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Peer reviewed: True, The biological processes underlying NK cell alloreactivity in haematopoietic stem cell transplantation (HSCT) remain unclear. Many different models to predict NK alloreactivity through KIR and MHC genotyping exist, raising ambiguities in its utility and application for clinicians. We assessed 27 predictive models, broadly divided into six categories of alloreactivity prediction: ligand-ligand, receptor-ligand, educational, KIR haplotype-based, KIR matching and KIR allelic polymorphism. The models were applied to 78 NGS-typed donor/recipient pairs undergoing allogeneic HSCT in genoidentical (n=43) or haploidentical (n=35) matchings. Correlations between different predictive models differed widely, suggesting that the choice of the model in predicting NK alloreactivity matters. For example, two broadly used models, educational and receptor-ligand, led to opposing predictions especially in the genoidentical cohort. Correlations also depended on the matching fashion, suggesting that this parameter should also be taken into account in the choice of the scoring strategy. The number of centromeric B-motifs was the only model strongly correlated with the incidence of acute graft-versus-host disease in our set of patients in both the genoidentical and the haploidentical cohorts, suggesting that KIR-based alloreactivity, not MHC mismatches, are responsible for it. To our best knowledge, this paper is the first to experimentally compare NK alloreactivity prediction models within a cohort of genoidentical and haploidentical donor-recipient pairs. This study helps to resolve current discrepancies in KIR-based alloreactivity predictions and highlights the need for deeper consideration of the models used in clinical studies as well as in medical practice.

The conserved CD94/NKG2A inhibitory receptor is expressed by nearly all human and ∼50% of mouse uterine natural killer (uNK) cells. Binding human HLA-E and mouse Qa-1, NKG2A drives NK cell education, a process of unknown physiological importance influenced by HLA-B alleles. Here, we show that NKG2A genetic ablation in dams mated with wild-type males caused suboptimal maternal vascular responses in pregnancy, accompanied by perturbed placental gene expression, reduced fetal weight, greater rates of smaller fetuses with asymmetric growth, and abnormal brain development. These are features of the human syndrome pre-eclampsia. In a genome-wide association study of 7,219 pre-eclampsia cases, we found a 7% greater relative risk associated with the maternal HLA-B allele that does not favor NKG2A education. These results show that the maternal HLA-B→HLA-E→NKG2A pathway contributes to healthy pregnancy and may have repercussions on offspring health, thus establishing the physiological relevance for NK cell education. VIDEO ABSTRACT.

INTRODUCTION: Preeclampsia (PE) is a human specific pregnancy-related syndrome of unknown etiology that affects 2-8 % of pregnancies. Polymorphism in maternal Killer Cell Immunoglobulin-like Receptors (KIRs) and the ligand fetal Human Leukocyte Antigen-C (HLA-C) may predispose pregnant mothers for PE due to defective trophoblast invasion into the maternal decidua. Our study aimed to investigate the association between maternal KIR and fetal HLA-C polymorphism and PE in Ethiopian pregnant women. METHODS: We included a total of 288 (157 controls and 131 PE cases) in a case-controls study at Adama Regional Referral Hospital, Ethiopia. The KIR and HLA-C genotyping was done using traditional polymerase chain reaction on genomic DNA extracted form maternal venous and cord blood followed by 2% agarose gel electrophoresis. RESULTS: The statistical associations between variables were evaluated using Pearson's Chi-square test. P < 0.05, with 95 % confidence interval was considered statistically significant. A significant association was observed between the KIR2DS1 and PE, with a higher frequency (60.5 %) of the gene in the control group. Similarly, a significant association was observed between KIR AA genotype and PE, with a higher frequency (38.2 %) of this genotype in the PE group. Ethiopians share the same risk genotype for PE as seen in previous African and European studies, namely homozygosity of a maternal KIR AA genotype. However, Ethiopians differ from other East African populations by sharing the same protective KIR2DS1 gene as Europeans.

Malaria is one of the deadliest infectious diseases in the world. Immune responses to Plasmodium falciparum malaria vary among individuals and between populations. Human genetic variation in immune system genes is likely to play a role in this heterogeneity. Natural killer (NK) cells produce inflammatory cytokines in response to malaria infection, kill intraerythrocytic Plasmodium falciparum parasites by cytolysis, and participate in the initiation and development of adaptive immune responses to plasmodial infection. These functions are modulated by interactions between killer-cell immunoglobulin-like receptors (KIRs) and human leukocyte antigens (HLAs). Therefore, variations in KIR and HLA genes can have a direct impact on NK cell functions. Understanding the role of KIRs and HLAs in immunity to malaria can help to better characterize antimalarial immune responses. In this review, we summarize the different KIRs and HLAs associated with immunity to malaria thus far.

