Evidence that the KIR2DS5 gene codes for a surface receptor triggering natural killer cell function

In this study, after immunization with NK cells from a KIR2DS5(+) donor and screening on cell transfectants expressing different members of the killer immunoglobulin-like receptor (KIR) family, we generated a mAb, DF200, reacting with several KIR2D receptors including KIR2DL1/L2/L3, KIR2DS1/S2 and KIR2DS5. By the analysis of peripheral blood NK cells and in vitro derived NK cell clones, we have demonstrated for the first time that KIR2DS5 is expressed at the cell surface in discrete subsets of NK cells and, after DF200 mAb-mediated engagement, can induce both cytotoxicity and cytokine release. Using co-transfection and co-immunoprecipitation, we found that KIR2DS5 associates with the DAP12 signaling polypeptide. Finally, soluble KIR2DS5-Fc fusion protein does not bind to cell transfectants expressing different HLA-C alleles, suggesting that, if KIR2DS5 does recognize HLA-C molecules, this may only occur in the presence of certain peptides.     

Positive association of Bx genotype,KIR2L5, KIR2DS5 and full-length KIR2DS4 with the risk of meningioma

BackgroundNK cells as a part of innate immune system, are controlled by a set of activating and inhibitory KIR receptors (aKIR, iKIR) which are implicated in tumor microenvironment immunity through a variety of activating and inhibitory immune signals. KIRs are multi gene family receptors that differ in the number and type of genes among individuals. In the current research we determined the KIRs genes and genotypes impact on predisposition to meningioma development in Iranians.MethodsSequence-specific primers-polymerase chain reaction (SSP-PCR) was performed for genotyping of 16 KIRs in 159 meningioma cases and 362 age and sex matched healthy controls (CNs) at Shiraz Institute for Cancer Research.ResultsComparison of the KIR genotypes frequencies between cases and controls disclosed a highly significant increase in Bx genotype, CxTx subset and Cen AB and Tel AB in meningioma cases and a decrease in AA genotype, C4Tx subset and Cen AA, Tel AA, Tel BB in healthy controls.Among all 16 KIR genes, the carriers of KIR2DL5 and KIR2DS5 constituted a much greater proportion in meningioma than control group. Comparison of carrier frequencies of KIR2DS4 variants between case and controls revealed a higher frequency of KIR2DS4 full length (KIR2DS4fl) in meningioma cases and a lower frequency of KIR2DS4 deleted variant (KIR2DS4del) in controls. Furthermore, the simultaneous presence of 2DS5, 2DS4fl, CenAB, TelAB and absence of 2DS4del, CenAA, TelAA, TelBB, magnify the risk of developing meningioma substantially (OR ≈ 23). Altogether, 41 distinct KIR genotypes were characterized in 521 subjects. Among them, some individuals were characterized by seven peculiar genotypes that the linkage disequilibrium between KIR2DS2-KIR2DL2 and KIR2DL5-KIR2DS3-KIR2DS5 has not been detected. The carriers of certain genotypes with presence of as KIR2DL5 and absence of KIR2DS3, KIR2DS5 constituted a much higher proportion in meningioma than control group which increase the risk of meningioma up to 72 times.ConclusionThis case- control study suggests carriers of Bx genotype, KIR2DL5, KIR2DS5, 2DS4fl, ≥ 4 iKIR, CxTx subset as well as Cen AB and Tel AB are associated with an increased risk of developing meningioma whereas carrying KIR2DS4del, AA, C4TX genotypes and Cen AA, Tel AA, Tel BB reduce the genetic predisposition for meningioma.     

KIR2DS2 and KIR2DS4 promoter hypomethylation patterns in patients undergoing hematopoietic cell transplantation (HCT)

The killer cell Ig-like receptor (KIR)–MHC class I pathway is an integral part of natural killer cell immunity, and its role in host protection from both cancer and infection is important. In addition, we have shown elevated KIR2DS2 and 2DS4 expression in PBMCs of patients undergoing hematopoietic cell transplantation (HCT) [1]. Since all inhibitory KIR promoters are known to be heavily methylated, the question asked here is how and when KIR2DS2 and 2DS4 promoters had changed their methylation profile in association with HCT.     

