KIR2DL3 and KIR2DL1 show similar impact on licensing of human NK cells

Killer cell immunoglobulin‐like receptor/HLA class I (KIR/HLA‐I) combinations are associated with disease risk, implicating functional roles for NK cells (NKCs) or KIR⁺ T cells. KIR/HLA‐I interactions can act through inhibition of NKC activation by target cells and NKC licensing for greater intrinsic responsiveness. We compared licensing conferred by the weaker, HLA‐C group 1/KIR2DL3, and the stronger, HLA‐C group 2/KIR2DL1, inhibitory combinations. The “rheostat model” predicts weaker licensing by HLA‐C1/KIR2DL3 interactions than HLA‐C2/KIR2DL1. We analyzed degranulation in NKC subsets expressing single and multiple receptors for HLA‐I. NKG2A had the strongest licensing impact, while KIR2DL3, KIR2DL1, and KIR3DL1 were weaker, and not significantly different to each other. Presence of one or two matched HLA‐C allotypes did not alter licensing of KIR2DL3⁺ and KIR2DL1⁺ NKC. Coexpression of activating KIR2DS1 disarmed KIR2DL3⁺ and KIR2DL1⁺ NKC to a similar extent. KIR3DL1 and NKG2A combined for more enhanced licensing of double‐positive NKC than the combination of KIR2DL3 and KIR2DL1. Thus, KIR2DL3 and KIR2DL1 have similar capacity to license NKC, suggesting that inhibitory signal strength and amount of available HLA‐C ligands do not correlate with NKC licensing. Altogether, our results show that the basis for disease associations of HLA‐C and KIR2DL likely encompasses factors other than licensing.     

Hematopoietic stem cell transplantation: Improving alloreactive Bw4 donor selection by genotyping codon 86 of KIR3DL1/S1

KIR3DL1 is a natural killer (NK) cell receptor that recognizes the Bw4 epitope of human leukocyte antigen (HLA) class I molecules. Following hematopoietic stem cell transplantation for patients lacking Bw4, KIR3DL1‐expressing NK cells from Bw4‐positive donors can be alloreactive and eliminate tumor cells. However, KIR3DL1 alleles having T instead of C at nucleotide 320 (encoding leucine 86 instead of serine 86) are not expressed on the cell surface. Thus, not all individuals testing positive for KIR3DL1 are optimal donors for Bw4‐negative recipients. Therefore, we developed a method for genotyping codon 86, which was validated by its perfect correlation with NK cell phenotype for 100 donors of diverse KIR3DL1/S1 genotype. We typed 600 donors and found that ～12.2% had the KIR3DL1 gene, but did not express cell‐surface KIR3DL1. By contrast, high‐expressing allotypes were identified when haplotypes from four families with duplicated KIR3DL1/S1 genes were characterized at high resolution. Identifying donors who have KIR3DL1 but lack cell‐surface KIR3DL1 would refine donor selection. With this technique, the number of individuals identified who may not be optimal donors for Bw4‐negative patients increases by threefold, when compared with standard methods. Taken together, we propose that allele typing of killer cell Ig‐like receptor (KIR) polymorphisms should become a standard practice when selecting donors.     

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.     

江苏汉族人群杀伤细胞免疫球蛋白样受体及其特异性配体HLA分析

目的:分析杀伤细胞免疫球蛋白样受体(KIR)及其特异性配体HLAⅠ类分子在江苏地区汉族人群的分布特点。方法:分别利用real-time PCR法和PCR-SSP方法对173例无血缘关系的江苏汉族健康人群进行KIR和HLA-Cw,HLA-Bw4分型,并分析KIR/HLA配对组合的类型和数量。结果:江苏汉族人群中,>92%的个体同时表达四种抑制性KIR(iKIR)(2DL1,2DL2/3,3DL1,3DL2)。2DL2/HLA-C1,2DL3/HLA-C1,2DL1/HLA-C2,3DL1/Bw4的频率分别依次分别为0.243,0.971,0.457,0.590;2DS1/HLA-C2,2DS2/HLA-C1的频率为0.162,0.231。54.3%的个体只表达2DL1而不表达相应的配体HLA-C2,32.9%的个体只表达3DL1而缺乏配体HLA-Bw4,另有5.8%的个体只表达HLA-Bw4而不表达3DL1。27.7%的个体同时表达3种iKIR/HLA,26%的个体同时表达两种iKIR/HLA,25.4%的个体只遗传一个iKIR/HLA配对,未发现3种iKIR/HLA同时缺失的个体。结论:在江苏汉族人群中,存在KIR与HLA表型分离现象,抑制性KIR/HLA配对表达高于刺激性KIR/HLA配对,约1/4个体只表达单个iKIR/HLA配对。     

