A hybrid qPCR/SNP array approach allows cost efficient assessment of KIR gene copy numbers in large samples.
ABSTRACT: Killer Immunoglobulin-like Receptors (KIRs) are surface receptors of natural killer cells that bind to their corresponding Human Leukocyte Antigen (HLA) class I ligands, making them interesting candidate genes for HLA-associated autoimmune diseases, including type 1 diabetes (T1D). However, allelic and copy number variation in the KIR region effectively mask it from standard genome-wide association studies: single nucleotide polymorphism (SNP) probes targeting the region are often discarded by standard genotype callers since they exhibit variable cluster numbers. Quantitative Polymerase Chain Reaction (qPCR) assays address this issue. However, their cost is prohibitive at the sample sizes required for detecting effects typically observed in complex genetic diseases.We propose a more powerful and cost-effective alternative, which combines signals from SNPs with more than three clusters found in existing datasets, with qPCR on a subset of samples. First, we showed that noise and batch effects in multiplexed qPCR assays are addressed through normalisation and simultaneous copy number calling of multiple genes. Then, we used supervised classification to impute copy numbers of specific KIR genes from SNP signals. We applied this method to assess copy number variation in two KIR genes, KIR3DL1 and KIR3DS1, which are suitable candidates for T1D susceptibility since they encode the only KIR molecules known to bind with HLA-Bw4 epitopes. We find no association between KIR3DL1/3DS1 copy number and T1D in 6744 cases and 5362 controls; a sample size twenty-fold larger than in any previous KIR association study. Due to our sample size, we can exclude odds ratios larger than 1.1 for the common KIR3DL1/3DS1 copy number groups at the 5% significance level.We found no evidence of association of KIR3DL1/3DS1 copy number with T1D, either overall or dependent on HLA-Bw4 epitope. Five other KIR genes, KIR2DS4, KIR2DL3, KIR2DL5, KIR2DS5 and KIR2DS1, in high linkage disequilibrium with KIR3DL1 and KIR3DS1, are also unlikely to be significantly associated. Our approach could potentially be applied to other KIR genes to allow cost effective assaying of gene copy number in large samples.
Project description:The Behçet's disease (BD)-associated human leukocyte antigen (HLA) allele, HLA-B*51 (B*51), encodes a ligand for a pair of allelic killer immunoglobulin-like receptors (KIR) present on cytotoxic cells-KIR3DL1, which inhibits their cytotoxicity, and KIR3DS1, which activates their cytotoxic activity. We tested whether KIR-regulated mechanisms contribute to BD by testing for association of KIR3DL1/KIR3DS1 genotypes with disease in 1799 BD patients and 1710 healthy controls from Turkey, as well as in different subsets of individuals with HLA-type-defined ligands for the KIR3D receptors. HLA types were imputed from single nucleotide polymorphism genotypes determined with the Immunochip. The presence of inhibitory KIR3DL1 or activating KIR3DS1 alleles did not differ significantly between cases and controls (KIR3DL1: 92.9% vs 93.4%, Pdominant=0.55; KIR3DS1: 42.7% vs 41.0%, Pdominant=0.29). The KIR3DL1/KIR3DS1 alleles were also present at similar frequencies among cases and controls bearing HLA-B with a Bw4 motif; HLA-B with a Bw4 motif with isoleucine at position 80; and HLA-B*51. Our results suggest that pathogenic mechanisms associated with HLA-B*51 do not primarily involve differential interactions with KIR3DL1 and KIR3DS1 receptors. However, due to the complexity of this locus (that is, sequence variation and copy number variation), we cannot exclude a role for other types of KIR variation in the pathogenesis of BD.
