妊娠期uNK、pNK细胞NKG2A与NKG2D的表达及其生物学意义的研究

目的:研究妊娠期妇女子宫NK细胞(uNK细胞)与外周血NK细胞(pNK细胞)表面NKG2A和NKG2D及其相应配体的表达,探讨uNK细胞表面NKG2A和NKG2D的不平衡表达与母胎界面所形成的免疫耐受关系。方法:采用流式细胞术检测30例孕6～9周的正常妊娠妇女uNK细胞和pNK细胞NKG2A、NKG2D的表达状况;RTPCR技术检测绒毛膜组织HLAE、MICA的表达。结果:子宫NK细胞NKG2A的表达显著高于外周血NK细胞,二者分别为(97.86±1.75)%与(33.35±10.92)%;子宫NK细胞NKG2D的表达水平与外周血NK细胞相近,分别为(93.21±4.52)%与(97.80±1.72)%,滋养层组织仅检测到HLAEmRNA的表达。结论:妊娠期子宫NK细胞表面高表达抑制性受体NKG2A,同时滋养层组织表达相应的配体HLAE,这可能是维持母胎界面免疫耐受的重要因素。     

卵巢肿瘤患者自然杀伤细胞活化性受体NKG2D与其配体MICA表达的初步研究

目的:研究卵巢癌、良性卵巢肿瘤患者外周血NK细胞表面活化性受体NKG2D的表达及局部组织中相应配体MICA的表达情况,并结合临床病理因素分析探讨宿主NK细胞受体NKG2D在抗卵巢癌中的作用及其与肿瘤免疫逃逸的关系。方法:对4 2例卵巢癌、2 3例良性卵巢肿瘤及2 0例正常妇女,采用流式细胞术检测外周血NK细胞NKG2D的表达状况,RT PCR技术检测在上述部分相应组织标本中MICA的表达。结果:恶性、良性卵巢肿瘤患者及正常人外周血NK细胞NKG2D的表达水平分别为( 94 2 3±6 0 2 ) %、( 98 70±0 98) %、( 98 6 1±1 5 9) % ,恶性组与另两组之间比较,差异有统计学意义(P <0 0 5 ) ;相应配体MICA在卵巢癌组织中的表达率较良性卵巢肿瘤中明显增高,差异有统计学意义(P <0 0 1) ;在卵巢癌病人是否绝经、不同组织类型、分化程度、手术分期及是否淋巴转移等各组临床病理情况下,其表达率未见明显差异(P >0 0 5 )。结论:卵巢恶性肿瘤患者外周血NK细胞活性降低,其活化性受体NKG2D表达的下降是NK细胞活性下降的原因之一。NKG2D的配体MICA的基因表达可能与卵巢癌的恶性转化有一定的相关性,卵巢癌的免疫逃逸可能与NKG2D表达下调及其配体MI CA的表达升高有关     

乏氧对人外周血NK细胞NKG2A、NKG2D及CD44表达的影响

目的： 观察乏氧微环境对人外周血自然杀伤细胞(NK)表面自然杀伤细胞2族成员A(NKG2A)、自然杀伤细胞2族成员D(NKG2D)及CD44分子表达的影响,探讨乏氧抑制NK细胞杀伤活性的分子机制。方法: 采用密度梯度离心法分离健康人外周血单个核细胞（PBMC）,贴壁去除单核细胞获得外周血淋巴细胞（PBL）,分别置常氧（21％O2）、乏氧（1％O2）以及有或无人重组白细胞介素2(rhIL-2)（1×106 U/L）刺激条件下培养16 h,流式细胞术（FCM）检测不同 NK细胞亚群 NKG2A、NKG2D以及CD44分子的表达。结果: 常氧条件,人外周血CD3-CD56+NK细胞NKG2A、NKG2D表达的阳性率分别为16.16%和78.45%,乏氧条件下二者表达的阳性率分别为15.16%和71.08%;rhIL-2上调NKG2A和NKG2D的表达,乏氧不影响 rhIL-2对NKG2D、 NKG2A的上调作用;rhIL-2显著上调NK细胞CD44的表达,乏氧抑制CD44的表达（P<0.05）。结论: 乏氧下调外周血NK细胞表面受体NKG2D及CD44的表达,但对NKG2A的表达无显著影响。由此提示,NKG2D及CD44分子可能在乏氧引起的NK细胞杀伤活性抑制中具有重要作用。     

膜型/分泌型MICA对NK细胞受体NKG2D的相反调节效应及其对NK细胞受体谱的影响

目的　观察膜型和分泌型MICA对NK细胞受体表达的影响 ,以探讨NK细胞抗肿瘤活化机制及肿瘤细胞表达MICA分子的意义。方法　用MTT法测定人NK细胞系 (NK92 )的细胞毒活性 ;用RT PCR或FACS检测NK细胞受体 (NKG2D ,NKG2A B ,KIR2DL1,KIR2DS1)及NKG2D的识别配体MICA的表达。结果　肿瘤细胞表面的MICA分子可上调NKG2D的表达 ,下调抑制性受体NKG2A B和KIR2DL1的表达 ;而分泌型MICA (sMICA)分子对NKG2D及抑制性受体的表达均有抑制作用。结论　膜型MICA分子可上调NKG2D的表达 ,激发NK细胞对肿瘤细胞的细胞毒效应 ;分泌型MICA分子则通过降低NKG2D的表达下调机体的抗肿瘤免疫效应 ,肿瘤细胞分泌sMICA分子为肿瘤发生免疫逃逸的机制之一。     