Investigation of killer cell immunoglobulin-like receptor KIR2DL4 diversity by sequence-based typing in Chinese population

Human killer cell immunoglobulin-like receptors (KIRs) play an important role in controlling natural killer (NK) cell function. Here, polymerase chain reaction sequence-based typing (PCR-SBT) procedures identifying alleles of the KIR2DL4 gene have been established. The method was designed around the specific amplification of exon 3 to exon 5 and exon 7 to exon 9 of the KIR2DL4 gene and produce discrimination of KIR2DL4 alleles. Genomic DNAs from 83 healthy unrelated Chinese Han individuals were typed for KIR2DL4 alleles by this method. Each sample was assigned to the putative KIR2DL4 allele combination according to the nucleotide polymorphism profiles of all KIR2DL4 alleles. Twenty-one different genotypes and seven KIR2DL4 alleles were observed in the population, with KIR2DL4*00102 having the highest frequency, 0.5. Five individuals bear a recombinant allele KIR3DP*004 that associated with three putative KIR2DL4 alleles. Our data demonstrated that the established PCR-SBT method for KIR2DL4 allele typing was reliable, and Chinese Han population is distinct in KIR2DL4 allele frequencies in comparison to some other populations.     

Assessment of fidelity and utility of the whole-genome amplification for the clinical tests offered in a histocompatibility and immunogenetics laboratory

Increasing emphasis on the use of molecular tests in a histocompatibility and immunogenetics laboratory (HIL) poses a potential problem of lack of sufficient DNA to perform multiple genetic analyses. In this study, we report the feasibility, fidelity and utility of multiple displacement amplification (MDA) method to perform whole-genome amplification (WGA) to generate DNA specimens that can be analyzed by multiple molecular techniques and can be used for different clinical tests offered by an HIL. The MDA-generated DNA when compared with the native DNA showed 100% congruency in genotyping of 37 genes/loci using multiple downstream molecular techniques: sequence-based typing and sequence-specific primer-based typing for 5 human leukocyte antigen (HLA) class I and II genes (HLA-A, B, C, DRB1 and DQB1), luminex-based sequence-specific oligonucleotide (SSO) genotyping for a panel of 16 killer immunoglobulin-like receptor (KIR) genes and automated fragment size analysis for a panel of 15 short tandem repeat (STR) loci and amelogenin gene. For post-allogeneic hematopoietic cell transplantation (HCT) chimerism analysis, MDA-generated DNA appeared useful for enriching pre-transplant DNA but not for enriching post-transplant chimeric DNA. Overall, our results show that MDA-based WGA could generate DNA of high yield and fidelity that can be used for various clinical tests and research purposes.     

