KIR genes and HLA class I ligands in a Caucasian Brazilian population with colorectal cancer

Colorectal cancer (CRC) can occur anywhere in the colon or rectum and represents the third most common cancer in the world in both sexes. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study was to evaluate the association between the KIR genes and HLA ligands in patients with colorectal cancer and healthy controls. We examined the polymorphism of 16 KIR genes and their HLA ligands in 154 caucasoid CRC patients and 216 controls. When both groups were compared, no significant differences were found for HLA ligands and KIR genes after Bonferroni correction. However, the Bx haplotypes (heterozygous and homozygous for the haplotype B) were more frequent in controls, when compared with patients. These findings suggest that individuals with Bx haplotypes could have some protection to colorectal cancer. The hypothesis is not related with the presence of a special KIR gene and HLA ligand related to the disease, but to the presence of several activating genes in the individuals with no better action of one in relation to other. Further studies to confirm this observation are warranted.     

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。     

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.     

Association of killer cell immunoglobulin-like receptor genotypes with susceptibility to endometriosis

PROBLEM: Endometriosis is an immune-related chronic inflammatory disease with a polygenic predisposition. The aim of this study was to investigate whether polymorphisms in killer cell immunoglobulin-like receptors (KIRs) is responsible, in part, for genetic susceptibility to endometriosis. METHOD OF STUDY: The KIRs genotype was determined in 186 patients with endometriosis and 165 control women using polymerase chain reaction with sequence-specific primers. RESULTS: The frequency of KIR3DS1 was significantly decreased in patients compared with controls (32% versus 44%, P=0.028). KIR data were analyzed using a model comprised of three large groups, in which a gradient of activation/inhibitory potential derived from the combination of KIR and human leukocyte antigen (HLA) ligand genes was taken into account. The frequency of inhibitory KIRs/HLA-class I combination genotypes was significantly higher in patients than in controls (chi2=6.010, 2 df, P=0.0496). CONCLUSION: Our results suggest that polymorphism in KIRs may be associated with susceptibility for endometriosis.     

内蒙古蒙古族人群杀伤细胞免疫球蛋白样受体基因簇多态性研究

目的 研究中国蒙古族人群杀伤细胞免疫球蛋白样受体(killer cell immunoglobulin-like receptors,KIR)基因的携带频率、KIR基因型及其遗传规律.方法 采集90名内蒙古农牧区蒙古族个体的血样,应用序列特异性引物聚合酶链反应(polymerase chain reaction-sequence specific primer,PCR-SSP)方法分别扩增KIR基因簇16个基因,依据实验结果计算各基因的携带频率并查询样本的基因型,和已报道的24个其他人群的相关数据进行主成分分析、计算Nei氏遗传距离并绘制遗传树.结果 (1)蒙古族人群KIR 2DL2、2DS2携带率高于蒙古利亚人,低于高加索人.(2)蒙古族KIR基因单倍型AA为37.78%,高于高加索人,低于蒙古利亚人.(3)系统进化树显示蒙古利亚人和高加索人分别聚类,蒙古族人群则介于两者之间.结论 蒙古族其成因似与受到高加索人和蒙古利亚人的双重影响有关,表现为介于高加索人和蒙古利亚人之间的中间形式.

Abstract：

Objective To investigate the killer cell immunoglobulin-like receptor (KIR) gene frequencies and genotypes distributions in the Inner Mongolian population. Methods Ninety genomic DNA samples were extracted from blood samples of randomly chosen Mongolian individuals. Gene-specific PCR amplification was used to identify genes present or absent for 16 KIR loci. KIR genotype distributions were obtained and compared to that of 24 populations published in literatures using principal component analysis by SAS8.0 software. Genetic tree was constructed by the calculate Nei's genetic distance. Results (1) The frequency of KIR 2DL2,2DS2 in Mongolian individual is higher than that in north Mongoloid and less than that in Caucasian. (2) Haplotype AA was identified in 37.78% of individuals, which is higher than that in north Mongoloid and lower than that in Caucasian. (3) Mongolian was considered between north Mongoloid and Caucasian by principal component and genetic tree analysis. Conclusion Mongolian might be affected by the north Mongoloid and Caucasian, and showed intermediate between the two populations.     