Genome-wide association studies of the related chronic inflammatory bowel diseases (IBD) known as Crohn's disease and ulcerative colitis have shown strong evidence of association to the major histocompatibility complex (MHC). This region encodes a large number of immunological candidates, including the antigen-presenting classical human leukocyte antigen (HLA) molecules. Studies in IBD have indicated that multiple independent associations exist at HLA and non-HLA genes, but they have lacked the statistical power to define the architecture of association and causal alleles. To address this, we performed high-density SNP typing of the MHC in >32,000 individuals with IBD, implicating multiple HLA alleles, with a primary role for HLA-DRB1*01:03 in both Crohn's disease and ulcerative colitis. Noteworthy differences were observed between these diseases, including a predominant role for class II HLA variants and heterozygous advantage observed in ulcerative colitis, suggesting an important role of the adaptive immune response in the colonic environment in the pathogenesis of IBD.

The killer cell immunoglobulin‐like receptor (KIR) KIR2DS2 induces NK cell activation upon ligation and in genetic studies is associated with protection against certain cancers and viral infections. One of the difficulties in understanding KIR2DS2 has been that ligands have been hard to define. In part, this is because the high sequence homology between KIR2DS2 and KIR2DL3/KIR2DL2 has made it difficult to make antibodies that specifically detect NK cells expressing KIR2DS2. Using transfected NKL cells and primary human samples, we report the identification of a novel antibody combination which allows identification of NK cells with relatively high expression of KIR2DS2. This separation is sufficient to examine primary human NK cell activation in response to KIR2DS2 specific ligands.

Although myelin oligodendrocyte glycoprotein is a candidate autoantigen in multiple sclerosis, its function remains unknown. In humans, mRNA expressed by the myelin oligodendrocyte glycoprotein gene is alternatively spliced resulting in at least nine unique protein isoforms. In this study, we investigated the sub-cellular localisation and membrane trafficking of six isoforms by cloning them into mammalian expression vectors. Confocal microscopy revealed that these protein products are expressed in different cellular compartments. While two full-length isoforms (25.6 and 25.1) are expressed at the cell surface, three alternatively spliced forms (22.7, 21.0 and 20.5) have a more intracellular distribution, localising to the endoplasmic reticulum and/or endosomes. Isoform 16.3, which lacks a transmembrane domain, is secreted. A switch in the sub-cellular localisation of myelin oligodendrocyte glycoprotein may have profound effects on receptor:ligand interactions and consequently the function of the protein. The structural features of the alternative isoforms and their differential, sub-cellular expression patterns could dictate the exposure of major immunogenic determinants within the central nervous system. Our findings highlight myelin oligodendrocyte glycoprotein splicing as a factor that could be critical to the phenotypic expression of multiple sclerosis.

In sub-Saharan Africans, maternal mortality is unacceptably high, with >400 deaths per 100,000 births compared with

AIM: To investigate influence of human leukocyte antigen (HLA) and killer immunoglobuline-like receptor (KIR) genotypes on risks of acute rejection (AR) after liver transplantation (LTX). METHODS: In this retrospective study we included 143 adult donor-recipient pairs with a minimum of 6 mo follow-up after LTX for whom DNA was available from both donor and recipients. Clinical data, all early complications including episodes and severity of AR and graft/patient survival were registered. The diagnosis of AR was based on clinical, biochemical and histological criteria. All suspected episodes of AR were biopsy confirmed. Key classical HLA loci (HLA-A, HLA-B, HLA-C and HLA-DRB1) were genotyped using Sanger sequencing. 16 KIR genes were genotyped using a novel real time PCR approach which allows for determination of the diploid copy number of each KIR gene. Immunohistochemical staining for T (CD3), B (CD20) and natural killer (NK) cells (CD56 and CD57) were performed on liver biopsies from 3 different patient groups [primary sclerosing cholangitis (PSC), primary biliary cirrhosis and non-autoimmune liver disease], 10 in each group, with similar grade of AR. RESULTS: Fourty-four (31%) patients were transplanted on the basis of PSC, 40% of them had AR vs 24% in the non-PSC group (P = 0.04). No significant impact of donor-recipient matching for HLA and KIR genotypes was detected. In the overall recipient population an increased risk of AR was detected for HLA-B*08 (P = 0.002, OR = 2.5; 95%CI: 1.4-4.6), HLA-C*07 (P = 0.001, OR = 2.4; 95%CI: 1.4-4.0) and HLA-DRB1*03 (P = 0.03, OR = 1.9; 95%CI: 1.0-3.3) and a decreased risk for HLA-DRB1*04 (P = 0.001, OR = 0.2; 95%CI: 0.1-0.5). For HLA-B*08, HLA-C*07 and DRB1*04 the associations remained evident in a subgroup analysis of non-PSC recipients (P = 0.04, P = 0.003 and P = 0.02, respectively). In PSC recipients corresponding P values were 0.002, 0.17 and 0.01 for HLA-B*08, HLA-C*07 and DRB1*04, respectively. A dosage effect of AR prevalence according to the PSC associated HLA alleles was also notable in the total recipient population. For HLA-B*08 the frequency of AR was 56% in HLA-B*08 homozygous recipients, 39% in heterozygous recipients and 21% in recipients lacking HLA-B*08 (P = 0.02). The same was observed for the HLA-C*07 allele with AR in 57%, 27% and 18% in recipients being homozygous, heterozygous and lacking HLA-C*07 respectively (P = 0.003). Immunohistochemical analysis showed similar infiltration of T, B and NK cells in biopsies with AR in all three groups. CONCLUSION: We found significant associations between the PSC-associated HLA-B*08, HLA-C*07, HLA-DRB1*03 and HLA-DRB1*04 alleles and risk of AR in liver transplant recipients.

