Establishment of Q-PCR method for qualitative testing of the presence or absence of KIR genes / 中国输血杂志

【Objective】 To establish a method for qualitative detection of the presence or absence of all KIR genes by quantitative polymerase chain reaction(Q-PCR). 【Methods】 Based on the polymorphism of high-resolution level KIR alleles in Chinese population and the IPD-KIR database, KIR gene-specific primers were designed to amplify all the 16 KIR genes and 2DS4-Normal and 2DS4-Deleted subtypes by Q-PCR. Meanwhile, one negative control and one positive control specific amplifying human growth hormone (HGH) gene fragment were set to monitor the false positive and false negative results in PCR amplification, respectively. A total of 302 samples with known KIR genotype previously identified by KIR PCR-SSP commercial kit were randomly selected for blind inspection to verify the reliability of KIR Q-PCR method established by authors. 【Results】 The results of 300 samples detected by our KIR Q-PCR method were consistent with the known results, but two samples showed inconsistent results. One sample was negative for 2DS5 by Q-PCR but positive by PCR-SSP, another sample was positive for 2DS1 by Q-PCR but negative by PCR-SSP. The two doubtful samples were genotyped by sequencing-based typing (PCR-SBT) for 2DS5 and 2DS1, respectively. PCR-SBT results confirmed that the results of Q-PCR test was correct. 【Conclusion】 The KIR Q-PCR method established in this paper can provide accurate and reliable results for testing the presence or absence of KIR genes.

Expert consensus on testing of killer-cell immunoglobulin-like receptor genes / 中国输血杂志

The killer-cell immunoglobulin-like receptor(KIR) gene family exhibits complex genetic diversity. In addition to allele level polymorphisms, KIR genes show extra diversity at haplotype content as well as copy number variation. In order to standardize the KIR gene testing, the Working Party on Histocompatibility, Chinese Society of Blood Transfusion (CSBT)and Shenzhen Blood Center organized experts to discuss and reach a consensus on KIR gene testing based on KIR-related literature and practical experience. The present consensus has summarized the techniques for identifying the diversity of KIR genes, quality control, testing report and its applications, aiming to improve the accuracy of KIR gene testing results.

Relationship between killer cell immunoglobulin-like receptor genes and immune reconstitution failure in human immunodeficiency virus infection/acquired immunodeficiency syndrome patients after anti-retroviral therapy / 中华传染病杂志

Objective:To analyze the relationship between killer cell immunoglobulin-like receptor ( KIR) genes and immune reconstitution failure in human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) patients after anti-retroviral therapy (ART). Methods:HIV/AIDS patients receiving ART for ≥1 year who attended the AIDS outpatient clinics of Wuming Hospital of Guangxi Medical University and People′s Hospital of Mashan from May 2007 to December 2019 were included. Patients were divided into immune reconstitution failure group and full immune reconstitution group. Polymerase chain reaction with sequence specific primers (PCR-SSP) was used to detect KIR genotypes in all subjects, and the genotype frequency (PF) of 16 KIR genotypes was calculated. Statistical analysis was conducted using chi-square test. Multivariate logistic regression was used to analyze the relationship between KIR genotypes and immune reconstitution failure.Results:There were 102 patients with HIV/AIDS, including 44 immunological non-responders and 58 immunological responders. The PF of KIR2 DL5 in immune reconstitution failure group was 59.09%(26/44), which was higher than 36.21%(21/58) in full immune reconstitution group, and the difference was statistically significant ( χ2=5.27, P=0.022). Multivariate logistics regression analysis showed that KIR2 DL5 was associated with immune reconstitution failure when adjusted for age and baseline CD4 + T cell count. Positive expression of KIR2 DL5 may be a risk factor for immune reconstitution failure (adjusted odds ratio (a OR)=2.431, 95% confidence interval 1.012 to 5.844, P=0.047). Conclusions:Positive expression of KIR2 DL5 may be related to immune reconstitution failure in HIV/AIDS patients after ART.