Project description:NK cell activity is regulated by the integration of positive and negative signals. One important source of these signals for human NK cells is the killer Ig-like receptor (KIR) family, which includes both members that transduce positive and those that generate negative signals. KIR3DL1 inhibits NK cell activity upon engagement by its ligand HLA-Bw4. The highly homologous KIR3DS1 is an activating receptor, which is implicated in the outcome of a variety of pathological situations. However, unlike KIR3DL1, direct binding of KIR3DS1(+) cells to HLA has not been demonstrated. We analyzed four key amino acid differences between KIR3DL1*01502 and KIR3DS1*013 to determine their role in KIR binding to HLA. Single substitutions of these residues dramatically reduced binding by KIR3DL1. In the reciprocal experiment, we found that the rare KIR3DS1 allotype KIR3DS1*014 binds HLA-Bw4 even though it differs from KIR3DS1*013 at only one of these positions (position 138). This reactivity was unexpectedly dependent on residues at other variable positions, as HLA-Bw4 binding was lost in receptors with KIR3DL1-like residues at both positions 199 and 138. These data provide the first evidence, to our knowledge, for the direct binding of KIR3DS1(+) cells to HLA-Bw4 and highlight the key role for position 138 in determining ligand specificity of KIR3DS1. They also reveal that KIR3DS1 reactivity and specificity is dictated by complex interactions between the residues in this region, suggesting a unique functional evolution of KIR3DS1 within the activating KIR family.
Project description:The function of natural killer (NK) cells is controlled by several activating and inhibitory receptors, including the family of killer-immunoglobulin-like receptors (KIRs). One distinctive feature of KIRs is the extensive number of various haplotypes generated by the gene content within the KIR gene locus as well as by highly polymorphic members of the KIR gene family, namely KIR3DL1/S1. Within the KIR3DL1/S1 gene locus, KIR3DS1 represents a conserved allelic variant and displays other unique features in comparison to the highly polymorphic KIR3DL1 allele. KIR3DS1 is present in all human populations and belongs to the KIR haplotype group B. KIR3DS1 encodes for an activating receptor featuring the characteristic short cytoplasmic tail and a positively charged residue within the transmembrane domain, which allows recruitment of the ITAM-bearing adaptor molecule DAP12. Although HLA class I molecules are thought to represent natural KIR ligands, and HLA-Bw4 molecules serve as ligands for KIR3DL1, the ligand for KIR3DS1 still needs to be identified. Despite the lack of formal evidence for an interaction of KIR3DS1 with HLA-Bw4-I80 or any other HLA class I subtype to date, a growing number of associations between the presence of KIR3DS1 and the outcome of viral infections have been described. Especially, the potential protective role of KIR3DS1 in combination with HLA-Bw4-I80 in the context of HIV-1 infection has been studied intensively. In addition, a number of recent studies have associated the presence or absence of KIR3DS1 with the occurrence and outcome of some malignancies, autoimmune diseases, and graft-versus-host disease (GVHD). In this review, we summarize the present knowledge regarding the characteristics of KIRD3S1 and discuss its role in various human diseases.
Project description:Several studies described an association between killer-cell immunoglobulin-like receptor (KIR)/HLA gene combinations and clinical outcomes in various diseases. In particular, an important combined role for KIR3DS1 and HLA-B Bw4-I80 in controlling viral infections and a higher protection against leukemic relapses in donor equipped with activating KIRs in haplo-HSCT has been described. Here, we show that KIR3DS1 mediates positive signals upon recognition of HLA-B*51 (Bw4-I80) surface molecules on target cells and that this activation occurs only in Bw4-I80neg individuals, including those carrying particular KIR/HLA combination settings. In addition, killing of HLA-B*51 transfected target cells mediated by KIR3DS1+/NKG2A+ natural killer (NK) cell clones from Bw4-I80neg donors could be partially inhibited by antibody-mediated masking of KIR3DS1. Interestingly, KIR3DS1-mediated recognition of HLA-B*51 could be better appreciated under experimental conditions in which the function of NKG2D was reduced by mAb-mediated blocking. This experimental approach may mimic the compromised function of NKG2D occurring in certain viral infections. We also show that, in KIR3DS1+/NKG2A+ NK cell clones derived from an HLA-B Bw4-T80 donor carrying 2 KIR3DS1 gene copy numbers, the positive signal generated by the engagement of KIR3DS1 by HLA-B*51 resulted in a more efficient killing of HLA-B*51-transfected target cells. Moreover, in these clones, a direct correlation between KIR3DS1 and NKG2D surface density was detected, while the expression of NKp46 was inversely correlated with that of KIR3DS1. Finally, we analyzed KIR3DS1+/NKG2A+ NK cell clones from a HLA-B Bw4neg donor carrying cytoplasmic KIR3DL1. Although these clones expressed lower levels of surface KIR3DS1, they displayed responses comparable to those of NK cell clones derived from HLA-B Bw4neg donors that expressed surface KIR3DL1. Altogether these data suggest that, in particular KIR/HLA combinations, KIR3DS1 may play a role in the process of human NK cell education.