Killer Immunoglobulin-like Receptors (KIR) are important surface receptors of Natural Killer cells. KIRs mediate the fate of target cells based on the composite inhibiting/activating signal generated on binding to their corresponding HLA (Human Leukocyte Antigen) class I ligands. Type 1 diabetes (T1D) is an autoimmune disease strongly associated with genetic variation in the HLA region, primarily with Class II genes, but also with HLA Class I loci including HLA- Bw4, an epitope grouping both HLA-A and HLA-B alleles into HLA-Bw4-80I and HLA-Bw4- 80T subgroups. Amongst the 17 known genes of the KIR region, characterised by alternative haplotypes of variable gene copy numbers, KIR3DL1 is the only one proven to bind in-vitro with both HLA-Bw4 epitopes. Its activating counterpart, KIR3DS1, putatively binds only to the HLA-Bw4-80I epitope. KIR3DL1/S1 is therefore a suitable T1D candidate gene but has not been studied in GWAS as SNP arrays can only genotype normal copy number regions. So far, insufficiently powered PCR-based genotyping studies of less than 600 individuals, have failed to detect convincing association of T1D with presence or absence of KIR3DL1/S1. For the first time, we are able to test for association in a sample size of 6,058 cases and 4,733 controls, twenty-fold larger than any previous study, by using KIR3DL1/S1 copy number calls from a recently developed KIR qPCR assay to impute copy number from ImmunoChip, a custom Illumina genotyping chip with dense coverage of autoimmune regions. The KIR3DL1/S1 copy numbers from qPCR were predicted by clustering jointly on the ΔCt values of KIR3DL1 and KIR3DS1. The imputed KIR3DL1/S1 copy numbers from ImmunoChip were predicted from raw SNP signals, Log R Ratio and B Allele Frequency, in the KIR3DL1/S1 region, using a nearest neighbour classifier trained on 749 case and 727 control samples analysed both with qPCR and on Immunochip (2.04% misclassification error rate in leave-one-out cross-validation). The association of KIR3DL1/S1 copy number with T1D was tested in all samples and on the appropriate HLA-Bw4 subsets. The interaction effect between KIR3DL1/S1 and HLA-Bw4 was tested with a case-only analysis. We can confirm from our study with a substantially increased sample size, that copy number variation in KIR3DL1/S1 is not an important modifier of T1D risk (OR < 1.1 at 95% confidence for common genotypes), either alone or dependently on HLA-Bw4 epitope, and that furthermore, there is no evidence of statistical interaction effect between HLA-Bw4 and KIR3DL1/S1 in T1D.

