HLA-G3蛋白能够抑制多克隆NK细胞的杀伤功能

目的 研究HLA－G3蛋白对NK细胞杀伤功能的抑制作用。方法 用RT－PCR方法扩增HLA－G3和HLA－G1的cDNA，并将其克隆到真核表达载体pcDNA3中，采用脂质体转染法将其转入K562细胞，用免疫荧光法确定其在细胞表面表达，细胞毒试验研究其对NK细胞杀伤功能的抑制作用。结果 HLA－G3蛋白和HLA－G1表达在细胞表面，以PBL细胞为效应细胞的细胞毒实验表明K562－G1细胞和K562－G3细胞的细胞裂解率都比K562细胞裂解率低。结论 HLA－G3蛋白能够抑制多克隆NK细胞的杀伤功能。     

Can HLA-G polymorphisms predict the development of cardiac allograft vasculopathy?

A 14 bp insertion/deletion polymorphism in exon 8 of the HLA-G gene is associated with mRNA stability and HLA-G expression. In cardiac transplantation, the 14 bp deletion polymorphism plays an important role in the expression of HLA-G and is associated with fewer episodes of cellular rejection. We investigated the association between the 14 bp insertion/deletion HLA-G polymorphism and cardiac allograft vasculopathy (CAV) post heart transplantation. There were no statistically significant differences in the presence of the three HLA-G genotypes (−14 bp/−14 bp, +14 bp/−14 bp, +14 bp/+14 bp) between patients without CAV and patients with CAV at 1 year (p = 0.61) or 5 years (p = 0.76) post-transplant. We found no correlation between HLA-G genotypes and CAV progression from baseline to 5 years post-transplant (p = 0.55). HLA-G polymorphism appears to play an important role as a genetic indicator for cellular rejection post-transplant; however, it is not a reliable marker to identify patients at risk of CAV.     

A combination of single nucleotide polymorphisms in the 3′untranslated region of HLA-G is associated with preeclampsia

Reduced expression of human leukocyte antigen-G (HLA-G) has been linked to onset of preeclampsia. Associations have also been reported between preeclampsia and single nucleotide polymorphisms (SNP) in the 3′-untranslated region (UTR) of the HLA-G gene. However, there are conflicting results between studies. This studied examined whether a SNP, by itself or in combination with other SNPs, in the 3′UTR of the HLA-G gene is associated with an increased risk of preeclampsia. Placenta samples were obtained from 47 preeclamptic and 68 control cases. DNA was extracted, and the 3′UTR was sequenced and analyzed for nine polymorphisms using different genetic models of inheritance. Four of these polymorphisms have never been analyzed for an association with preeclampsia. Disputing existing reports, preeclamptic cases were suggestively associated with a G/G-genotype at SNP +3187 (p < 0.05). Several SNP combinations were more prevalent in preeclampsia cases. Following corrections for multiple testing, one SNP combination (+3027C/C and +3187G/G) was significantly more prevalent in preeclampsia cases using co-dominant, additive, and dominant models (p < 0.001). Taken together with the current literature, the data suggests that HLA-G 3′UTR SNP-pair associations, and not individual SNPs, could be useful in a predictive test for the susceptibility to preeclampsia.     

Meta-analysis of the relationship between 14bp insertion/deletion polymorphism of HLA-G gene and susceptibility to systemic lupus erythematosus

