceptibility to childhood ALL and Significant functional heterogeneity among KIR2DL1 alleles and a pivotal role of confers an elevated risk for late relapse. Blood 2014; 124: 2248–2251. arginine 245. Blood 2009; 114: 5182–5190. 19 Guinan KJ, Cunningham RT, Meenagh A, Dring MM, Middleton D, Gardiner CM. 29 Andersson S, Fauriat C, Malmberg JA, Ljunggren HG, Malmberg KJ. KIR acquisition Receptor systems controlling natural killer cell function are genetically stratified in Europe. Genes Immun 2010; 11:67–78. probabilities are independent of self-HLA class I ligands and increase with cellular – 20 Uhrberg M, Parham P, Wernet PDefinition of gene content for nine common KIR expression. Blood 2009; 114:95 104. group. B haplotypes of the Caucasoid population: KIR haplotypes contain 30 Beziat V, Liu LL, Malmberg JA, Ivarsson MA, Sohlberg E, Bjorklund AT et al. NK cell between seven and eleven KIR genes. Immunogenetics 2002; 54:221–229. responses to cytomegalovirus infection lead to stable imprints in the human KIR – 21 Beziat V, Traherne J, Malmberg JA, Ivarsson MA, Bjorkstrom NK, Retiere C et al. repertoire and involve activating KIRs. Blood 2013; 121: 2678 2688. Tracing dynamic expansion of human NK-cell subsets by high-resolution analysis 31 Davidson CL, Li NL, Burshtyn DN. LILRB1 polymorphism and surface phenotypes of KIR repertoires and cellular differentiation. Eur J Immunol 2014; 44: 2192–2196. of natural killer cells. Hum Immunol 2010; 71: 942–949. 22 Pando MJ, Gardiner CM, Gleimer M, McQueen KL, Parham P. The protein made 32 Jiang W, Johnson C, Jayaraman J, Simecek N, Noble J, Moffatt MF et al. from a common allele of KIR3DL1 (3DL1*004) is poorly expressed at cell surfaces Copy number variation leads to considerable diversity for B but not A haplotypes due to substitution at positions 86 in Ig domain 0 and 182 in Ig domain 1. of the human KIR genes encoding NK cell receptors. Genome Res 2012; 22: J Immunol 2003; 171: 6640–6649. 1845–1854.

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