Killer Immunoglobulin-like Receptors (KIR) are important surface receptors of Natural Killer cells. KIRs mediate the fate of target cells based on the composite inhibiting/activating signal generated on binding to their corresponding HLA (Human Leukocyte Antigen) class I ligands. Type 1 diabetes (T1D) is an autoimmune disease strongly associated with genetic variation in the HLA region, primarily with Class II genes, but also with HLA Class I loci including HLA- Bw4, an epitope grouping both HLA-A and HLA-B alleles into HLA-Bw4-80I and HLA-Bw4- 80T subgroups. Amongst the 17 known genes of the KIR region, characterised by alternative haplotypes of variable gene copy numbers, KIR3DL1 is the only one proven to bind in-vitro with both HLA-Bw4 epitopes. Its activating counterpart, KIR3DS1, putatively binds only to the HLA-Bw4-80I epitope. KIR3DL1/S1 is therefore a suitable T1D candidate gene but has not been studied in GWAS as SNP arrays can only genotype normal copy number regions. So far, insufficiently powered PCR-based genotyping studies of less than 600 individuals, have failed to detect convincing association of T1D with presence or absence of KIR3DL1/S1. For the first time, we are able to test for association in a sample size of 6,058 cases and 4,733 controls, twenty-fold larger than any previous study, by using KIR3DL1/S1 copy number calls from a recently developed KIR qPCR assay to impute copy number from ImmunoChip, a custom Illumina genotyping chip with dense coverage of autoimmune regions. The KIR3DL1/S1 copy numbers from qPCR were predicted by clustering jointly on the ΔCt values of KIR3DL1 and KIR3DS1. The imputed KIR3DL1/S1 copy numbers from ImmunoChip were predicted from raw SNP signals, Log R Ratio and B Allele Frequency, in the KIR3DL1/S1 region, using a nearest neighbour classifier trained on 749 case and 727 control samples analysed both with qPCR and on Immunochip (2.04% misclassification error rate in leave-one-out cross-validation). The association of KIR3DL1/S1 copy number with T1D was tested in all samples and on the appropriate HLA-Bw4 subsets. The interaction effect between KIR3DL1/S1 and HLA-Bw4 was tested with a case-only analysis. We can confirm from our study with a substantially increased sample size, that copy number variation in KIR3DL1/S1 is not an important modifier of T1D risk (OR < 1.1 at 95% confidence for common genotypes), either alone or dependently on HLA-Bw4 epitope, and that furthermore, there is no evidence of statistical interaction effect between HLA-Bw4 and KIR3DL1/S1 in T1D.

The Killer-cell Immunoglobulin-like Receptor (KIR) gene complex has considerable biomedical importance. Patterns of polymorphism in the KIR region include variability in the gene content of haplotypes and diverse structural arrangements. Droplet digital PCR (ddPCR) was used to identify different haplotype motifs and to enumerate KIR copy number variants (CNVs). ddPCR detected a variety of KIR haplotype configurations in DNA from well-characterized cell lines. Mendelian segregation of ddPCR-estimated KIR2DL5 CNVs was observed in Gambian families and CNV typing of other KIRs was shown to be accurate when compared to an established quantitative PCR method.

The killer cell immunoglobulin-like receptor (KIR) KIR2DS2 induces natural killer (NK) cell activation upon ligation and in genetic studies is associated with protection against certain cancers and viral infections. One of the difficulties in understanding KIR2DS2 has been that ligands have been hard to define. In part, this is because the high sequence homology between KIR2DS2 and KIR2DL3/KIR2DL2 has made it difficult to make antibodies that specifically detect NK cells expressing KIR2DS2. Using transfected NK cell line (NKL) cells and primary human samples, we report the identification of a novel antibody combination which allows identification of NK cells with relatively high expression of KIR2DS2. This separation is sufficient to examine primary human NK cell activation in response to KIR2DS2 specific ligands.

Killer cell immunoglobulin-like receptors (KIRs), expressed on natural killer cells and T cells, have considerable biomedical relevance playing significant roles in immunity, pregnancy and transplantation. The $\textit{KIR }$ locus is one of the most complex and polymorphic regions of the human genome. Extensive sequence homology and copy number variation makes $\textit{KIR }$s technically laborious and expensive to type. To aid the investigation of $\textit{KIR }$s in human disease we developed a high-throughput, multiplex real-time polymerase chain reaction method to determine gene copy number for each $\textit{KIR }$ locus. We used reference DNA samples to validate the accuracy and a cohort of 1698 individuals to evaluate capability for precise copy number discrimination. The method provides improved information and identifies $\textit{KIR }$ haplotype alterations that were not previously visible using other approaches., This work was funded by the Medical Research Council (MRC) and the Wellcome Trust with partial funding from the National Institute of Health (NIH) Cambridge Biomedical Research Centre and NIH Research Blood and Transplant Research Unit (NIHR BTRU) in Organ Donation and Transplantation at the University of Cambridge in collaboration with Newcastle University and in partnership with NHS Blood and Transplant (NHSBT)., This is the final version of the article. It first appeared from BioMed Central via https://doi.org/10.1186/s13073-016-0358-0

Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell-mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8+ T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1-, hepatitis C virus (HCV)-, and human T cell leukemia virus type 1 (HTLV-1)-infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8+ T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections.