Distribution of KIR genes in the Czech population

Killer cells immunoglobulin-like receptors (KIRs) are a family of inhibitory and activating receptors expressed mainly by natural killer (NK) cells and few subsets of T lymphocytes. KIRs regulate NK cells' activity through interactions with specific HLA class I molecules and other yet unknown ligands presented on target cells. At present, 17 KIR genes and pseudogenes have been identified. As the number of KIR genes in different haplotypes varies, a wide range of genotypes in different ethnic populations may be observed. In our study, 125 healthy non-related Czech individuals were KIR typed both by sequence-specific primers and by sequence-specific oligonucleotide KIR genotyping methods. Thirty-eight different genotypes were observed in the Czech population and all 16 KIR genes known to date were found. Framework genes KIR 3DL3, KIR 2DL4, KIR 3DL2 and the pseudogene KIR 3DP1 were present in all individuals. The most frequent non-framework KIR genes detected in the Czech population were: KIR 2DL1 (95%), KIR 3DL1 (94%), KIR 2DS4 (92%) and the pseudogene 2DP1 (94%). Human leucocyte antigen (HLA)-C typing demonstrated prevalence of the C1/C2 heterozygosity (43%) and C1 homozygosity (41%) over the C2 heterozygosity. One hundred and twenty individuals from our panel carried at least one inhibitory KIR for the corresponding HLA-C group found in the genotype. Gene frequencies and found genotypes demonstrated similarity of the Czech population's KIR repertoire with the KIR repertoires of other Caucasian populations studied before.     

Genetic profile of KIR and HLA in southern Chinese Han population

KIR and their HLA ligands are encoded by two of the most diverse gene families in the human genome. The function of KIR on the NK cell is highly dependent on the normal expression of class I HLA on the target cell. Previous population studies in southern Chinese have been focused on the KIR framework genes and genotypes but little is known about the compound profiles of KIR/HLA. The present study examined 503 unrelated individuals from southern Chinese Han population for the polymorphism of KIR and class I HLA genes. All 16 KIR genes were detected in the study population and the four framework genes KIR3DL2, 3DL3, 3DP1, and 2DL4 were present in all individuals. Thirty unique KIR gene profiles were found reflecting a rather limited number of KIR haplotypes in this population. KIRAA1 was the most common profile observed in 54.7% of the samples. Among the AA1 individuals, 15.6% were homozygous for the deleted KIR2DS4. Haplotype A (74.8%) was more common than haplotype B (25.2%). HLA-C1 was a much more common ligand for 2D KIRs than C2. Bw4-80I, Bw4-80T, and the Bw4-bearing HLA-A alleles were detected at similar frequencies. The matched KIR+HLA pairs 2DL2/3+C1 (98.1%), 3DL1+Bw4 (73.3%), 3DL2+A3/11 (60.0%) were the most common ones whereas 3DS1+Bw4-80I was the least common (9.4%). A total of 193 unique compound profiles of KIR–HLA were identified in 480 informative individuals, 130 of the profiles being detected only once. The study provided a comprehensive analysis of the KIR/HLA profiles in southern Chinese in regards of the presence/absence of KIR genes, HLA ligands, matched KIR+HLA pairs, and KIR/HLA compound profiles. The results could help to better understand the role played by KIR/HLA interaction in associated diseases and clinical transplantation in southern Chinese.     

Distribution of killer cell immunoglobulin-like receptors in the Greek population

The interaction between killer cell immunoglobulin-like receptors (KIR) expressed on natural killer (NK) cells, and human leukocyt antigen (HLA) molecules expressed on target cells is known to regulate the cytolytic activity. A wide range of KIR genotypes is observed in the population, as the number of KIR loci can vary. In the present study we have determined the frequencies and combinations of 13 KIR genes and two CD94:NKG2 receptor genes and their distribution in the two haplotype groups in a panel of 233 unrelated healthy Greek Caucasians. We have typed genomic DNA for the presence of the putative KIR loci KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1 using modified polymerase chain reaction sequence-specific primers. The frequency of KIR loci combined with the linkage disequilibrium values suggest that the Greek population shares several general features with other Caucasoid populations studied before, but still distinguishes itself by the increased or decreased frequency of several alleles. The majority of the 45 different KIR genotypes seen in Greeks have been observed in Caucasoid populations studied before. Nevertheless, two of these genotypes, although met in other populations, have not been found in Caucasians before. One individual possesses a novel profile with no KIR inhibitory gene. The A haplotypes remain the most prevalent, with AA1 being the most common genotype, and the number of inhibitory KIRs being more variable than the number of noninhibitory KIRs in most haplotypes.     