导入NKG5—SV基因增强细胞杀伤活性的研究

目的研究免疫效应蛋白NKG5的拼接异构体基因NKG5-SV导入外周血自然杀伤细胞(NK细胞)对其杀伤活性的影响.方法Percoll细胞分离液分离外周血NK细胞,流式细胞仪检测分离后CD56阳性细胞浓度.以逆转录病毒为载体将NKG5-SV基因和对照基因LacZ导入NK细胞,Northernblot检测NKG5-SV的表达,K562细胞杀伤实验检测病人NK细胞转染NKG5基因前后和正常人NK细胞的NK活性.结果Percoll分离后的淋巴细胞CD56阳性细胞为77.44%,分离前为15.88%.病人NK细胞经NKG5-SV基因转染后NKG5-SVmRNA表达增高.病人NK细胞、NK-LacZ细胞、NK-NKG5-SV细胞和正常人NK细胞对K562的杀伤活性分别为5.96%±0.38%、6.03%±0.42%(P＞0.05)、27.67%±0.18%(P＜0.01)、30.33%±0.83%(P＜0.01).结论免疫效应蛋白基因导入NK细胞,可增强NK细胞的杀伤活性.     

妊娠早期子宫微环境中蜕膜、滋养层及子宫NK细胞基因表达的研究

目的 检测妊娠早期子宫微环境中蜕膜、滋养层及子宫NK（uNK）细胞基因表达变化，为揭示妊娠中母胎免疫屏障及胚胎发育与植入等过程中的生理与病理机制提供新的研究靶点。方法 首先用免疫磁珠（MACS）技术分离及纯化uNK细胞，流式细胞仪检测其纯度，RT-PCR技术检测与uNK细胞的侵袭、促血管形成等生物学活性有关基因的转录水平，同时检测蜕膜与滋养层组织相应的配体表达状况。结果 通过MACS技术检测，所获得的NK细胞纯度较高，大于95％，RT-PCR检测发现妊娠早期uNK细胞可表达多种与侵袭和血管形成等功能有关的生物活性分子，同时在蜕膜与滋养层组织中检测到相应受体的表达。结论 妊娠早期uNK细胞是一群具有强大调节功能的NK细胞亚群，在调控滋养层细胞的侵袭、母胎界面血管重建与免疫耐受形成中可能起重要的作用。     

传染性单核细胞增多症患儿NKG2D表达变化初探

目的 观察传染性单核细胞增多症(infectious mononucleosis,IM)患儿自然杀伤(NK)细胞和CD8+T细胞NKG2D表达,探讨导致Epstein-Barr病毒(EBV)感染免疫功能紊乱的可能机制.方法 传染性单核细胞增多症患儿29例,同龄健康对照组25例.流式细胞术检测外周血NK细胞、CD8+T细胞表面激活性受体NKG2D及抑制性受体NKG2A表达,CD14+单核细胞(MC)表面NKG2D配体MHC Ⅰ相关分子A(MICA)与人巨细胞病毒蛋白UL16的结合蛋白1(ULBP-1)表达;酶联免疫吸附试验(EHSA)检测血浆游离MICA (sMICA)、IL-7、IL-12、IL-15、IFN-γ及TGF-β等细胞因子浓度.结果 (1)IM组患儿NK细胞及CD8+T细胞表面NKG2D表达明显低于对照组(P＜0.05),其中3例拟诊EBV-相关噬血细胞综合征(EBV-HLH)患儿表达下调最为显著;(2)IM组患儿CD14+ MC MICA与ULBP-1表达与对照组相比差异无统计学意义(P＞0.05);(3)IM患儿细胞因子IL-15与TGF-β较对照组降低,IL-7、IL-12、IFN-γ及sMICA较对照组升高;(4)IM患儿NK细胞NKG2A表达明显高于对照组(P＜0.05),CD8+T细胞NKG2A表达与对照组相比无明显差异(P＞0.05).结论 EBV感染患儿NK细胞、CD8+T细胞NKG2D表达过度下调可能是导致免疫功能紊乱的原因之一,IL-15及IL-12等细胞因子调控失衡,sMICA血浓度增高等多种因素可能与其NKG2D表达下调有关.     