Killer immunoglobulin-like receptor and human leukocyte antigen expression as immunodiagnostic parameters for pelvic endometriosis

PROBLEM: We investigated host immunologic responses to endometriosis by comparing immune cell surface antigens in peripheral blood (PB) and peritoneal fluid (PF) from women with endometriosis with those in PB and PF from other patients. METHOD OF STUDY: Japanese women with endometriosis (n = 56) were compared with controls with other laparoscopic diagnoses (n = 68). PB and PF were collected at the time of laparoscopy for flow cytometry. RESULTS: No significant difference in phenotypic parameters of T cells (CD3, CD4, and CD8), B cells (CD19), natural killer (NK) cells (CD56), or monocytes/macrophages (CD14) was seen between women with and without endometriosis. However, increased killer immunoglobulin-like receptor (CD158a) expression by NK cells and decreased human leukocyte antigen (HLA)-ABC and -DR expression by macrophages, all suggesting decreased functional activation were found in endometriosis. These markers showed significant association with endometriosis by odds ratio, logistic regression, and decision tree analyses. CONCLUSIONS: Increased CD158a(+) NK cells in PB and PF indicated decreased NK cell cytotoxicity in endometriosis, while decreased HLA expression on PF macrophages suggested impaired antigen presentation. Thus, aberrant immune responses by NK cells and macrophages may represent risk factors for endometriosis.     

Expression of killer immunoglobulin-like receptors on peripheral blood NK cell subsets of women with recurrent spontaneous abortions or implantation failures

PROBLEM: Decidual natural killer (NK) cells express inhibitory receptors (killer immunoglobulin-like receptors, KIRs), which bind to ligands on trophoblast cells (human leucocyte antigen, HLA-C). This interaction appears to block NK cytotoxicity against trophoblast cells. In this study, we investigated the expression of inhibitory and activating receptors in peripheral blood NK cells of women with recurrent spontaneous abortion (RSA) or implantation failures. METHOD OF STUDY: CD56(dim)/CD16(+), CD56(bright)/CD16(-) NK cells and CD56(+)/CD3(+) NKT cells of women with RSA or in vitro fertilization (IVF) failures and normal controls were analyzed for the expression of CD158a, CD158b inhibitory KIRs or CD161-activating receptors, by flow cytometric analysis. RESULTS: CD158a and CD158b inhibitory receptor expression by CD56(dim)/CD16(+) and CD56(bright)/CD16(-) NK cells were significantly decreased, and CD161-activating receptor expression by CD56(+)/CD3(+) NKT cells was significantly increased in women with implantation failures when compared with normal controls. CONCLUSIONS: An imbalance between inhibitory and activating receptor expression was found in NK cells of women with implantation failures. This imbalance may explain the adverse reproductive outcome.     

树突状细胞上调MHC Ⅰ类相关抗原A表达对NK细胞活性的影响

目的:观察单核细胞、未成熟和成熟树突状细胞(DCs)表达MHC Ⅰ类相关抗原A(MICA)的情况,以及DCs表达MICA后对NK细胞活性的影响.方法:用流式细胞术分别检测单核细胞、未成熟DC(iDCs)以及LPS、TNF-α、CD40L、IL-15和IFN-α分别刺激的成熟DCs表面MICA的表达,并观察DCs表达MICA后对NK细胞表达CD69、细胞毒活性和分泌IFN-γ的影响.最后用流式细胞术检测重组NKG2D/Fc蛋白和抗IL-12单克隆抗体(mAb)对DCs激活NK细胞的影响.结果:单核细胞不表达MICA,iDCs低表达MICA.LPS、TNF-α和CD40L对DCs表达MICA无影响;但IFN-α和IL-15可促进DC上调MICA表达.DCs表达MICA后可促进NK细胞表达CD69、分泌IFN-γ、杀伤K562细胞.NKG2D/Fe蛋白可抑制NK细胞的杀伤活性和分泌IFN-γ;而抗IL-12mAb仅抑制IFN-γ分泌.结论:MICA在DCs表面的表达受局部微环境影响,且DCs表达MICA后可增强NK细胞的活性.     