Killer cell immunoglobulin-like receptor genes in Spanish multiple sclerosis patients

Killer cell immunoglobulin-like receptors (KIRs) are regulators of cytolytic activity of natural killer and certain T cells through interactions with human leukocyte antigen (HLA) class I ligands. KIRs have been shown to contribute to the pathogenesis of several autoimmune diseases, but their role in multiple sclerosis (MS) is still unclear. Here we determined the influence of KIR genes and their HLA class I ligands on susceptibility to MS and on the response to interferon-beta treatment in a Spanish population. KIR and HLA genotyping were performed in 200 MS patients and 200 controls. Significantly higher frequencies were found for KIR2DL5 and KIR3DS1 genes in MS patients and the carriage of the KIR2DL1 gene was associated with a higher progression index. Moreover, the frequency of the HLA-Bw4 motif was significantly reduced in MS patients. The KIR2DL1 and HLA-C2 matches were more frequent in MS patients, whereas the KIR3DL1 and HLA-Bw4 matches were more frequent in healthy controls. Nevertheless, non significant associations were found between all the KIR genes and therapeutic response to interferon-beta. Our results confirm that the carriage of HLA-Bw4 is a protective factor in MS and suggest that KIR2DL5 and KIR3DS1 may have a predisposing role in the disease.     

Diversity distributions of killer cell immunoglobulin-like receptor genes and their ligands in the Chinese Shaanxi Han population

In the present study, 17 killer cell immunoglobulin-like receptors (KIR) genes and KIR ligands (human leukocyte antigen [HLA] -A and -B) were detected by using a polymerase chain reaction-sequence-specific primer (PCR-SSP) method in 104 unrelated healthy Han individuals living in Shaanxi province, China. The observed carrier frequencies of the 12 KIR genes ranged from 0.14 to 0.96. KIR2DL4, 3DL2, 3DL3, 2DP1 and 3DP1 were found to be present in every individual. A total of 51 different KIR gene profiles were identified, in which 11 gene profiles exclusively belonged to the study population. Neighbor-joining phylogenetic tree between the studing population and its neighboring ethnic groups was constructed using the observed carrier frequencies of 13 KIR loci. The phylogenetic tree shows that the Shaanxi Han population, Han populations in different regions, Yi, Japanese, and Koreans were in the same cluster. KIR/HLA relationships show that KIR3DS1(-)/3DL1(+)/Bw4(+) was the most common association in the population. In conclusion, the present study findings reveal the high polymorphism of KIRs in the Shaanxi Han population, demonstrate the KIR/HLA association in the study population, and enrich the KIR and HLA gene resources. The obtained KIR data will further the understanding of genetic relationships among populations in different geographic areas, and assist in answering questions regarding KIR/HLA relationships.     

Functional advantage of educated KIR2DL1+ natural killer cells for anti‐HIV‐1 antibody‐dependent activation

Evidence from the RV144 HIV‐1 vaccine trial implicates anti‐HIV‐1 antibody‐dependent cellular cytotoxicity (ADCC) in vaccine‐conferred protection from infection. Among effector cells that mediate ADCC are natural killer (NK) cells. The ability of NK cells to be activated in an antibody‐dependent manner is reliant upon several factors. In general, NK cell‐mediated antibody‐dependent activation is most robust in terminally differentiated CD57⁺ NK cells, as well as NK cells educated through ontological interactions between inhibitory killer immunoglobulin‐like receptors (KIR) and their major histocompatibility complex class I [MHC‐I or human leucocyte antigen (HLA‐I)] ligands. With regard to anti‐HIV‐1 antibody‐dependent NK cell activation, previous research has demonstrated that the epidemiologically relevant KIR3DL1/HLA‐Bw4 receptor/ligand combination confers enhanced activation potential. In the present study we assessed the ability of the KIR2DL1/HLA–C2 receptor/ligand combination to confer enhanced activation upon direct stimulation with HLA‐I‐devoid target cells or antibody‐dependent stimulation with HIV‐1 gp140‐pulsed CEM.NKr‐CCR5 target cells in the presence of an anti‐HIV‐1 antibody source. Among donors carrying the HLA‐C2 ligand for KIR2DL1, higher interferon (IFN)‐γ production was observed within KIR2DL1⁺ NK cells than in KIR2DL1^– NK cells upon both direct and antibody‐dependent stimulation. No differences in KIR2DL1⁺ and KIR2DL1^– NK cell activation were observed in HLA‐C1 homozygous donors. Additionally, higher activation in KIR2DL1⁺ than KIR2DL1^– NK cells from HLA–C2 carrying donors was observed within less differentiated CD57^– NK cells, demonstrating that the observed differences were due to education and not an overabundance of KIR2DL1⁺ NK cells within differentiated CD57⁺ NK cells. These observations are relevant for understanding the regulation of anti‐HIV‐1 antibody‐dependent NK cell responses.     