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Allogeneic hematopoietic stem cell transplantation (aHSCT) is a lifesaving therapy for hematological malignancies. For years, a fully matched HLA donor was a requisite for the procedure. However, new immunosuppressive strategies have enabled the recruitment of viable alternative donors, particularly haploidentical donors. Over 95% of patients have at least two potential haploidentical donors available to them. To identify the best haploidentical donor, the assessment of new immunogenetic criteria could help. To this end, the clinical benefit of KIR genotyping in aHSCT has been widely studied but remains contentious. This review aims to evaluate the importance of KIR-driven NK cell alloreactivity in the context of aHSCT and explain potential reasons for the discrepancies in the literature. Here, through a non-systematic review, we highlight how the studies in this field and their respective predictive models or scoring strategies could be conceptually opposed, explaining why the role of NK cells remains unclear in aHCST outcomes. We evaluate the limitations of each published prediction model and describe how every scoring strategy to date only partly delivers the requirements for optimally effective NK cells in aHSCT. Finally, we propose approaches toward finding the optimal use of KIR genotyping in aHSCT for a unified criterion for donor selection.

Killer immunoglobulin-like receptors (KIRs) are a family of receptors expressed on Natural killer (NK) cells. The extensive polymorphism of KIR is involved in the immune responses of NK cells and influences dengue infections. We investigated the diversity of KIR copy numbers in dengue-infected patients from northeastern Thailand. Copy numbers of KIRs were determined by quantitative polymerase chain reaction in 137 dengue-infected patients, comprising 63 dengue fever (DF) and 74 dengue hemorrhagic fever (DHF). The distribution of KIRs was observed to be between 0 and 4 copies. The KIR AA genotype with heterozygous KIR2DS4D/WT was the most common in dengue patients, 25.4% DF and 23% DHF. Forty KIR profiles were determined in dengue patients, including 31 usual, 6 expanded, and 3 contracted profiles. Investigation of KIR copy number and dengue severity indicated that two copies of KIR2DL3 combined with HLA-C1C1 associated with an increased risk of DHF (OR 2.32, 95% CI 1.159-4.624, P = 0.016), whereas one copy of KIR2DL2 and KIR2DL3 together with HLA-C1C1 associated with a reduced risk of DHF (OR 0.17, 95% CI 0.058-0.482, P < 0.001). The outcomes of this study will contribute to the understanding of KIR complexity and innate immune responses in dengue infections.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

Natural killer (NK) cells are innate lymphocytes that eliminate infected and transformed cells. They discriminate healthy from diseased tissue through killer cell Ig-like receptor (KIR) recognition of HLA class I ligands. Directly impacting NK cell function, KIR polymorphism associates with infection control and multiple autoimmune and pregnancy syndromes. Here we analyze KIR diversity of 241 individuals from five groups of Iranians. These five populations represent Baloch, Kurd, and Lur, together comprising 15% of the ethnically diverse Iranian population. We identified 159 KIR alleles, including 11 not previously characterized. We also identified 170 centromeric and 94 telomeric haplotypes, and 15 different KIR haplotypes carrying either a deletion or duplication encompassing one or more complete KIR genes. As expected, comparing our data with those representing major worldwide populations revealed the greatest similarity between Iranians and Europeans. Despite this similarity we observed higher frequencies of KIR3DL1 * 001 in Iran than any other population, and the highest frequency of HLA-B*51, a Bw4-containing allotype that acts as a strong educator of KIR3DL1 * 001 + NK cells. Compared to Europeans, the Iranians we studied also have a reduced frequency of 3DL1 * 004, which encodes an allotype that is not expressed at the NK cell surface. Concurrent with the resulting high frequency of strong viable interactions between inhibitory KIR and polymorphic HLA class I, the majority of KIR-A haplotypes characterized do not express a functional activating receptor. By contrast, the most frequent KIR-B haplotype in Iran expresses only one functional inhibitory KIR and the maximum number of activating KIR. This first complete, high-resolution, characterization of the KIR locus of Iranians will form a valuable reference for future clinical and population studies.