Polymorphism of KIR2DL4 gene among northern Chinese Han population / 中国输血杂志

【Objective】 To investigate the polymorphism of KIR2DL4 gene in northern Chinese Han population. 【Methods】 A total of 327 DNA samples were isolated by magnetic beads from unrelated individuals of northern Chinese Han population. The coding sequence (CDS) of KIR2DL4 were amplified using four pairs of KIR2DL4-specific PCR primers developed by our own KIR sequencing-based typing patent, and each exon of KIR2DL4 carried by the four PCR amplicons was then subjected to DNA Sanger sequencing in both directions. The genotype of each sample was assigned using the Assign 4.7 software. 【Results】 Among 327 individuals, 8 different kinds of KIR2DL4 alleles were detected, with observed frequencies ranked as KIR2DL4*00102 [77.1%(252/327)], *00501 [35.8%(117/327)], *011 [20.2%(66/327)], *00602 [12.5%(41/327)], *00801 [8.6%(28/327)], *00103 [4.9%(16/327)], *00503 [1.5%(5/327)] and *00504 [0.9%(3/327)]. In this study, the 10A type alleles which can encode a full membrane-bound receptor include 2DL4*00102, *00103, *00501, *00503, *00504 and *00602, whereas the 9A type alleles which produce truncated forms of receptor include 2DL4*00801 and *011. The observed frequencies for 10A and 9A type KIR2DL4 alleles were 97.6% (319/327) and 27.8% (91/327), respectively. The ratio of 10A to 9A type was 3.5: 1. The observed frequencies of KIR2DL4 alleles in northern Chinese Han population showed no significant difference compared with southern Chinese Han population (P>0.05). 【Conclusion】 The allelic diversity of KIR2DL4 elucidated in the present study can provide valuable data for KIR-associated disease studies and human evolution.

Research progress on thecorrelation between killer cell immunoglobulin-like receptor and tumor / 中国输血杂志

The killer cell immunoglobulin-like receptor (KIR) is located in 100-200kb region of the leukocyte receptor complex (LRC) on chromosome 19. KIR, mainly expresses on the surface of natural killer (NK) cells, is divided into inhibitory and activated receptor in function. Tumor cells could evade the killing of NK cells by regulating down the expression of HLA molecules on the cell surface. KIR molecules regulate the killing effect of NK cells by combining with human leukocyte antigen (HLA) to conduct inhibitory and active signals. In recent years, KIR and its immune regulation on tumors have become a research hotspot. The article reviews the research progress of KIR and its association with tumors, especially the correlation between KIR and high incidence of nasopharyngeal carcinoma (NPC) in Guangdong and Guangxi of China.

Establishment of a RT-PCR assay for detection of mRNA expression of KIR2DS1 gene on NK cell surface / 临床检验杂志

Objective@#To establish a real-time PCR (RT-PCR) assay for detecting mRNA expression of killer cell immunoglobulin-like receptor (KIR) 2DS1 gene( KIR2DS1 ) on the surface of natural killer (NK) cells, and evaluate its performance. @*Methods@#A total of 57 recipient-donor pairs of allogeneic hematopoietic stem cell transplantation (Allo-HSCT) were enrolled in this study. The specific primers and probe of KIR2DS1 gene were designed for Taqman-MGB fluorescence quantitative PCR detection system. The performance parameters of the detecting system, such as coincidence rate, repeatability, sensitivity, scope of application of the instrument and reproducibility of operation technicians were evaluated and validated. @*Results@#The KIR-SSO Genotyping Test was used as the gold standard. The results of 35 samples showed the accuracies of self-built method were all 100% for both of positive and negative KIR2DS1 . Three samples with high, median and low value of Ct values were used to verify the repeatability. The coefficients of variation of intra-assay and inter-assay were ranged from 0.09% to 0.46% and 0.71% to 1.13% respectively. The sensitivity of the established method was up to 10 2 copies/μL at least. The coefficients of variation of the three samples with sensitivity of 10 2 copies/μL were 5.37%, 2.71% and 5.51% in five repeated tests respectively. The regression analysis for the samples measured by ABI-7500 and LC-480 fluorescence quantitative PCR instrument showed regression equation was Y=0.973 6X+0.118 3 (R 2 =0.961 9, R 2 ＞0.95). The reproducibility of 10 samples with positive KIR2DS1 operated by two technicians showed that the biases were all less than ±5%. @*Conclusion@#A TaqMan-MGB real-time PCR assay for detection of mRNA expression of KIR2DS1 gene was established successfully with fine performance.