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.     

Molecular determinant-based typing of KIR alleles and KIR ligands

Killer cell immunoglobulin-like receptors (KIRs) regulate NK cell function. KIRs and their HLA ligands are highly polymorphic in nature with substantial allelic polymorphism. At present, there is a lack of an expedient method for KIR and HLA allele typing with relevant functional information. Here, we developed a single-nucleotide polymorphism (SNP) assay to type various allele groups of KIR2DL1 with distinct functional properties based on polymorphism at position 245. We also established a SNP assay to type different KIR ligands based on polymorphism at position 77 in HLA-C and position 83 in HLA-B and -A. Our SNP assays for KIR and KIR ligand typing are much cheaper and faster than existing high-resolution typing. Importantly, our high-throughput methods provide readouts that are informative in predicting NK cell activity in health, disease, and transplantation.     

NK receptor interactions with MHC class I molecules in pregnancy

Both HLA class I molecules and their receptors on Natural Killer cells, the KIR molecules, are highly polymorphic. It is generally believed that this variation is driven in response to the role of these receptors and counter-receptors in resistance to disease. Uterine NK cells are the major maternal leukocyte population present within the decidua, and they express KIR2D receptors for HLA-C, the only polymorphic class I molecule on trophoblast. Genetic and functional data suggest that the maternal KIR/fetal HLA-C interaction in pregnancy may affect the delivery of an optimal blood supply to mother and fetus. The drive for novelty in HLA-C and KIR2D allelic diversity may relate not only to survival from infections but also to reproductive success.     

Amoxicillin conjugates to HLA class I molecules and interferes with signalling through the ILT2/LIR-1/CD85j inhibitory receptor

BACKGROUND: Drugs behave as haptens and are recognized by specific T-cell receptors in the context of major histocompatibility complex (MHC) molecules in allergic subjects. Natural killer cell receptors (NKRs) are MHC class I-specific receptors that modulate the threshold of activation of immunocompetent cells. Amongst them, ILT2/LIR-1/CD85j is an inhibitory NKR widely distributed in several cell lineages and with a broad spectrum of recognition of human leucocyte antigen (HLA) class I ligands. METHODS: We have evaluated, at the biochemical and cellular level, the ability of amoxicillin (AX) conjugate to HLA class I molecules and to interfere with the inhibitory signal delivered by the HLA class I receptor ILT2/LIR-1/CD85j. RESULTS: We have detected AX bound to cell membrane proteins and in particular to HLA class I molecules. Preincubation with AX rendered target cells susceptible to NK cell-mediated lysis. In conjugation experiments, target cell-bound AX hampered tyrosine phosphorylation of ILT2/LIR-1/CD85j upon ligand recognition and the subsequent recruitment of SHP-1 phosphatase. CONCLUSION: Conjugation of AX to HLA class I molecules may mask HLA recognition by inhibitory receptors and attenuate the negative signal delivered by SHP-1 phosphatase, thus lowering the threshold of activation of effector cells.     

CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies

CD94 molecules have been suggested to function as inhibitory natural killer cell (NK) receptors involved in the recognition of HLA-B alleles sharing the Bw6 supertypic specificity. In this study, we show that CD94 molecules may play a more general role: they are also involved in the recognition of other HLA class I molecules, including HLA-C and at least some HLA-A alleles. The inhibitory effect mediated by CD94 molecules on NK cytolytic activity is lower in magnitude than that of bona fide inhibitory receptors such as p58 or p70. Distinct from the other human NK receptors involved in HLA class I recognition, CD94 is expressed on virtually all NK cells. In addition, it has been shown to be functionally heterogeneous since, in different clones, CD94 mediated either cell triggering or inhibition. Although NK cells expressing inhibitory CD94 molecules are usually characterized by a CD94^bright phenotype, there is no precise correlation between fluorescence intensity and inhibitory or activating function. Here, we describe two novel monoclonal antibodies (mAb) which selectively recognize inhibitory CD94 molecules and bind to a subset (variable in size among different donors) of CD94^bright cells. The use of these mAb allows the direct assessment of NK cells expressing inhibitory CD94 receptors both at the population and at the clonal level.     