Molecular characterisation of KIR2DS2*005, a fusion gene associated with a shortened KIR haplotype

KIR2DS2 is an activating homologue of KIR2DL2, an inhibitory killer-cell immunoglobulin-like receptor (KIR) that surveys expression of major histocompatibility complex-C allotypes bearing a C1 epitope. We have studied here its allele KIR2DS2*005, which shows a hybrid structure-it is identical to other KIR2DS2 alleles in the ectodomain, but has transmembrane and cytoplasmic regions identical to those of KIR2DS3(*)001, a short-tailed KIR of uncertain expression and function. Our results reveal that KIR2DS2*005 is a fusion gene-the product of an unequal crossing over by which the genes KIR2DS2 and KIR2DS3 recombined within a 400 base pair region of complete identity in intron 6. Also resulting from that recombination was a shortened KIR haplotype of the B group, in which three genes commonly linked to KIR2DS2 (KIR2DL2, KIR2DL5B and KIR2DS3) are deleted. Population studies indicate that KIR2DS2*005 is still associated to such haplotype, and it can be found in approximately 1.2% of Caucasoids. Using a combination of two monoclonal antibodies, we also demonstrate that KIR2DS2*005 encodes a molecule expressed on the surface of natural killer- and T-lymphocytes.     

The impact of KIR2DS4 gene on clinical outcome after hematopoietic stem cell transplantation

Killer cell immunoglobulin-like receptors (KIR) are a family of inhibitory/activating receptors expressed on NK cells. Interactions of KIR receptors with KIR ligands have been shown to modify hematopoietic stem cell transplantation (HSCT) outcome. The aim of this research was to determine the KIR2DS4 allele variants distribution among 111 patients with different hematological malignancy who underwent HSCT and their donors, and to evaluate KIR2DS4 alleles’ impact on HSCT outcome. The KIR gene frequency analysis showed a significantly higher incidence of full-length KIR2DS4 alleles among patients. The impact of KIR2DS4 alleles on transplantation outcomes revealed that donors’ full-length KIR2DS4 alleles is associated with lower overall survival rates, higher risk of GVHD and higher relapse incidence. The expression of full-length KIR2DS4 allele variants may contribute to a worse clinical outcome after HSCT. KIR typing for KIR2DS4 could be used as an additional criterion for selecting suitable donors in cases when more than one HLA identical donor is identified for a specific patient.     

Activating KIR2DS5 receptor is a risk for thyroid cancer

Natural killer (NK) cells are key members of innate immunity against tumor and infection. Killer cell immunoglobulin-like receptor (KIR) genes regulates NK cell function, which varies substantially between individuals in the number and type. Specific KIRs are associated with certain diseases. Herein we investigated if KIR genes are associated with thyroid cancer risk. Eighty-five patients with thyroid cancer were characterized for the presence and absence of 11 variable KIR genes using a gene-specific PCR typing system, and compared with our recently published healthy control data. Overall, a trend toward more activating KIR receptors was observed in thyroid cancer patients compared to the healthy controls. Particularly, the activating KIR2DS5 gene was significantly increased in patients compared to the controls. Additionally, three other genes (3DS1-2DL5-2DS1) that are strongly linked to KIR2DS5 at the telomeric region of the KIR gene complex [called Telomeric 4 (T4) gene cluster] were also more predominant in the thyroid cancer patient group than in healthy controls. A similar trend of having more of the T4 gene cluster was also reported in a previous study with cervical neoplasia. Together, these data suggest that specific activating KIR genes in cancer patients could generate a chronic inflammatory condition resulting in a tumor microenvironment that may favor tumor growth.     

A role for KIR gene variants other than KIR2DS1 in conferring susceptibility to psoriasis

Recently we described an association between psoriasis and KIR2DS1, a gene for a stimulatory natural killer cell receptor, in a Polish population. The association was independently reported among Japanese and confirmed in a U.S. population. Prompted by these findings, we reanalyzed data by a multivariate approach in search of possible effects of KIR genes other than KIR2DS1 (non-KIR2DS1). The methodology was based on a stratified analysis and multiple logistic regression. We found that the non-KIR2DS1 genes had joint effects comparable to or stronger than the effects of KIR2DS1 in both the fraction of explained variance (0.174 vs 0.204, respectively, for KIR2DS1 and non-KIR2DS1) and the statistical significance (p = 0.000008 vs p = 0.000001, respectively). When individual genes were considered, a decrease in KIR2DS5 among patients vs controls (OR = 0.2, pcor = 0.0005) and a decrease in KIR2DS3 restricted to KIR2DS1-positive individuals (OR = 0.2, pcor = 0.005) were evident. We also performed a multivariate analysis of the HLA-Cw genotypes but failed to demonstrate any effects in addition to the known association with HLA-Cw*06. We conclude that the effect of the KIR genes on psoriasis susceptibility is complex, extending beyond the association with KIR2DS1 and involving protective effects and interactions.     