Association between polymorphisms of killer cell immunoglobulin-like receptor gene and the risk of essential hypertension: a case-control study / 中华流行病学杂志

Objective To assess the association between killer cell immunoglobulin-like receptor (KIR) gene polymorphisms and the risk of hypertension in autoimmune mechanism.Methods We conducted a case-control study including 205 hypertensives and 205 controls matched with sex and age,from a community-based population.KIR genes of all subjects were genotyped by polymerase chain reaction with sequence-specific primers (PCR-SSP).Conditional logistic regression model and generalized multifactor dimensionality reduction (GMDR) method were used to estimate the association among KIR gene polymorphisms and the risk of hypertension.Results The genotypic frequencies of KIRs were not significantly different between the hypertensives and the control groups (P＞0.05).Among all the models of GMDR concerning the association between interactions of KIR genes and essential hypertension,the testing accuracy of the interaction between K/R2DS2 and KIR2DS3 was the highest (55.13％),with cross-validation consistency as 10/10 (P=0.054).Results from the conditional logistic regression showed that individuals with KIR2DS2 +:KIR2DS3-were significantly associated with an increased risk on hypertension (OR=2.555,95％CI:1.203-5.429,P=0.015).However,individuals with KIR2DS2 +:KIR2DS3 + were significantly associated with a reduced risk of hypertension (0R=0.268,95％ CI:0.088-0.815,P=0.020).Individuals with KIR2DS2-KIR2DS3 + did not seem to be associated with the risk of hypertension (0R=1.602,95％ C I:0.785-3.266,P=0.195),when compared to the KIR2DS2-KIR2DS3-group.Interactions between KIR2DS2 and KIR2DS3 were significantly associated with the risk of hypertension,after adjusted for BMI,smoking,drinking and family history of hypertension (OR=0.065,95％CI:0.013-0.317,P=0.001).Conclusion Individuals with KIR2DS2 and no KIR2DS3 were associated with the increased risk of hypertension.KIR2DS2 that coexisted with KIR2DS3 were associated with the reduced risk of hypertension.Antagonism between KIR2DS2 and KIR2DS3 might serve as a protect factor for hypertension.

Effects of methylated CpG islands in promoter region on the expression of KIR3DL1 protein / 中华微生物学和免疫学杂志

Objective To investigate the effects of methylated CpG islands in the promoter region on the expression of killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1).Methods Three voluntary unpaid blood donors carrying high expression allele KIR 3DL1*01502 and three donors carrying low expres-sion allele KIR3DL1*005 were recruited in this study .The nucleotide sequences and the methylated CpG islands in the promoter regions of KIR 3DL1*01502 allele and KIR3DL1*005 allele were analyzed .The NK cells expressing KIR3DL1*01502 and KIR3DL1*005 were respectively treated with 5-aza for the dem-ethylation of CpG islands within the promoters .The expression of KIR3DL1 protein on the surface of NK cells was measured with flow cytometer .Results Two differences at nucleotide sites -65 and -269 were detected within the promoter regions of KIR3DL1*01502 and KIR3DL1*005, resulting in two distinct CpG islands.The CpG islands within the promoter of KIR 3DL1*01502 allele were highly methylated .The ex-pression of KIR3DL1 protein on NK cells which carried KIR 3DL1*01502 allele was significantly increased after the demethylation of CpG islands .However , the treatment of demethylation had no significant effects on the expression KIR3DL1 protein on NK cells harboring KIR3DL1*005 allele.Conclusion The methylated CpG islands within the promoter of KIR 3DL1*01502 allele affected the antigen expression on the surface of NK cells.Different KIR3DL1 alleles might show different mechanisms in regulating antigen expression .