We performed a meta-analysis to examine the relationship between the human leukocyte antigen-G (HLA-G) 14 base pairs sequence (14bp) insertion (ins)/deletion (del) polymorphism to systemic lupus erythematosus (SLE). Eligible studies were extracted in PubMed, Embase, Cochrane Library and CNKI (Chinese) up to March 31, 2014. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were estimated to evaluate the strength of the association. Finally, 7 studies with 1864 cases and 2259 controls were involved in this meta-analysis. Overall, the HLA-G 14bp ins/del polymorphism was significantly associated with SLE susceptibility (ins vs. del: OR = 1.179, 95%CI = 1.037–1.341, P = 0.012; ins/ins vs. del/del: OR = 1.394, 95%CI = 1.153–1.684, P = 0.001; ins/del vs. del/del: OR = 1.199, 95%CI = 1.041–1.382, P = 0.012; ins/ins + ins/del vs. del/del: OR = 1.252, 95%CI = 1.097–1.430, P = 0.001). When stratified by ethnicity, significance was found in Asians (ins/ins vs. del/del: OR = 1.326, 95%CI = 1.001–1.756, P = 0.049) and Caucasians (ins/ins vs. del/del: OR = 1.454, 95%CI = 1.126–1.878, P = 0.004; ins/del vs. del/del: OR = 1.288, 95%CI = 1.051–1.579, P = 0.015; ins/ins + ins/del vs. del/del: OR = 1.340, 95%CI = 1.106–1.623, P = 0.003). Our results suggest that the HLA-G 14bp insertion allele might act as an increased risk against SLE. Besides, this is the first meta-analysis to report an association between the HLA-G 14bp ins/del polymorphism and SLE. Larger and well-designed studies are needed to further confirm these findings.     

Association between human leukocyte antigen-G genotype and success of in vitro fertilization and pregnancy outcome

To determine if a 14-bp deletion/insertion polymorphism in the 3'-untranslated region of exon 8 of the gene encoding human leukocyte antigen (HLA)-G in a homozygous form is associated with repeated, unsuccessful in vitro fertilization (IVF) treatments, and with increased risk of recurrent spontaneous abortions (RSA), 29 white women undergoing IVF treatments, 61 RSA women and 93 fertile controls were HLA-G genotype. The HLA-G genotype, homozygous for the presence of the 14 bp sequence in exon 8, was significantly associated with reduced fertility with respect to unsuccessful IVF treatments and increased risk of recurrent miscarriage (combined P < 0.01). The 14-bp insertion/deletion polymorphism is associated with differences in HLA-G mRNA alternative splicing and levels of HLA-G. This might affect a possible immunomodulatory role of HLA-G expression in both the mother and foetus during implantation and pregnancy.     

Association of HLA-G alleles and 3' UTR 14 bp haplotypes with recurrent miscarriage in Brazilian couples

Human leukocyte antigen (HLA)-G expression is restricted, expressed on trophoblast, with a major role in fetus acceptance. In addition to the 46 HLA-G alleles, the presence or absence of a 14 bp polymorphism located in the 3' UTR contributes to gene polymorphism that may influence both HLA-G mRNA stability and HLA-G isoform's splicing and consequently could play an immunomodulatory function in pregnancy. To elucidate the role of HLA-G polymorphism in pregnancy, HLA-G allele frequencies and the 14 bp polymorphisms were analyzed and compared in 60 couples with recurrent miscarriage (RM) and 68 fertile control couples. Two haplotypes showed a significant elevated frequency in patients (HLA-G*01:01:08/+14, p(c) < 0.0001 and HLA-G*01:04:01/-14, p(c) < 0.0001). The haplotype HLA-G*01:01:A/+14 exhibited a significant protective effect against RM in women (p(c) = 0.0238). Remarkably, significant differences in linkage disequilibrium were observed between patient and control groups. Two alleles showed a positive association with the +14 bp segment in RM patients and a strong negative association with fertile controls (HLA-G*01:01:08 = patients D' = 0.295-0.371; controls D' = -0.715 to -1.000; HLAG* 01:05N = patients D' = 0.728-1.000; controls D' = -1.000). HLA-G*01:04:01 showed a negative association with the 14 bp segment in patients and a positive association in controls (patients D' = -0.249 to - 0.674; controlss D' = 0.688-1.000). Our results suggest that haplotypic combinations of HLA-G alleles and the 14 bp segment may be associated with RM.     