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

The physiological functions of natural killer (NK) cells in human immunity and reproduction depend upon diverse interactions between killer cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands: HLA-A, HLA-B, and HLA-C. The genomic regions containing the KIR and HLA class I genes are unlinked, structurally complex, and highly polymorphic. They are also strongly associated with a wide spectrum of diseases, including infections, autoimmune disorders, cancers, and pregnancy disorders, as well as the efficacy of transplantation and other immunotherapies. To facilitate study of these extraordinary genes, we developed a method that captures, sequences, and analyzes the 13 KIR genes and HLA-A, HLA-B, and HLA-C from genomic DNA. We also devised a bioinformatics pipeline that attributes sequencing reads to specific KIR genes, determines copy number by read depth, and calls high-resolution genotypes for each KIR gene. We validated this method by using DNA from well-characterized cell lines, comparing it to established methods of HLA and KIR genotyping, and determining KIR genotypes from 1000 Genomes sequence data. This identified 116 previously uncharacterized KIR alleles, which were all demonstrated to be authentic by sequencing from source DNA via standard methods. Analysis of just two KIR genes showed that 22% of the 1000 Genomes individuals have a previously uncharacterized allele or a structural variant. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations and defining the precise HLA and KIR factors associated with disease. The methods are applicable to other highly polymorphic genes.

Leukocyte immunoglobulin-like receptors (LILR) are expressed mostly on myelomonocytic cells where they are mediators of immunological tolerance. Two LILR genes, LILRA3 and LILRA6, exhibit marked copy number variation. We assessed the contribution of these genes to atopic dermatitis (AD) by analysing transmission in 378 AD families. The data indicated that copies of LILRA6 were over-transmitted to affected patients. They are consistent with a contribution of LILR genes to AD. They could affect the equilibrium between activating and inhibitory signals in the immune response.

Infection by human cytomegalovirus (HCMV) leads to NKG2C-driven expansion of adaptive natural killer (NK) cells, contributing to host defense. However, approximately 4% of all humans carry a homozygous deletion of the gene that encodes NKG2C ($\textit{NKG2C}^{-/-}$). Assessment of NK cell repertoires in 60 $\textit{NKG2C}^{-/-}$ donors revealed a broad range of NK cell populations displaying characteristic footprints of adaptive NK cells, including a terminally differentiated phenotype, functional reprogramming, and epigenetic remodeling of the interferon (IFN)-$\gamma$ promoter. We found that both NKG2C$^{-}$ and NKG2C$^{+}$ adaptive NK cells expressed high levels of CD2, which synergistically enhanced ERK and S6RP phosphorylation following CD16 ligation. Notably, CD2 co-stimulation was critical for the ability of adaptive NK cells to respond to antibody-coated target cells. These results reveal an unexpected redundancy in the human NK cell response to HCMV and suggest that CD2 provides "signal 2" in antibody-driven adaptive NK cell responses.

Made available in DSpace on 2019-10-06T16:55:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Wisconsin Turfgrass Association National Institutes of Health Wellcome Trust Institute for National Strategic Studies Leukemia and Lymphoma Research Seventh Framework Programme Horizon 2020 Medical Research Council Canada National Institute on Drug Abuse National Institute of Child Health and Human Development Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell–mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8+ T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1–, hepatitis C virus (HCV)–, and human T cell leukemia virus type 1 (HTLV-1)–infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8+ T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections. Department of Medicine Imperial College London Faculty of Engineering São Paulo State University—UNESP Institute for Infection and Immunity St. George’s University of London International AIDS Vaccine Initiative Human Immunology Laboratory Clinical Research Department London School of Hygiene and Tropical Medicine Divi-sion of Infection and Immunity Cardiff University School of Medicine Nuffield Department of Medicine University of Oxford Oxford NIHR Biomedical Research Centre Immunology Division Department of Pathology University of Cambridge Cancer and Inflammation Program Leidos Biomedical Research Inc. Frederick National Laboratory for Cancer Research Johns Hopkins University Rho San Francisco Department of Public Health Faculty of Medicine University of Southampton Division of Cancer Epidemiology and Genetics National Cancer Institute Ragon Institute of MGH, MIT and Harvard Faculty of Engineering São Paulo State University—UNESP Wisconsin Turfgrass Association: 090323/Z/09/Z National Institutes of Health: 100326Z/12/Z Wellcome Trust: 103865Z/14/Z Institute for National Strategic Studies: 105609/Z/14/Z Leukemia and Lymphoma Research: 15012 Seventh Framework Programme: 317040 Horizon 2020: 695551 Medical Research Council Canada: G1001052 Medical Research Council Canada: J007439 National Institute on Drug Abuse: K24-AI118591 Medical Research Council Canada: MR/L018373/L Medical Research Council Canada: MR/M019829/1 Medical Research Council Canada: MR/P001602/1 National Institutes of Health: R01 DA13324 National Institute on Drug Abuse: R01-DA-12568 National Institute of Child Health and Human Development: R01-HD-41224 National Institute on Drug Abuse: U01-DA-036297