NK cell receptor gene of the KIR family with two IG domains but highest homology to KIR receptors with three IG domains

The killer cell inhibitory receptors (KIRs) are surface glycoproteins expressed by natural killer (NK) cells and some T cells. They recognize polymorphic human HLA class I molecules. Two families of KIRs have been identified and named p58 and p70. The p58 family of genes encode type I membrane proteins with two extracellular immunoglobulin (Ig) domains, while the p70 genes have three Ig domains. We here report the cloning and characterization of a novel KIR cDNA obtained from tumor cell lines with NK reactivity (YT and NK-92). This gene is also expressed in the normal cell line NK 3.3 and in NK cells obtained from some but not all normal donors. The clone, KIR103AS, has an open reading frame consistent with a KIR with two extracellular Ig domains, a transmembrane region and a 114 amino acid long cytoplasmic domain containing a single src homology 2 (SH2) binding motif. The membrane distal Ig domain of KIR 103AS has highest homology with the first Ig domain of p70 KIRs and differs significantly from the first Ig domain of p58 KIRs. The second, membrane proximal Ig domain of KIR103AS has similar and high homology with the membrane proximal Ig domains of both p70 and p58 KIRs. The extracellular domains of KIR 103AS therefore share characteristic features with both p70 and p58 genes: the domain structure is identical to p58 KIRs but the sequence homology matches closely with p70 KIRs. The putative transmembrane and cytoplasmic domains are distinctly different from all previously reported KIR cDNAs.     

KIR2DL2/S2 and HLA-C C1C1 genotype is associated with better response to treatment and prolonged survival of patients with non-small cell lung cancer in a Polish Caucasian population

Non-small cell lung carcinoma (NSCLC) is a multifactorial disease influenced by both environmental and genetic factors. Here, we examined whether the repertoire of genes encoding killer immunoglobulin-like receptors (KIR) and genes for their ligands, C1/C2 and Bw4, may affect a susceptibility to NSCLC and response to treatment. We typed 269 NSCLC patients and 690 healthy control individuals for KIR genes and for their ligands. KIR genes were not associated with NSCLC. C1C2 genotype was less frequent whereas both C1C1 and C2C2 homozygotes were more frequent in patients than in controls (χ(2)=7.73; df=2; p=0.021). Patients positive for KIR2DL2 and KIR2DS2 gene and homozygous for the C1 ligand were 6 times more likely to respond to treatment than those with other genotypes (p=0.034). In accordance with this, patients with the KIR2DL2+/KIR2DS2+, C1C1 genotype survived longer than others (p=0.0094). Median survival was 23months for KIR2DL2/2DS2/C1C1-positive patients, but only 10months for those with other genotypes.     

Killer immunoglobulin-like receptors (KIR2DL2 and/or KIR2DS2) in presence of their ligand (HLA-C1 group) protect against chronic myeloid leukaemia

We have analysed the frequency of killer immunoglobulin-like receptors (KIR) in cohorts of patients from Turkey with acute lymphocyte leukaemia ( n  = 52), acute myeloid leukaemia ( n  = 54) and chronic myeloid leukaemia (CML) ( n  = 52) and compared the results with 154 controls. We also examined the frequencies of human leucocyte antigen (HLA)-C groups, -Bw4, -Bw6 and where appropriate the combination of the KIR gene and its ligand. We found several statistically significant results between the patients and the controls. We proposed a model in CML of protection via KIR2DL2 and/or KIR2DS2 with the presence of the ligand HLA-C1 group and susceptibility via HLA-Bw4 homozygosity (i.e. absence of HLA-Bw6).     

