Deciphering the killer-cell immunoglobulin-like receptor system at super-resolution for natural killer and T-cell biology.

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.V.B. is supported by the French National Research Agency (ANR) (grant no. NKIR-ANR-13-PDOC-0025-01). P.J.N. and H.H. are supported by U.S. National Institutes of Health grant R01 AI17892. J.A.T. is supported by the European Research Council (ERC) and Medical Research Council (MRC)

Killer-cell Immunoglobulin-like Receptor gene linkage and copy number variation analysis by droplet digital PCR.

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

Polymorphism in killer cell immunoglobulin-like receptors and human leukocyte antigen-c and predisposition to preeclampsia in Ethiopian pregnant women population.

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

Mechanisms of Copy Number Variation and Hybrid Gene Formation in the KIR Immune Gene Complex

The fine-scale structure of the majority of copy number variation (CNV) regions remains unknown. The killer immunoglobulin receptor (KIR) gene complex exhibits significant CNV. The evolutionary plasticity of the KIRs and their broad biomedical relevance makes it important to understand how these immune receptors evolve. In this paper, we describe haplotype re-arrangement creating novel loci at the KIR complex. We completely sequenced, after fosmid cloning, two rare contracted haplotypes. Evidence of frequent hybrid KIR genes in samples from many populations suggested that re-arrangements may be frequent and selectively advantageous. We propose mechanisms for formation of novel hybrid KIR genes, facilitated by protrusive non-B DNA structures at transposon recombination sites. The heightened propensity to generate novel hybrid KIR receptors may provide a proactive evolutionary measure, to militate against pathogen evasion or subversion. We propose that CNV in KIR is an evolutionary strategy, which KIR typing for disease association must take into account

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes.

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

Killer cell immunoglobulin-like receptor (KIR) genes and their HLA-C ligands in a Ugandan population.

Killer cell immunoglobulin-like receptor (KIR) genes are expressed by natural killer cells and encoded by a family of genes exhibiting considerable haplotypic and allelic variation. HLA-C molecules, the dominant ligands for KIR, are present in all individuals and are discriminated by two KIR epitopes, C1 and C2. We studied the frequencies of KIR genes and HLA-C1 and C2 groups in a large cohort (n = 492) from Kampala, Uganda, East Africa and compared our findings with published data from other populations in sub-Saharan Africa (SSA) and several European populations. We find considerably more KIR diversity and weaker linkage disequilibrium in SSA compared to the European populations and describe several novel KIR genotypes. C1 and C2 frequencies were similar to other SSA populations with a higher frequency of the C2 epitope (54.9 %) compared to Europe (average 39.7 %). Analysis of this large cohort from Uganda in the context of other African populations reveals variations in KIR and HLA-C1 and C2 that are consistent with migrations within Africa and potential selection pressures on these genes. Our results will help understand how KIR/HLA-C interactions contribute to resistance to pathogens and reproductive success

Downregulation of HLA-I by the molluscum contagiosum virus mc080 impacts NK-cell recognition and promotes CD8+ T-cell evasion.

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

Imputation of KIR Types from SNP Variation Data.

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.This work was supported by the Australian National Health and Medical Research Council (NHMRC), Career Development Fellowship ID 1053756 (S.L.); by a Victorian Life Sciences Computation Initiative (VLSCI) grant number VR0240 on its Peak Computing Facility at the University of Melbourne, an initiative of the Victorian Government, Australia (S.L.); by the UK Multiple Sclerosis Society, grant 894/08 (S.S.); and by the Wellcome Trust and the MRC with partial funding from the National Institute of Health Cambridge Biomedical Research Centre (J.T., J.A.T.). Research at the Murdoch Childrens Research Institute was supported by the Victorian Government's Operational Infrastructure Support Program.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.ajhg.2015.09.00

Electrostatic Modifications of the Human Leukocyte Antigen-DR P9 Peptide-Binding Pocket and Susceptibility to Primary Sclerosing Cholangitis

The strongest genetic risk factors for primary sclerosing cholangitis (PSC) are found in the human leukocyte antigen (HLA) complex at chromosome 6p21. Genes in the HLA class II region encode molecules that present antigen to T lymphocytes. Polymorphisms in these genes are associated with most autoimmune diseases, most likely because they contribute to the specificity of immune responses. The aim of this study was to analyze the structure and electrostatic properties of the peptide-binding groove of HLA-DR in relation to PSC. Thus, four-digit resolution HLA-DRB1 genotyping was performed in 356 PSC patients and 366 healthy controls. Sequence information was used to assign which amino acids were encoded at all polymorphic positions. In stepwise logistic regressions, variations at residues 37 and 86 were independently associated with PSC (P = 1.2 × 10−32 and P = 1.8 × 10−22 in single-residue models, respectively). Three-dimensional modeling was performed to explore the effect of these key residues on the HLA-DR molecule. This analysis indicated that residue 37 was a major determinant of the electrostatic properties of pocket P9 of the peptide-binding groove. Asparagine at residue 37, which was associated with PSC, induced a positive charge in pocket P9. Tyrosine, which protected against PSC, induced a negative charge in this pocket. Consistent with the statistical observations, variation at residue 86 also indirectly influenced the electrostatic properties of this pocket. DRB1*13:01, which was PSC-associated, had a positive P9 pocket and DRB1*13:02, protective against PSC, had a negative P9 pocket. Conclusion: The results suggest that in patients with PSC, residues 37 and 86 of the HLA-DRβ chain critically influence the electrostatic properties of pocket P9 and thereby the range of peptides presented. (Hepatology 2011;53:1967-1976