International audience; Killer-cell Ig-like receptor (KIR) genes are inherited as haplotypes. They are expressed by NK cells and linked to outcomes of infectious diseases and pregnancy in humans. Understanding how genotype relates to phenotype is difficult because of the extensive diversity of the KIR family. Indeed, high-resolution KIR genotyping and phenotyping in single NK cells in the context of disease association is lacking. In this article, we describe a new method to separate NK cells expressing allotypes of the KIR2DL1 gene carried by the KIR A haplotype (KIR2DL1A) from those expressing KIR2DL1 alleles carried by the KIR B haplotype (KIR2DL1B). We find that in KIR AB heterozygous individuals, different KIR2DL1 allotypes can be detected in both peripheral blood and uterine NK cells. Using this new method, we demonstrate that both blood and uterine NK cells codominantly express KIR2DL1A and KIR2DL1B allotypes but with a predominance of KIR2DL1A variants, which associate with enhanced NK cell function. In a case-control study of pre-eclampsia, we show that KIR2DL1A, not KIR2DL1B, associates with increased disease risk. This method will facilitate our understanding of how individual KIR2DL1 allelic variants affect NK cell function and contribute to disease risk.

Killer Immunoglobulin-like Receptors (KIR) and their Human Leukocyte Antigen (HLA) ligands play a central role in immunity and human health. These molecules are encoded by gene families with copy number variation, extreme levels of sequence diversity and complex expression patterns. The rapid evolution of KIR and HLA genes and their associations with infectious diseases, pregnancy disorders, immunopathologies and outcome of cell transplantation has generated considerable in-terest from immunologists, geneticists and clinicians. Until recently, however, analyses have been stuck at low-level resolution, focussing primarily on presence or absence of KIR genes. This is changing with the advent of modern high throughput sequencing, cell phenotyp-ing, and bioinformatics. These developments allow high-resolution analysis and much deeper un-derstanding of KIR evolution and KIR function. The impending deluge of high dimensional data brings inevitably new challenges in analysis, interpretation and communication of re-sults, but the benefits are already tangible. The diversity of KIR across worldwide human popula-tions is being catalogued at the allele level. Structures of KIR molecules and their interactions with HLA-peptide complexes are being determined. How KIR modulate natural killer (NK) cell education is being defined. Ligands for activating KIR, elusive for many years, are being discovered. KIR gene complexes and their related receptor gene families are being characterised in animal models and livestock breeds. These advances are helping generate a more complete picture of the impact of KIR variation in health and disease and offer new opportunities for immunotherapy, as highlight-ed in a recent meeting*.

Summary Killer‐cell immunoglobulin‐like receptors (KIRs) are components of two fundamental biological systems essential for human health and survival. First, they contribute to host immune responses, both innate and adaptive, through their expression by natural killer cells and T cells. Second, KIR play a key role in regulating placentation, and hence reproductive success. Analogous to the diversity of their human leucocyte antigen class I ligands, KIR are extremely polymorphic. In this review, we describe recent developments, fuelled by methodological advances, that are helping to decipher the KIR system in terms of haplotypes, polymorphisms, expression patterns and their ligand interactions. These developments are delivering deeper insight into the relevance of KIR in immune system function, evolution and disease.

For most immune-mediated diseases, the main determinant of patient well-being is not the diagnosis itself but instead the course that the disease takes over time (prognosis)1,2,3. Prognosis may vary substantially between patients for reasons that are poorly understood. Familial studies support a genetic contribution to prognosis4,5,6, but little evidence has been found for a proposed association between prognosis and the burden of susceptibility variants7,8,9,10,11,12,13. To better characterize how genetic variation influences disease prognosis, we performed a within-cases genome-wide association study in two cohorts of patients with Crohn's disease. We identified four genome-wide significant loci, none of which showed any association with disease susceptibility. Conversely, the aggregated effect of all 170 disease susceptibility loci was not associated with disease prognosis. Together, these data suggest that the genetic contribution to prognosis in Crohn's disease is largely independent of the contribution to disease susceptibility and point to a biology of prognosis that could provide new therapeutic opportunities.