Cloning of NKG2-F,a new member of the NKG2 family of human natural killer cell receptor genes

The NKG2 family of genes encodes at least four different type II transmembrane molecules (NKG2-A, NKG2-B, NKG2-C and NKG2-E) which contain a C-lectin domain. These proteins have been shown to be covalently associated with CD94, another C-type lectin member. The heterodimers are involved in natural killer cell-mediated recognition of different HLA-allotypes. Here we describe the cloning of a new NKG2-related gene, termed NKG2-F, localized 25 kb from NKG2-A as well as its relationship with the previously described NKG2-D cDNA. Despite the similarities with the other NKG2 genes, NKG2-F encodes a putative protein which does not contain any lectin domain. However, a conserved 24-amino acid sequence, present in all members of the NKG2 family, suggests that NKG2-F is also able to form heterodimers with CD94.     

A High Dose of Intravenous Immunoglobulin Increases CD94 Expression on Natural Killer Cells in Women with Recurrent Spontaneous Abortion

Problem  A high dose of intravenous immunoglobulin (HIVIg) therapy is effective in various diseases such as autoimmune diseases, and also is expected to have efficacy in recurrent spontaneous abortion (RSA). The aim of this study was to understand immunological mechanisms of this therapy.
Method of study  By flowcytometric analyses, we examined phenotypic changes of a variety of immunological cells including natural killer (NK) cells, cytotoxic T cells, regulatory T cells and macrophages in peripheral blood of RSA women with HIVIg therapy ( n  = 8).
Results  Expression percentages of inhibitory CD94 on NK cells significantly ( P =  0.01) increased after the therapy (58.8 ± 21.4% versus 71.0 ± 17.6%).
Conclusion  Mechanisms of possible efficacy of HIVIg therapy for RSA may include enhancement of CD94 expression and subsequent suppression of NK cell cytotoxicity.     

Association of killer cell immunoglobulin-like receptor and human leucocyte antigen-Cw gene combinations with systemic lupus erythematosus

Killer cell immunoglobulin-like receptors (KIRs) are a diverse family of activating and inhibitory receptors expressed on natural killer (NK) cells and T cells, the genes of which show extreme polymorphism. Some KIRs bind to human leucocyte antigen (HLA) class I subgroups, and genetic interactions between KIR genes and their ligand HLA have been shown to be associated with several autoimmune diseases. The present study aimed to investigate whether the combinations of KIR genes and HLA-Cw ligands associate with the susceptibility of systemic lupus erythematosus (SLE). Polymerase chain reaction using sequence-specific primers was used to determine the genotypes of KIR genes and HLA-Cw alleles. We found that the frequencies of HLA-Cw07 were statistically significantly higher in the patient group than those in the control group (P = 0·009). KIR2DS1⁺HLA⁻Cw^Lys was more common in subjects with SLE compared to control subjects (P = 0·015). In addition, the frequency of KIR2DS1 was increased in SLE when KIR2DL1/HLA-Cw are absent, and the difference was significant (P = 0·001). KIR genotype and HLA ligand interaction may potentially influence the threshold for NK (and/or T) cell activation mediated through activating receptors, thereby contributing to the pathogenesis of SLE.     

Investigation of killer cell immunoglobulin-like receptor gene diversity in KIR3DL1 and KIR3DS1 in a transplant population

Several overlapping amplicons were used to obtain the sequence of genomic DNA covering most of the coding regions of KIR3DL1 and KIR3DS1 from a family and 77 bone marrow transplant patients and their unrelated donors. Alleles 3DL1*00101 and *002 were most frequently observed in addition to 12 other known 3DL1 alleles. A single 3DS1 allele, 3DS1*01301, was identified in the 31 of 32 individuals carrying this gene. Two new alleles, 3DL1*01702 and 3DS1*058, were characterized. Three samples appeared to carry the duplicated killer cell immunoglobulin-like receptor (KIR) haplotype observed in other studies based on the presence of 3DS1 and two 3DL1 alleles. Additionally, one sample appeared to carry a novel KIR haplotype containing one 3DL1 and two 3DS1 alleles.