Comparison of KIR gene content profiles revealed a difference between northern and southern Persians in the distribution of KIR2DS5 and its linked loci

Killer cell immunoglobulin-like receptors (KIR) are the key receptors of human natural killer (NK) cells that mount an early immune response against infection and tumors. The number and type of KIR genes are substantially variable between individuals and populations. Recently we reported KIR gene content diversity in a Persian population living in the southern province of Fars, which is comparable to that of European Caucasians. These results are consistent with the ethnic ancestry and affinity between Persians and Caucasians. Herein we analyzed another Persian population living in the northern province of Tehran and discovered an unexpected increase in the distribution of KIR2DS5 and its linked loci KIR3DS1, -2DS1, and -2DL5 in northern Persians compared with that reported in the southern Persian population. Although the geographic barriers may have limited the gene flow, the impact of the local environment on the natural selection of KIR2DS5 and its linked loci in the northern Persians cannot be completely ruled out. The difference in northern and southern populations in activating KIR gene content creates an appealing hypothesis that KIR2DS5-enriched northern Persians are more resistant to developing clinical conditions demonstrated to be associated with KIR2DS5, such as psoriasis vulgaris, endometriosis, ankylosing spondylitis, and acute rejection of kidney grafts, compared with those living in the southern part of the country.     

Studies on the expression of the deleted KIR2DS4*003 gene product and distribution of KIR2DS4 deleted and nondeleted versions in different populations

A KIR2DS4 deletion variant allele, previously identified through killer immunoglobulinlike receptor (KIR) polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) typing, was functionally investigated using an in vitro cell line model system and in vivo protein expression studies. The KIR2DS4 deletion variant has previously been found in 80% of individuals from Northern Ireland, indicating that it is present at a high incidence in this population. It differs from the normal KIR2DS4 sequence by a 22 bp deletion in exon 5, which causes a frame shift, yielding a truncated KIR2DS4 protein with loss of the transmembrane and cytoplasmic domains of the full-length KIR2DS4 protein. This study has determined that the deleted variant of KIR2DS4 is not anchored to the cell membrane but encodes a soluble form of the protein that is potentially secreted. The frequencies of the deleted and nondeleted versions were also determined in several world-wide populations. A trend was observed towards decreased frequencies of KIR2DS4 deleted variant occurrence in populations having KIR2DS4 as the only activating KIR gene.     

Influence of KIR gene diversity on the course of HSV-1 infection: resistance to the disease is associated with the absence of KIR2DL2 and KIR2DS2

Herpes simplex virus type 1 (HSV-1) causes lifelong latent infections in most humans. Periodical virus reactivations from latency in the neurons of sensitive ganglia lead to transport to mucocutaneous regions and productive replication, which results in recurrent inflammatory herpetic lesions or in asymptomatic virus shedding. The medical consequences of such lesions and the frequency of recurrences vary greatly in different subjects. Furthermore, many infected individuals never suffer manifestations of the disease, even when exposed to stimuli that trigger clinical recurrences in other humans. The origin of the variability in the clinical course of HSV-1 infection remains unexplained. Herpesviruses and other pathogens sabotage the expression of major histocompatibility complex class I molecules by infected cells, thus subverting T-cell-mediated immunity. Subversion of antigen presentation is counteracted by natural killer cells, which survey the human leukocyte antigen (HLA) expression by specific receptors. These include the killer cell immunoglobulin-like receptors (KIRs), which are encoded by a complex of extremely diverse and rapidly evolving genes. Here, we analyze the contribution of KIR gene diversity to the variable clinical course of HSV-1 infection by comparing the distribution of these genes in humans with clinical manifestations of the disease with that in asymptomatically infected donors. This study provides preliminary evidence that the receptors KIR2DL2 and KIR2DS2 predispose to symptomatic HSV-1 infection and favor the frequently recurring forms of the disease. Possible contribution of the 'HLA-C1' ligand to HSV-1 disease was not statistically supported. Because of an absolute genetic linkage between KIR2DL2 and KIR2DS2, we could not determine which receptor was primarily responsible for the observed association, but our results suggest that presence in the genome of KIR2DL2 and KIR2DS2 hinders an effective cellular response to HSV-1.     