Biphasic response of NK cells expressing both activating and inhibitory killer Ig-like receptors

NK cells can co-express inhibitory and activating killer Ig-like receptors (KIR) recognizing the same HLA class I ligand. We present evidence from experiments with NK cells expressing both activating (KIR2DS2) and inhibitory (KIR2DL2 and KIR2DL3) receptors that the activating KIR can function without apparent interference from the inhibitory KIR. These studies used CD158b mAb that is equally reactive with KIR2DS2, KIR2DL2 and KIR2DL3. First, we show using plastic-immobilized CD158b mAb that the activating KIR2DS2 is stimulated, resulting in NK cell division and degranulation. Second, we show using soluble CD158b mAb and FcRII (+) P815 cells that high concentrations of CD158b mAb trigger the inhibitory KIR, whereas low concentrations stimulate the activating KIR2DS2 resulting in NK cell division and cytolysis. These results demonstrate that the activating KIR2DS2 can function on cells co-expressing the inhibitory KIR2DL2 and/or KIR2DL3, indicating the potential for independent function of activating KIR with natural ligand.     

Combination of KIR 2DL2 and HLA‐C1 (Asn80) confers susceptibility to type 1 diabetes in Latvians

Killer immunoglobulin‐like receptors (KIRs) are known to modulate natural killer (NK) and NK T‐cell function by interacting with human leucocyte antigen (HLA) class I ligands on target cells. The aim of our study was to investigate the influence of KIR2D genes with their HLA‐C ligands in susceptibility to type 1 diabetes. A total of 98 type 1 diabetes patients and 70 healthy subjects from Latvia were typed for KIR genes and HLA‐C ligands using polymerase chain reaction‐based genotyping. The HLA C1+/C2+ combination was positively, and C1–/C2+ combination was negatively, associated with type 1 diabetes. Stratification analysis of KIR/HLA‐C ligand combinations showed 2DL2+/C1+, 2DL3+/C1+, and 2DS2+ /C1+ to be positively, and 2DL2–/C1– and 2DS2–/ C1– to be negatively, associated. The presence of 2DL2‐HLA‐C1 in the absence of 2DS1, 2DS2 confers maximum susceptibility. Absence of 2DL2 and HLA‐C1 along with absence of 2DS1 and 2DS2 confer maximum protection. A hypothetical model of KIR/ligand combinations on immune responses and type 1 diabetes susceptibility is proposed. Our results suggest that a combination KIR2DL2‐ HLA‐C1 plays a critical role in susceptibility or protection in Latvians against type 1 diabetes.     

Investigation of killer cell immunoglobulin-like receptors gene KIR3DL2 diversity and confirmation of KIR3DL2*015 in a Chinese population

Human killer cell immunoglobulin-like receptors (KIRs) are a subfamily of the immunoglobulin superfamily. Here, we established polymerase chain reaction-sequence-based typing (PCR-SBT) procedure to identify alleles of KIR3DL2 gene. The method was designed around the specific amplification of exon 3 to exon 4 and exon 8 to exon 9 of the KIR3DL2 gene. Genomic DNA from 104 healthy, unrelated Chinese Han individuals was typed for KIR3DL2 alleles. Each sample was assigned to the putative allele combination according to the sequences of all KIR3DL2 alleles. We observed 18 different genotypes and eight KIR3DL2 alleles in the population, with KIR3DL2*002 having the highest frequency of 0.558, and confirmed the new KIR3DL2*015 allele. Our data showed that the established PCR-SBT methods for KIR3DL2 allele typing were reliable, and Chinese Han population is distinct in KIR3DL2 allele frequencies.     