Study on the killer cell immunoglobulin-like receptor gene polymorphisms in leukemia patients and their siblings who have same HLA-A/B/DR typing / 中华微生物学和免疫学杂志

Objective To investigate the relationship between killer cell immunoglobulin-like receptors(KIR)and HLA by distribution of KIR gene in leukemia patients and their siblings who have same HLA-A/B/DR typing.Methods KIR genotypes were detected by PCR-SSP on 78 patients and their siblings who have same HLA-A/B/DR typing.Results There were 48.72%in 78 patients who had same KIR genotypes with their siblings while the 44.87%patients had different KIR genotypes with their siblings.There was no difference in frequency between patients and their siblings(P＞0.05).There were no differences in frequency among chronic myelocytic leukemia(CML),non acute lymphoblagtic leukemia(NALL)and acute lymphoblagtie leukemia(ALL)but the frequency of KIR2DS4 in CML was higher than others.Condusion The KIR gene and HLAⅠ antigen are heredity independently and relatively stable.The factor of disease has little effect on KIR gene.

Killer cell immunoglobulin-like receptor gene polymorphism in Hashimoto′s thyroiditis patients / 中华微生物学和免疫学杂志

Objective To investigate the relationship of the killer cell immunoglobulin-like receptor (KIR) gene polymorphism with Hashimoto′s thyroiditis(HT). Methods One hundred HT patients and 260 randomly matched healthy controls were enrolled to detect the KIR genotype. The genomic DNA were extracted, and 15 selected KIR genes, KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1 and pseudogene KIR2DP1, were determined by a polymerase chain reaction using sequence-specific primers (PCR-SSP). Results The frequency of KIR2DL5 gene was significantly lower of the patient group than that of the control group (0.200 vs 0.312, RR=0.64, P<0.01). Conclusion There may be an association between pathogenesis of HT and KIR2DL5 gene.

HLA Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

Much of the success of hematopoietic stem cell transplantation (HSCT) has been due to the ability to overcome posttransplant complications, by performing HLA genotypically matched or even mismatched sibling donor HSCT or unrelated donor HSCT. Based on the promising results with using vigorously T-cell-depleted high-dose peripheral blood stem cell transplants by the Perugia University group, several methods to eradicate refractory leukemic cells have been employed worldwide. However, there are limitations of the existing data regarding haploidentical HSCT, that is, the small numbers of patients and the heterogeneous patient populations in most of the published series. Also, researchers have not exactly demonstrated the effect of natural killer (NK) cell alloreactivity in various settings of allogeneic HSCT. In reality, haploidentical HSCT is possible without T-cell depletion. However, it isn't clear whether reduced-intensity HSCT from a haploidentical related donor or a mismatched unrelated donor is feasible. Anyhow, successfully overcoming the major histocompatibility barriers with using related or unrelated donors means that virtually all patients would have an immediately available donor for desperately needed HSCT. The full potential of haploidentical HSCT may be ultimately achieved through a better understanding of the transplant immunology, including the Korean specificity of killer cell immunoglobulin-like receptor polymorphism. Further study and better support from Korean government insurance coverage that would offer HSCT to more patients in need of transplant and cultivating an optimal NK alloreaction without detrimental complications is urgently required.