Association of KIR2DS4 and its variant KIR1D with syphilis in a Chinese Han population

Syphilis is a sexually transmitted infection caused by the Treponema pallidum subspecies pallidum spirochaete bacterium. The killer immunoglobulin-like receptor (KIR) gene family encodes cell surface receptors that are found on natural killer (NK) cells and certain T-cell subsets. NK cells are fast-acting effector lymphocytes of innate immunity that respond to infection. The activity of NK cells depends on the dynamic balance of activating and inhibitory signals that are transmitted through respective receptors including KIRs. KIR2DS4 is the only activating KIR gene in KIR haplotype A. KIR1D is a partial deletion KIR2DS4 variant encoding protein devoid of transmembrane region. Up to now, there is no knowledge of association of KIR2DS4 and its variant KIR1D with syphilis in a population that belongs to KIR gene haplotype A. Polymerase chain reaction with sequence-specific primers (PCR-SSP) method was used to genotype KIR genes in 190 patients with syphilis and 192 healthy controls. The gene frequencies of KIR2DS4 and KIR1D were analysed for the association with syphilis in patients and healthy controls who belong to KIR gene haplotype A. The gene frequency of KIR1D/KIR1D in patients with syphilis classified as haplotype A was 16.85% and was significantly higher than that in the control group (6.59%) (P = 0.032). However, there was no significant difference for the gene frequencies of KIR2DS4/KIR2DS4 and KIR2DS4/KIR1D between the two groups (P > 0.05). KIR1D/KIR1D was found in association with susceptibility to syphilis in the Chinese Han population that belongs to KIR gene haplotype A.     

KIR2DL2/S2 and KIR2DS5 in alcoholic cirrhotic patients undergoing liver transplantation

IntroductionThe molecular mechanisms underlying alcoholic liver fibrosis and cirrhosis are not completely understood. Hepatic fibrosis involves the interplay of diverse cells and factors, including hepatic stellate cells (HSCs), Kupffer, NK cells, and T-lymphocyte subsets. Killer-cell immunoglobulin-like receptors (KIR) are membrane receptors involved in mediation between NK and activated HSCs, regulating NK cell function through their interaction with HLA-I molecules. The aim of this study was to analyse the genetic association between KIR genes and the susceptibility to or protection from alcoholic cirrhosis (AC) in a cohort of male AC patients undergoing liver transplantation (LT) with and without concomitant viral infections.Material and methodsKIR genotyping was performed in nuclear DNA extracted from 281 AC patients and compared with 319 male controls.ResultsSignificant differences between total AC patients and healthy controls were only found in the case of KIR2DL2 and KIR2DS5. KIR2DL2 was significantly underrepresented in non-viral AC patients (52.6% vs. 63.3%; p = 0.015), while patients heterozygous for KIR2DL2 were also underrepresented in the non-viral AC group compared with controls (p = 0.034). KIR2DS5 was overrepresented in this group compared with healthy controls (p = 0.002). All these observations were only evident in AC patients older than 54 years old.ConclusionsOur data suggest a contrary effect of KIR2DL2 and KIR2DS5 in AC patients older than 54 years, in whom the presence of KIR2DL2 appears to be protective against AC, whereas the presence of KIR2DS5 seems to promote the fibrotic process, particularly in patients with no associated viral infection.     

Investigation of killer cell immunoglobulin-like receptor gene diversity, KIR2DL1 and KIR2DS1

Polymorphism in the alleles of the killer cell immunoglobulin-like receptor 2DL1 and 2DS1 genes has been investigated by the development of polymerase chain reaction-sequence-specific oligonucleotide probing systems. The methods have been applied to 77 Northern Irish families, establishing allele frequencies from the unrelated parents. Additionally, cell line DNA from individuals and CEPH families of the 13th International Histocompatibility Workshop panel were investigated. Eight of the reported KIR2DL1 alleles and only the KIR2DS1*002 allele were identified in the groups studied. Two individuals were positive for three alleles of KIR2DL1, and a putative variant of KIR2DL1*001 was observed. Results also indicated an inherited KIR2DL1/2DS1 splice variant present in a haplotype with several core loci absent, in two families.     