HLA-G mRNA差异表达对细胞膜表面HLA-G异构体分子表达的影响

目的 探讨HLA-G异构体(HLA-G1～6)mRNA差异表达对HLA-G分子在细胞表面表达的影响.方法 通过RT-PCR方法分析卵巢癌细胞株HO-8910、H0-8910PM、OVCAR-3,白血病细胞株Jurkat、K562、HL60、MUTZ-1,绒癌细胞株JEG-3、JAR内HLA-G异构体mRNA的表达种类,采用流式细胞术分析上述细胞株细胞表面及细胞内HLA-G分子的分布及表达水平.结果 阳性对照JEG-3细胞内表达HLA-G1～6 mRNA,阴性对照JAR不表达HLA-G1～6 mRNA.HLA-G1 mRNA在HO-8910、HO-8910PM、OVCAR-3、MUTZ-1、Jurkat细胞内表达.除阳性对照JEG-3外,其他细胞均不表达HLA-G2 mRNA;表达HLA-G3 mRNA的细胞有HO-8910、HO-8910PM、K562、HL60、MUTZ-1、OVCAR-3、Jurkat;表达HLA-G4 mRNA的细胞有HO-8910、HO-8910PM、HL60、Jurkat;表达HLA-G5mRNA的细胞为Jurkat.FACS分析显示在JEG-3、HO-8910PM、Jurkat细胞膜表面表达HLA-G分子,而除JAR细胞外,其他细胞内均表达HLA-G分子.结论 HLA-G2、-G3、-G4、-G5、-G6均不能在细胞表面表达,而HIJA-G1分子则能在特定的细胞表面表达.     

Association of the maternal 14-bp insertion polymorphism in the HLA-G gene in women with recurrent spontaneous abortions

Human leukocyte antigen (HLA)-G has been postulated as an important immunotolerant molecule in maintaining fetal-maternal relationship. Recent reports indicated that the 14-bp deletion/insertion polymorphism in exon 8 of HLA-G gene influences HLA-G mRNA stability and isoform splicing patterns, thus modulating the levels of HLA-G expression. This might play an immunomodulatory role of HLA-G during implantation and pregnancy. In the present study, 109 unrelated fertile control women and 79 women who had experienced recurrent spontaneous abortion (RSA) were genotyped for the 14-bp insertion/deletion polymorphism. No significant difference was observed in the distribution of 14-bp insertion/deletion genotype between controls and the RSA group. However, a greater number of 14-bp insertion alleles exist in the RSA group than in the controls.     

The HLA-G low expressor genotype is associated with protection against bipolar disorder

Implication of immune processes in bipolar disorder (BD) has recently gained increasing attention. Tolerogenic molecules, among which HLA-G plays a prominent role, mediate the modulation of such processes. The HLA-G locus is characterized by a high number of polymorphisms including a functionally relevant 14 base pair (bp) insertion/deletion (Ins/Del) allele affecting the HLA-G expression. Here, we analyzed the distribution of this polymorphism in 561 BD patients and 161 healthy and found that the HLA-G 14bp Ins/Ins genotype was significantly more prevalent in healthy controls than in patients (corrected p; pc = 0.032) and that the prevalence of such protective genotype is lower among patients born during the winter season as compared to those born in other periods (pc = 0.006). Possible mechanisms between low HLA G expression and resistance to infections as well as potential relationships between infections in early life and susceptibility to BD are discussed.     

膜结合型HLA-G1～G4分子的克隆表达及对NK细胞杀伤功能的影响

目的 探讨膜结合型HLA-G1～G4异构体的表达及对NK细胞杀伤功能的影响.方法 通过基因克隆及转染,分别建立稳定表达HLA-G1～G4抗原的人绒癌JAR细胞株.采用RTPCR、流式细胞术、Western blot及免疫细胞化学法分析、鉴定转染细胞中HLA-G的mRNA及蛋白表达.通过加载HLA-G高亲和性KIPAQFYIL抗原肽,观察对HLA-G表达的影响.LDH释放法检测HLA-G1～G4表达对NK细胞杀伤活性的影响.结果 RT-PCR、Western blot及免疫细胞化学结果显示,HLA-G1～G4/pVITRO2-mcs重组质粒成功转染HLA-G表达阴性的人绒癌JAR细胞株.FACS分析显示HLA-G1抗原能在JAR-HLA-G1细胞株表面表达,HLA-G2～G4抗原不能有效到达细胞表面.体外杀伤试验发现表达HLA-G1～G4抗原的细胞均能抑制NK细胞的杀伤活性(P＜0.05);加载HLA-G高亲和性KIPAQFYIL抗原肽对HLA-G表达无明显影响,对NK细胞杀伤抑制程度也未见明显改变.结论 HLA-G1～G4能够明显抑制NK细胞的杀伤活性,提示不同膜结合型HLA-G异构体分子均能作为免疫耐受分子,具备免疫调节功能.     