Killer cell immunoglobulin-like receptors (KIRs), expressed on natural killer cells and T cells, have considerable biomedical relevance playing significant roles in immunity, pregnancy and transplantation. The KIR locus is one of the most complex and polymorphic regions of the human genome. Extensive sequence homology and copy number variation makes KIRs technically laborious and expensive to type. To aid the investigation of KIRs in human disease we developed a high-throughput, multiplex real-time polymerase chain reaction method to determine gene copy number for each KIR locus. We used reference DNA samples to validate the accuracy and a cohort of 1698 individuals to evaluate capability for precise copy number discrimination. The method provides improved information and identifies KIR haplotype alterations that were not previously visible using other approaches. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0358-0) contains supplementary material, which is available to authorized users.

Objective: Natural killer (NK) cells are important mediators of liver inflammation in chronic liver disease. The aim of this study was to investigate why liver transplants (LTs) are not rejected by NK cells in the absence of human leukocyte antigen (HLA) matching, and to identify a tolerogenic NK cell phenotype.Design: phenotypic and functional analyses on NK cells from 54 LT recipients were performed, and comparisons made with healthy controls. Further investigation was performed using gene expression analysis and donor:recipient HLA typing.Results: NK cells from non-HCV LT recipients were hypofunctional, with reduced expression of NKp46 (pConclusions: LT is associated with transcriptional and functional changes in NK cells, resulting in reduced activation. NK cell tolerance occurs upstream of major histocompatibility complex (MHC) class I mediated education, and is associated with deficient STAT4 phosphorylation. STAT4 therefore represents a potential therapeutic target to induce NK cell tolerance in liver disease.

Classical human leukocyte antigens (HLA) genes confer the strongest, but not the only, genetic susceptibility to type 1 diabetes. Killer cell immunoglobulin-like receptors (KIR), on natural killer (NK) cells, bind ligands including class I HLA. We examined presence or absence, with copy number, of KIR loci in 1698 individuals, from 339 multiplex type 1 diabetes families, from the Human Biological Data Interchange, previously genotyped for HLA. Combining family data with KIR copy number information allowed assignment of haplotypes using identity by descent. This is the first disease study to use KIR copy number typing and unambiguously define haplotypes by gene transmission. KIR A1 haplotypes were positively associated with T1D in the subset of patients without the high T1D risk HLA genotype, DR3/DR4 (odds ratio=1.29, P=0.0096). The data point to a role for KIR in type 1 diabetes risk in late-onset patients. In the top quartile (age of onset>14), KIR A2 haplotype was overtransmitted (63.4%, odds ratio=1.73, P=0.024) and KIR B haplotypes were undertransmitted (41.1%, odds ratio=0.70, P=0.0052) to patients. The data suggest that inhibitory 'A' haplotypes are predisposing and stimulatory 'B' haplotypes confer protection in both DR3/DR4-negative and late-onset patient groups.

Large population studies of immune system genes are essential for characterizing their role in diseases, including autoimmune conditions. Of key interest are a group of genes encoding the killer cell immunoglobulin-like receptors (KIRs), which have known and hypothesized roles in autoimmune diseases, resistance to viruses, reproductive conditions, and cancer. These genes are highly polymorphic, which makes typing expensive and time consuming. Consequently, despite their importance, KIRs have been little studied in large cohorts. Statistical imputation methods developed for other complex loci (e.g., human leukocyte antigen [HLA]) on the basis of SNP data provide an inexpensive high-throughput alternative to direct laboratory typing of these loci and have enabled important findings and insights for many diseases. We present KIR∗IMP, a method for imputation of KIR copy number. We show that KIR∗IMP is highly accurate and thus allows the study of KIRs in large cohorts and enables detailed investigation of the role of KIRs in human disease.

The killer cell immunoglobulin-like receptors (KIR) mediate human natural killer (NK) cell cytotoxicity via activating or inhibiting signals. Although informative and functional haplotype patterns have been reported, most genotyping has been performed at resolutions that are structurally ambiguous. In order to leverage structural information given low-resolution genotypes, we performed experiments to quantify the effects of population variations, reference haplotypes, and genotyping resolutions on population-level haplotype frequency estimations as well as predictions of individual haplotypes. We genotyped 10,157 unrelated individuals in 5 populations (518 African American[AFA], 258 Asian or Pacific Islander [API], 8,245 European[EUR], 1,073 Hispanic[HIS], and 63 Native American[NAM]) for KIR gene presence/absence (PA), and additionally half of the AFA samples for KIR gene copy number variation (CNV). A custom EM algorithm was used to estimate haplotype frequencies for each population by interpretation in the context of three sets of reference haplotypes. The algorithm also assigns each individual the haplotype pairs of maximum likelihood. Generally, our haplotype frequency estimates agree with similar previous publications to within