Killer cell immunoglobulin-like receptor genes in Spanish multiple sclerosis patients

Killer cell immunoglobulin-like receptors (KIRs) are regulators of cytolytic activity of natural killer and certain T cells through interactions with human leukocyte antigen (HLA) class I ligands. KIRs have been shown to contribute to the pathogenesis of several autoimmune diseases, but their role in multiple sclerosis (MS) is still unclear. Here we determined the influence of KIR genes and their HLA class I ligands on susceptibility to MS and on the response to interferon-beta treatment in a Spanish population. KIR and HLA genotyping were performed in 200 MS patients and 200 controls. Significantly higher frequencies were found for KIR2DL5 and KIR3DS1 genes in MS patients and the carriage of the KIR2DL1 gene was associated with a higher progression index. Moreover, the frequency of the HLA-Bw4 motif was significantly reduced in MS patients. The KIR2DL1 and HLA-C2 matches were more frequent in MS patients, whereas the KIR3DL1 and HLA-Bw4 matches were more frequent in healthy controls. Nevertheless, non significant associations were found between all the KIR genes and therapeutic response to interferon-beta. Our results confirm that the carriage of HLA-Bw4 is a protective factor in MS and suggest that KIR2DL5 and KIR3DS1 may have a predisposing role in the disease.     

High-throughput genotyping of KIR2DL2/L3, KIR3DL1/S1, and their HLA class I ligands using real-time PCR

Killer immunoglobulin-like receptors (KIRs) expressed on natural killer cells are critical components of innate immunity. Interactions between KIRs and their human leukocyte antigen (HLA) ligands have been shown to influence autoimmune and infectious disease course in defined populations. However, the low throughput and high cost of current methods impede confirmation of the universality of these findings. To support large epidemiology surveys, we developed a high-throughput real-time polymerase chain reaction-based assay to identify carriers of KIR3DL1, KIR3DS1, KIR2DL2, and KIR2DL3 and their HLA ligands. The platform performed with 100% sensitivity and specificity in detection of carrier and non-carrier on reference samples. The application of this platform will further clarify the nature and impact of the KIR–HLA epistatic interaction on disease course in large global population-based studies.     

Increased frequency and function of KIR2DL1–3+ NK cells in primary HIV‐1 infection are determined by HLA‐C group haplotypes

The acquisition and maintenance of NK‐cell function is mediated by inhibitory killer‐cell immunoglobulin‐like receptors (KIRs) through their interaction with HLA class I molecules. Recently, HLA‐C expression levels were shown to be correlated with protection against multiple outcomes of HIV‐1 infection; however, the underlying mechanisms are poorly understood. As HLA‐C is the natural ligand for the inhibitory receptors KIR2DL1 and KIR2DL2/3, we sought to determine whether HLA‐C group haplotypes affect NK‐cell responses during primary HIV‐1 infection. The phenotypes and functional capacity of NK cells derived from HIV‐1‐positive and HIV‐1‐negative individuals were assessed (N = 42 and N = 40, respectively). HIV‐1 infection was associated with an increased frequency of KIR2DL1–3⁺ NK cells. Further analysis showed that KIR2DL1⁺ NK cells were selectively increased in individuals homozygous for HLA‐C2, while HLA‐C1‐homozygous individuals displayed increased proportions of KIR2DL2/3⁺ NK cells. KIR2DL1–3⁺ NK cells were furthermore more polyfunctional during primary HIV‐1 infection in individuals also encoding for their cognate HLA‐C group haplotypes, as measured by degranulation and IFN‐γ and TNF‐α production. These results identify a novel relationship between HLA‐C and KIR2DL⁺ NK‐cell subsets and demonstrate that HLA‐C‐mediated licensing modulates NK‐cell responses to primary HIV‐1 infection.