Project description:<h4>Objective</h4>To explore genes of the killer-cell immunoglobulin-like receptor (KIR) and of the HLA ligand and their relationship with the outcome of metastatic colorectal cancer (mCRC) patients treated with first-line 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI).<h4>Methods</h4>A total of 224 mCRC patients were screened for KIR/HLA typing. The determination of the KIR/HLA combinations was based upon the gene content and variants. Genetic associations with complete response (CR), time to progression (TTP) and overall survival (OS) were evaluated by calculating odds and hazard ratios. Multivariate modeling with prognostic covariates was also performed.<h4>Results</h4>For CR, the presence of KIR2DL5A, 2DS5, 2DS1, 3DS1, and KIR3DS1/HLA-Bw4-I80 was associated with increased CR rates, with median ORs ranging from 2.1 to 4.3, while the absence of KIR2DS4 and 3DL1 was associated with increased CR rates (OR 3.1). After univariate analysis, patients that underwent resective surgery of tumor, absence of KIR2DS5, and presence of KIR3DL1/HLA-Bw4-I80 showed a significant better OS (HR 1.5 to 2.8). Multivariate analysis identified as parameters independently related to OS the type of treatment (surgery; HR 2.0) and KIR3DL1/HLA-Bw4-I80 genotype (HR for T-I80 2.7 and for no functional KIR/HLA interaction 1.8). For TTP, no association with KIR/HLA genes was observed.<h4>Conclusion</h4>This study, for the first time, evidences that the genotyping for KIR-HLA pairs are found predictive markers associated with complete response and improves overall survival prediction of FOLFIRI treatment response in metastatic colorectal cancer. These results suggest a role of the KIR/HLA system in patient outcome, and guide new research on the immunogenetics of mCRC through mechanistic studies and clinical validation.
Project description:A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.
Project description:Killer Immunoglobulin-like Receptors (KIR) are a family of receptors expressed on natural killer (NK) and T-cell subsets. KIR3DL1 is a highly polymorphic receptor that binds to groups of HLAA and HLA-B allotypes that express the Bw4 epitope. The variation in KIR3DL1 allotypes manifests at a number of levels. Most dramatically, a common allelic variant encodes an activating rather than an inhibitory receptor (KIR3DS1). In addition, sequence variants can affect both the frequency of expression within the NK cell population and the intensity of expression on a given cell. KIR3DL1 polymorphism also influences the interaction with HLA-Bw4 molecules, due to contacts with the HLA molecule itself and sensitivity to the presented peptide. A body of evidence from genetic association studies supports the biological significance not only of the interaction of KIR3DL1 with HLA-Bw4 but also the functional variation seen with different KIR3DL1 and HLA allotypes. In this review, we discuss our current understanding of KIR3DL1 function and our recent insights from the structure of the KIR3DL1 in complex with HLA. In addition, we will summarize our current understanding of KIR3DS1, including its ligand specificity and its role in immune responses.