HLA-G非编码区单核苷酸多态性在复发性流产患者中的表达及对影响多因素Logistic分析研究

目的探讨HLA-G非编码区单核苷酸多态性在复发性流产患者中的表达及对影响多因素Logistic分析研究。方法选择2016年6月-2018年12月我院正常妊娠且无流产史患者作为对照组,选择期间复发性流产患者(URSA)作为观察组其中对照组30例,观察组20例。采用聚合酶链反应(PCR)检测HLA-G非编码区14bp基因多态性和逆转录-聚合酶链反应(RT-PCR),比较两组人群绒毛组织HLA-G 14bp插入/缺失基因型及绒毛组织中HLA-G mRNA的相对表达量及对多因素Logistic分析的影响。结果观察组-14bp/+14bp的杂合子基因频率为65.00%,对照组-14bp/+14bp的杂合子基因频率为43.33%,观察组高于对照组,差异有统计学意义(P<0.05);插入纯合子基因频率为+14bp/+14bp为10%。对照组基因频率为26.67%,与对照组比较有统计学意义(P<0.05);观察组HLA-G mRNA水平显着高于对照组(t=9.658,P=0.000)(P<0.05);HLA-G 蛋白在观察组里呈强表达,在对照组里呈弱表达,差异有统计学意义(P<0.05);HLA-G非编码区单核苷酸多态性也是导致患者复发性流产的一个重要因素,对于复发性流产多因素Logistic分析研究提供一个新的研究方向。结论 HLA-G非编码区-14bp/+14bp的杂合子基因大大增加了复发性流产患者的发病几率,同时复发性流产影响HLA-G mRNA及HLA-G 蛋白的正常表达。对复发性流产多因素Logistic分析提供一个新的研究方向。     

NK cytolysis is dependent on the proportion of HLA-G expression

The suppressive functions of HLA-G to various immune cells have been well established. The proportion of HLA-G expression in malignant lesion cells was found from negative to 100%. However, effects for the different proportion of HLA-G expression on the cytolysis of NK cells remain to be explored. In this study, NK cytolysis to the various proportion of HLA-G1 expression on leukemia cell line K562 was investigated. Analysis of NK cell cytotoxicity was by detecting the NK cell surface CD107a expression. Data showed that NK cell cytolysis could be inhibited by the HLA-G1 expression and in a manner of HLA-G1 expression proportion dependent manner (r = 0.925, p = 0.008). Our study provided further understanding for the roles of HLA-G1 expression in malignant cell immune escaping from NK cells.     

Association of 14-bp insertion/deletion polymorphism of HLA-G gene with idiopathic recurrent miscarriages in infertility center patients in Yazd,Iran

HLA-G is supposed to play a pivotal role in tolerance of the semi-allogeneic graft in pregnancy by inhibiting the cytotoxic functions of T and NK cells. A 14-bp insertion and/or deletion polymorphism in exon-8 has a possible role in HLA-G expression. The present study analyzed the 14-bp insertion/deletion polymorphism in normal pregnancy and recurrent miscarriage patients in order to discover a possible correlation between the 14-bp polymorphism and recurrent miscarriage (RM). In this study, genomic DNA from 200 RM patients and 200 normal fertile control individuals using the routine salting out method were isolated. Exon-8 of HLA-G gene of the two groups were amplified using polymerase chain reaction and analyzed by electrophoresis on 10% non-denaturing polyacrylamide gel electrophoresis containing ethidium bromide and visualized under ultraviolet light. HLA-G allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test. The results showed that there was a difference in allelic frequencies of 14-bp insertion polymorphism between fertile controls and RM patients; the frequency of +14 bp/?14?bp heterozygotes was significantly higher in RM patients as compared with fertile controls. Furthermore, the frequency of +14-bp insertion allele was significantly higher in those with RM as compared with normal fertile controls. From the findings here, it was concluded that a 14-bp insertion/deletion polymorphism in exon 8 could play a possible role in recurrent miscarriages. These results might ultimately be of significance for clinicians and those involved in understanding infertility and RM.     