Duplication, mutation and recombination of the human orphan gene KIR2DS3 contribute to the diversity of KIR haplotypes

The KIR2DS3 gene is an activating homologue of the inhibitory killer-cell immunoglobulin-like receptors (KIR) that recognize HLA-C molecules, enabling NK cells to survey the normal function of endogenous antigen presentation. The genetics of KIR2DS3 is complicated by the existence of alleles with similar coding sequences that map to different regions of the KIR complex in chromosome 19, or whose location in this complex is unknown. Here, by studying the family segregation of the KIR alleles 2DS3*001, *002 and *003N, and the distribution of these in unrelated individuals, we demonstrate the existence of two paralogous KIR2DS3 genes that can be inherited separately or, as it happens frequently in Caucasoids due to linkage disequilibrium, together. Each KIR2DS3 gene is almost invariably associated in its 5' end to a different copy of KIR2DL5, a gene previously shown to be duplicated in humans. KIR2DL5 and KIR2DS3 thus form two highly homologous gene clusters situated in the centromeric and the telomeric intervals of KIR haplotypes. Recombination between those clusters is the likely origin of new haplotypes, characterized in this study, which harbour further duplications or deletions of multiple KIR genes. Our results help understand the genetics of KIR2DS3 and the diversity of human KIR genotypes.     

Diversity of the killer cell immunoglobulin-like receptor gene KIR2DS4 in the Chinese population

Human killer cell immunoglobulin-like receptors are a subfamily of the immunoglobulin superfamily, which map to the leukocyte receptor complex on chromosome 19. Here, we established polymerase chain reaction-sequence-based typing (PCR-SBT) procedures to identify alleles of the KIR2DS4 gene. The method was designed around the specific amplification of exons 4-5 of the KIR2DS4 gene. Genomic DNA from 105 healthy unrelated Chinese Han individuals were typed for the KIR2DS4 alleles. Each sample was assigned the KIR2DS4 alleles combination, consistent with the pairwise combinations of sequences of all the known KIR2DS4 alleles. We observed eleven different genotypes and four KIR2DS4 alleles in the population, with the KIR2DS4*00101 having the highest frequency, 0.576, and also confirmed the new KIR2DS4*007 allele. Our data demonstrated that the established PCR-SBT method for the KIR2DS4 allele typing was reliable.     

A common KIR2DS4 deletion variant in the human that predicts a soluble KIR molecule analogous to the KIR1D molecule observed in the rhesus monkey

A KIR2DS4 deletion variant allele, previously identified through KIR PCR-SSOP typing studies, was characterized, alongside a normal KIR2DS4 allele, by cDNA cloning and sequencing and its prevalence in the population determined using a deletion specific probe. The KIR2DS4 deletion variant was found in 72 of the 90 individuals screened and differed from the normal KIR2DS4 sequence by a single 22 bp deletion in exon 5. The deletion causes a frameshift predicting a truncated KIR2DS4 protein with a significantly altered D2 domain that would be secreted due to the loss of the transmembrane/cytoplasmic domains. Parallels with a recent study in the rhesus monkey highlighting access to the same open reading frame as the deletion variant, also predicting a soluble KIR molecule, are drawn.     

Investigation of killer cell immunoglobulin-like receptor (KIR) gene diversity: KIR2DL2, KIR2DL5 and KIR2DS5

Human killer cell immunoglobulin-like receptor (KIR) genes are important for restraining natural killer cytotoxicity toward cells with autologous human leukocyte antigen (HLA) while targeting cells lacking or expressing low levels of self-HLA molecules. KIR gene content and alleles vary across individual genomes and populations, requiring specialized laboratory tools for their characterization. Here, we detail methods based on sequence-specific polymerase chain reaction amplification and oligonucleotide probe hybridization to identify alleles of KIR2DL2, KIR2DL5A, KIR2DL5B and KIR2DS5. Allele frequencies for a Northern Irish population of 354 individuals typed with this system are given, along with results from 132 cell lines from the International Histocompatibility Workshop that cover many world populations. This information complements published reports by our laboratory for allele-level typing of other KIR members, totaling 12 of the 17 known genes. These methods are allowing us to characterize KIR haplotypes in our population.     