Project description:In patients with neuroblastoma (NB), treatment with anti-GD2 monoclonal antibody (mAb) directs natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against tumor cells. However, tumor cytotoxicity is attenuated by ligation of inhibitory killer immunoglobulin-like receptors (KIRs) by HLA class I molecules. KIR3DL1 polymorphism influences its ability to engage HLA-Bw4 ligands. We tested the hypothesis that poorly interacting combinations of KIR3DL1 and HLA ligands are more permissive of mAb-mediated antitumor effect.KIR3DL1 and HLA-B subtyping were performed with a multiplex intermediate-resolution polymerase chain reaction assay for a cohort of 245 patients who were treated with antibody 3F8 for high-risk NB. Patient outcomes were analyzed according to expected degree of interaction between KIR3DL1 and HLA-B subtypes and grouped as strong, weak, or noninteractors. A comparison of NK response to 3F8 mAb opsonized NB cells between strong- and noninteracting donors was performed by flow cytometry.KIR3DL1 and HLA-B subtype combinations associated with noninteraction as a result of lack of receptor expression [KIR3DL1(-)], failure of interaction with inhibitory ligands [KIR3DS1(+)], or absence of KIR ligands resulted in significantly improved overall and progression-free survival. Patients with KIR3DL1 and HLA-B subtype combinations that were predictive of weak interaction had superior outcomes compared with those that were predictive of strong interaction; however, both groups were inferior to those with noninteracting subtype combinations. In vitro analysis of 3F8-mediated ADCC showed that KIR3DL1(-) and 3DS1(+) NK cells were insensitive to inhibition by HLA-Bw4-expressing NB targets.We conclude that KIR3LD1 and HLA-B allele combinations can have a prognostic impact on patient survival after treatment with anti-GD2 mAb that relies on NK-ADCC. The survival advantage seen in noninteracting combinations supports the therapeutic disinhibition of individuals with strongly interacting KIR and ligand pairs.
Project description:Decline of peak viremia during acute HIV-1 infection occurs before the development of vigorous adaptive immunity, and the level of decline correlates inversely with the rate of AIDS progression, implicating a potential role for the innate immune response in determining disease outcome. The combined expression of an activating natural killer (NK) cell receptor, the killer immunoglobulin-like receptor (KIR) 3DS1, and its presumed ligand, human leukocyte antigen (HLA)-B Bw4-80I, has been associated in epidemiological studies with a slow progression to AIDS. We examined the functional ability of NK cells to differentially control HIV-1 replication in vitro based on their KIR and HLA types. NK cells expressing KIR3DS1 showed strong, significant dose- and cell contact-dependent inhibition of HIV-1 replication in target cells expressing HLA-B Bw4-80I compared with NK cells that did not express KIR3DS1. Furthermore, KIR3DS1+ NK cells and NKLs were preferentially activated, and lysed HIV-1 infected target cells in an HLA-B Bw4-80I-dependent manner. These data provide the first functional evidence that variation at the KIR locus influences the effectiveness of NK cell activity in the containment of viral replication.
Project description:BACKGROUND: Natural killer (NK) cells have emerged as pivotal players in innate immunity, especially in the defense against viral infections and tumors. Killer immunoglobulin-like receptors (KIRs)--an important recognition receptor expressed on the surface of NK cells--regulate the inhibition and/or activation of NK cells after interacting with human leukocyte antigen (HLA) class I ligands. Various KIR genes might impact the prognosis of many different diseases. The implications of KIR-HLA interaction in HIV disease progression remains poorly understood. METHODS: Here, we studied KIR genotypes, mRNA levels, HLA genotypes, CD4+ T cell counts and viral loads in our cohort of Human Immunodeficiency Virus (HIV)-infected individuals, a group that includes HIV long-term nonprogressors (LTNPs) and typical progressors (TPs). RESULTS: We found that the frequency of KIR3DS1/L1 heterozygotes with HLA-Bw4-80I gene was much higher in LTNPs than in TPs (P?=?0.001) and that the KIR3DL1 homozygotes without HLA-Bw4-80I gene had higher viral loads and lower CD4+ T cell counts (P?=?0.014 and P?=?0.021, respectively). Our study also confirmed that homozygosity for the HLA-Bw6 allele was associated with rapid disease progression. In addition to the aforementioned results on the DNA level, we observed that higher level expression of KIR3DS1 mRNA was in LTNP group, and that higher level expression of KIR3DL1 mRNA was in TP group. CONCLUSIONS: Our data suggest that different KIR-HLA genotypes and different levels of transcripts associate with HIV disease progression.