Association of KIR (Killer Cell Immunoglobulin-like Receptor) Genotype with Psoriasis in Korean Population

BACKGROUND: Psoriasis is a multifactorial autoimmune skin disease with a pathogenesis that has remained obscure. Recently, T cells bearing natural killer receptors (NKRs) were precisely and strongly targeted as new putative pathogenic immunocytes in psoriasis. Among NKRs, killer cell immunoglobulin-like receptor (KIR) is the major molecule recognizing HLA class I allotypes and might be closely related to psoriasis. METHODS: To investigate the association of KIR genotype and patients with psoriasis in Korean, we defined the 14 KIR genotypes in 96 patients with psoriasis and 86 healthy controls using PCR-SSP methods. RESULTS: The frequencies of KIR2DS4 and KIR3DL1 were significantly decreased in psoriasis compared with controls (RR=0.21, p or =30 years) respectively, these phenomena were similarly observed independent of groups divided (type I: RR=0.26, p<0.005; type II: RR=0.14, p<0.0006). When the patients were divided into subgroups according to the age of onset and family history, the frequencies of KIR2DS4, KIR3DL1, and KIR2DS3 were significantly decreased in type I compared with type II psoriasis (3DL1, 2DS4: p<0.004; 2DS3: p<0.04) and were significantly decreased in psoriasis without family history compared to with family history (3DL1, 2DS4: p<0.007; 2DS3: p<0.05). The frequency of haplotype combination BB was significantly increased in psoriasis compared with controls (RR=2.74, p<0.009). CONCLUSION: These results suggest that KIR genotype is a factor for the occurrence and development of psoriasis and in future how combinations of HLA and KIR genes influence psoriasis needs to be defined.

Antitumor effect of allogeneic NK cells on human multi-drug resistant nasopharyngeal carcinoma cells subcutaneously transplanted in BALB/c nude mice / 中国肿瘤生物治疗杂志

Objective:To study the inhibitory effect of allogeneic natural killer(NK) cells on subcutaneously transplanted human multi-drug resistant nasopharyngeal carcinoma cells(CNE2/DDP) in BALB/c nude mice.Methods:Human leucocyte antigen(HLA) genotypes of CNE2/DDP cells and the genotypes of inhibitory killer cell immunoglobulin-like receptor(KIR) in NK cells(isolated from 3 healthy persons by immuno-magnetic microbead technique) were analyzed by PCR-SSP.Twelve BALB/c nude mice were evenly divided into 2 groups:the control group and the treatment group.Mice in the treatment group were injected subcutaneously with 1?106 CNE2/DDP cells together with 3?107 NK cells via the tail veins;mice in the control group were injected with 1?106 CNE2/DDP cells subcutaneously.The tumor formation time,tumor formation rate and changes of tumor size were observed.Three weeks after tumor formation,all the mice were killed and human NK cells in peripheral blood were analyzed by flow cytometry;the tumors were weighed and the tumor inhibitory rates were calculated.Results:The HLA genotypes of CNE2/DDPcells were A2,24,B18,35,Cw4,and 7;the KIR genotypes of the 3 healthy persons were KIR2DL1,KIR2DL3,KIR3DL1,and KIR3DL2.There were mismatches between the KIRs expressed in NK cells and HLA class Ⅰ molecules expressed in the CNE2/DDP cells.NK cells obviously inhibited the growth of CNE2/DDP xenograft in nude mice.The tumor formation periods of control group and NK cell group were(17.17?1.17) d and(24.83?1.47) d,respectively(P

The gene frequencies of HLA-Cw in Guangdong Han population and its significance / 中国免疫学杂志

Objective:To explore the gene frequencies of HLA-Cw and to analyze the recognition characteristic between HLA-Cw and KIR in Guangdong Han population.Methods:An auto semi-quantitative PCR-RSSO method was adopted to detect the HLA-Cw genotypes of a sample of 122 bone marrow donors.Results:The gene frequencies of HLA-CW 03,07,01,08,04,14,15,12,06,05,16 were 0.237 1 , 0.215 9 , 0.175 2 , 0.112 9 , 0.050 5 , 0.041 9 , 0.041 9 , 0.037 6 , 0.033 3 , 0.008 2 , 0.004 1 respectively.HLA-Cw 02,04,05,06 belong to “group 1” recognizing KIR-2DL1/2DS1.HLA-Cw 01,03,07,08 belong to “group 2” recognizing KIR-2DL2/2DL3;2DS2/2DS3.There was a significant difference between the two groups in total HLA-Cw gene frequencies in Guangdong Han population( P