Isolation of a novel KIR2DL3-specific mAb: comparative analysis of the surface distribution and function of KIR2DL2, KIR2DL3 and KIR2DS2

In recent years an increasing number of sequences coding for new KIRs have been described. However, the limited availability of mAbs with unique KIR specificities has hindered an exhaustive assessment of their actual function, HLA-specificity, expression at the cell surface and distribution in different cell populations. In this study we report the generation of a novel mAb (ECM41) specific for KIR2DL3 molecules. By the use of cell transfectants expressing one or other KIR we show that this reagent allows discrimination of KIR2DL3 from other GL183 mAb-reactive molecules such as KIR2DL2 and KIR2DS2. Moreover we show that this novel mAb can be used to assess the surface expression and distribution of KIR2DL3 in different polyclonal NK populations and in NK cell clones. Along this line, we were able to analyze the HLA class I specificity of NK clones expressing either KIR2DL3 or KIR2DL2, two inhibitory receptors that were so far serologically undistinguishable. Finally, the combined use of GL183 and ECM41 mAbs in redirected killing assays allowed us to investigate the functional outcome of the simultaneous engagement of KIR2DL3 and KIR2DS2 in NK cell clones co-expressing KIRs that display opposite (inhibitory vs activating) function.     

杀伤细胞免疫球蛋白样受体2DS4及其变体KIR1D与梅毒的关联性研究

目的 探讨杀伤细胞免疫球蛋白样受体(killer cell immunogloblin-like receptor,KIR)基因KIR2DS4及其变体KIR1D与属于KIR基因单体型A (haplotype A)的梅毒(syphilis)患者的关联性.方法 采用序列特异性引物聚合酶链反应(PCR-SSP)法,分析192例无血缘关系的健康人和190例梅毒患者KIR基因的单体型,在归类于单体型A的梅毒患者和健康人群中分析KIR2DS4和KIR1D基因与梅毒的关联.结果 在192例健康者中,含有KIR2DS4基因的个体为187例,其中有91例归类于纯合子(homozygous)单体型A,占48.7% (91/187);在190例梅毒患者中,出现KIR2DS4基因的个体为181例,其中有89例归类于纯合子单体型A,占49.2% (89/181).在归属于单体型A的人群中,KIR1D/KIR1D在梅毒病例组的基因型频率为16.9%,显著高于对照组(6.6%,P＝0.032),而KIR2DS4/KIR2DS4和KIR2DS4/KIR1D基因型频率在两组间差异均无统计学意义(P>0.05).结论 KIR1D/KIR1D可能与纯合子单体型A中的个体的梅毒发生相关联.

Abstract：

Objective To investigate the killer cell immunoglobulin-like receptor (KIR) genes, KIR2DS4 and its variant KIR1D for an association with syphilis in the comparison between syphilis patients and unrelated healthy subjects. Methods One hundred and ninety syphilis patients and 192 unrelated healthy subjects were performed to determine the KIR genotypes by PCR-SSP method. The gene frequencies of KIR2DS4 and KIR1D were analyzed for an association with syphilis in the patients and healthy people who belonged to KIR gene haplotype A. Results Of 192 healthy individuals, 187 were identified with a KIR2DS4 gene. And 91 individuals were classified as homozygous haplotype A with the percent of 48.7% (91/187) in 187 KIR2DS4 positive individuals. Of 190 syphilis patients, 181 were identified with a KIR2DS4 gene. And 89 individuals were classified as homozygous haplotype A with the percent of 49.2% (89/181) in 181 KIR2DS4 positive individuals. The frequency of KIR1D/KIR1D in syphilis patients classified as haplotype A was 16.9%, and was significantly higher than that in the control group (6.6%, P=0.032). However, there was no significant difference for the frequencies of KIR2DS4/KIR2DS4 and KIR2DS4/KIR1D between the two groups (P>0.05). Conclusion KIR1D/KIR1D might be associated with syphilis in the comparison between syphilis patients and unrelated healthy controls who were classified as homozygous haplotype A.