Analysis of HLA-G gene polymorphism and protein expression in invasive breast ductal carcinoma

The human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule predominantly expressed in trophoblastic placental cells to protect the fetus during pregnancy. However, evidence has shown that this molecule may be implicated in the immune escape mechanism of tumor cells. Thus, the aim of this study was to evaluate the frequency of 14-bp insertion/deletion HLA-G polymorphism, as well as the expression of this molecule in patients with invasive breast ductal carcinoma (IDC). A significant association between the expression of HLA-G and the presence of metastasis in lymph nodes (p = 0.01) was observed and the expression of HLA-G was significantly higher in patients with shorter survival time (p = 0.03). The analysis suggests that the polymorphism observed in patients with IDC may be inducing a higher expression of the HLA-G molecule, which may possibly contribute to shorter survival time and a worse clinical prognosis for such patients.     

Human leukocyte antigen (HLA)-G during pregnancy part II: Associations between maternal and fetal HLA-G genotypes and soluble HLA-G

Human leukocyte antigen (HLA)-G is a class Ib molecule with restricted tissue distribution expressed on the extra-villous trophoblast and seems to have immunomodulatory functions during pregnancy. Studies have linked HLA-G polymorphisms to pregnancy complications such as preeclampsia and recurrent miscarriage. Levels of soluble HLA-G (sHLA-G) in blood plasma from non-pregnant donors seem to be associated with these polymorphisms. In the current study, we have genotyped 246 mothers and their offspring for HLA-G polymorphisms in the 3′-untranslated region (3′UTR) and measured sHLA-G in maternal blood plasma samples from gestational week 20 and at term, as well as in fetal umbilical cord blood samples. This is the first large study simultaneously performing HLA-G genotyping of mother and offspring and measuring sHLA-G in both maternal and umbilical cord blood. The results showed that increasing numbers of 14 bp ins (rs66554220) alleles in the mother–child genotype combinations were associated with higher maternal sHLA-G levels at term when restricting the analysis to 14 bp ins/del heterozygous mothers (p = 0.015). Furthermore, increasing numbers of 14InsG haplotypes (14 bp ins/del and +3142C/G (rs1063320) polymorphism) in mother–child genotype combinations were associated with higher levels of sHLA-G at term in heterozygous 14DelC/14InsG mothers (p = 0.005). In conclusion, the results indicate that there is an association between combined feto-maternal HLA-G genotypes and sHLA-G levels in maternal blood plasma.     

HLA-G polymorphism and in vitro fertilization failure in a Polish population

To investigate whether human leukocyte antigen-G ( HLA-G ) gene polymorphism is associated with in vitro fertilization (IVF) failure, we sequenced exons 2–4 of the HLA-G gene in 50 couples with three or more IVFs (including 10 couples with five or more IVFs) and 58 control fertile couples from a Polish population. Of the 10 different HLA-G alleles identified in our study subjects, neither allele was found to be associated with IVF. We also genotyped 50 couples with IVF and 71 control couples for the −725C>G variant in the promoter region and the 14 bp insertion or deletion polymorphism in the 3' untranslated region of the HLA-G gene. The frequency of −725GG or GC genotype in women with IVF and in control fertile women was similar [26% vs 25.3%; odds ratio (OR) = 1.0; P  = 1.0]. The 14 bp ins/ins or ins/del genotype was more common in women with IVF than in control women (76.9% vs 59.1%; OR 2.4; P  = 0.03), but the difference was not significant after Bonferroni correction for multiple comparisons. The frequency of the ins/ins or ins/del genotype was particularly high (90%) in women who experienced five or more IVFs (OR = 6.2; P  = 0.08), but again, the excess was not statistically significant, possibly because of small sample sizes. These results are in line with functional studies that show lower levels of HLA-G mRNA and protein related to the HLA-G allele including the 14 bp sequence and suggest that the insertion allele may be associated with an increased risk of IVF.     

β2-Microglobulin-free HLA-G activates natural killer cells by increasing cytotoxicity and proinflammatory cytokine production

Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class-I molecule and plays a role in tissue specific immunoregulation. Many studies have addressed functional aspects of β2-microglobulin (β2m)-associated HLA-G1. β2m-free HLA-G has been found in human placental cytotrophoblasts and pancreatic β cells although its function remains unclear. In the present study, we investigated the function of β2m-free HLA-G by transfecting HLA-G1 and -G3 into human β2m deficient rat pancreatic β cell carcinoma (BRIN-BD11) cells. RT-PCR and western blots studies confirmed high expression of HLA-G1 and -G3 in -G1 and -G3 transfectants, respectively. HLA-G1 and -G3 were detected mainly in intracellular compartments of BRIN-BD11 transductants by confocal fluorescent microscopy and flow cytometry. Functional analysis revealed that β2m-free HLA-G promoted xenogeneic cytotoxic lysis of BRIN-BD11 cells by natural killer (NK) cells and increased production of IL-1β, TNF-α, and IFN-γ. Stimulation of cytotoxic lysis was impaired by blocking the MAPK and DNA-PKcs pathways in NK cells. Importantly, treatment with 33mAb, a KLR2DL4 receptor agonist, induced NK-mediated cytotoxic lysis of BRIN-BD11 cells transfected with a mock vector. Our data suggest that β2m-free HLA-G activates NK cells via engagement of KLR2DL4 receptors.     

14 bp deletion polymorphism in the HLA-G gene is a risk factor for idiopathic dilated cardiomyopathy in a Chinese Han population

Human leukocyte antigen (HLA) has been reported to be associated with the pathogenesis of autoimmune-associated idiopathic dilated cardiomyopathy (IDC). However, the HLA-G in this context is limited. In the current study, a total of 117 IDC patients and age and sex matched 401 unrelated healthy controls in a Chinese Han population were HLA-G genotyped for the 14 bp insertion and deletion polymorphism. IDC patients showed markedly increased frequencies of -14 bp/-14 bp genotype [Pc = 0.00049, odds ratio (OR) = 2.17] and -14 bp alleles (Pc = 4.1 x 10(-5), OR = 1.97) when compared with healthy controls. Whereas the frequencies of +14 bp/+14 bp genotype (Pc = 0.0036, OR = 0.35) and +14 bp alleles (Pc = 4.1 x 10(-5), OR = 0.51) were significantly lower in IDC. These data, for the first time, indicated that 14 bp insertion/deletion polymorphism in HLA-G gene could be a genetic risk factor for the susceptibility to IDC.     

HLA-G haplotype structure shows good conservation between different populations and good correlation with high,normal and low soluble HLA-G expression

HLA-G molecule has considerable impact in various clinical fields, therefore many studies attempted to predict its expression based on HLA-G genotype. These studies have focused on polymorphisms in either the coding region or in one of the two untranslated regions (UTR) of the gene. The aim of our study was to determine if HLA-G haplotype defined based on SNPs 5′ and 3′UTR could be used to predict soluble HLA-G expression in unstimulated individuals. Our findings showed that HLA-G haplotype structure was well conserved between distant populations and that the defined haplotypes were correlated with high, normal and low HLA-G soluble secretors. In conclusion, we showed that this genotyping strategy based on the use of a few selected SNPs rather than isolated SNP analysis allows reliable HLA-G expression in all populations. This strategy could be useful in a number of clinical settings, e.g., predicting graft compatibility immunogenetic laboratories.