青海高原藏族胃癌患者自然杀伤细胞活化性受体D及其配体MICA表达的研究

目的： 研究高原藏族胃癌患者外周血NK细胞表面活化性受体D(NKG2D)的表达及局部肿瘤组织和癌旁正常组织中相应配体MICA 的表达,探讨宿主NKG2D 在抗胃癌中的作用及其与肿瘤免疫逃逸的关系。方法: 对33例高原藏族胃癌患者及20 例健康人,采用流式细胞术检测外周血NKG2D 的表达状况,RT-PCR 和免疫组织化学技术检测在局部肿瘤组织和癌旁正常组织标本中MICA 的表达。结果: 高原藏族胃癌患者外周血NKG2D 的表达为(13.47 ±5.26)%，明显低于正常组（32.62±10.08)%，2组之间差异显著(P<0．05); 藏族胃癌组织MICA mRNA的表达水平显著高于癌旁正常组织（P<0.05）。胃癌组织MICA蛋白表达在癌旁正常组织中为(21.21%,7/33),胃癌组织阳性率为(78.79%，26/33),高分化组(60.00%,3/5),中分化组(72.73%,8/11),低分化组(88.24%,15/17),组间差异显著(P<0.05),而与肿瘤的大小、性别、年龄、淋巴结转移无关（P>0.05）。 Spearman相关分析显示，MICA表达水平与mRNA的表达水平显著正相关（r=0.903,P<0.01）。结论: 高原藏族胃癌的免疫逃逸可能与NKG2D 表达下调及其配体MICA 的表达升高有关，高原藏族胃癌患者外周血NK细胞活性降低,其NKG2D 表达的下降是NK细胞活性下降的原因之一。NKG2D配体MICA 的基因表达可能与高原胃癌患者的恶性转化有一定的相关性。     

白血病患者NK细胞表面NKG2D受体及其配体MICA/B的研究

目的:研究白血病患者NK细胞表面NKG2D受体及其配体MICA/B的表达,探讨白血病细胞逃逸NK细胞杀伤的机制.方法:采用流式细胞术检测NK细胞表面NKG2D受体和骨髓有核细胞表面MICA/B配体.结果:治疗前组和完全缓解组NKG2D受体的表达均较健康组低(P＜0.05);且完全缓解组低于治疗前组(P＜0.05);治疗前组和完全缓解组MICA/B配体的表达均低于增生性贫血组(P＞0.50);完全缓解组与治疗前组比较差异无显著性(P＞0.05).结论:白血病患者体内NKG2D-MICA/B介导的NK细胞功能受抑,这可能导致白血病细胞逃逸NK细胞的细胞毒作用;白血病化疗后完全缓解时其体内NKG2D-MICA/B介导的NK细胞功能仍未恢复,且较治疗前更低.     

NKG2D配体表达及其介导NK细胞抗肝细胞癌作用的研究进展

人自然杀伤细胞家族2成员D(NKG2D)配体包括主要组织相容性复合体Ⅰ类肽相关序列A(MICA)、MICB、UL16结合蛋白(ULBP),均定位于6号染色体上;由胞外结构域、跨膜区和胞质结构域三部分组成,在肝细胞癌等多种肿瘤细胞既能单独表达又可共同表达。其与NKG2D相互结合的亲和力从高到低依次为ULBP1、MICA、MICB,通过激活磷脂酰肌醇3激酶(PI3K)等信号通路,使自然杀伤(NK)细胞分泌肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)、颗粒酶增加,并使肝癌细胞Fas配体(Fas L)、肿瘤坏死因子相关凋亡诱导配体(TRAIL)表达上调,促进靶细胞溶解。诱导肝癌细胞表达MICA、MICB、ULBP1,可显著增强NK细胞对其的敏感性,多途径发挥对肝癌细胞的细胞毒性效应。     

HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells

Non-classical MHC class I molecule HLA-E is the ligand for CD94/NKG2 NK cell receptors. Surface expression of HLA-E requires binding of specific HLA class I leader sequences. The uterine mucosa in early pregnancy (decidua) is infiltrated by large numbers of NK cells, which are closely associated with placental trophoblast cells. In this study we demonstrate that trophoblast cells express HLA-E on their cell surface in addition to the previously reported expression of HLA-G and HLA-C. Furthermore, we show that the vast majority of decidual NK cells bind to HLA-E tetrameric complexes and this binding is inhibited by mAb to CD94. Thus, recognition of fetal HLA-E by decidual NK cells may play a key role in regulation of placentation. The functional consequences of decidual NK cell interaction were investigated in cytotoxicity assays using polyclonal decidual NK cells. The overall effect of CD94/NKG2 interaction with HLA-E is inhibition of cytotoxicity by decidual NK cells. However, since decidual NK cells are unable to kill trophoblast even in the presence of mAb to MHC class I molecules and NK cell receptors, HLA-E interaction with CD94/NKG2 receptors may regulate other functions besides cytolysis during implantation.     

Cell-mediated immunity imbalance in patients with intrahepatic cholestasis of pregnancy

Decidual lymphocytes may mediate fetal trophoblast recognition and regulate maternal immune reaction and play an essential role in the maintenance of normal pregnancy. The aim of this study was to compare the percentage of T cells, natural killer （NK） cells and natural killer T （NKT） cells within decidual parietalis of normal pregnant controls （NP） and patients with intraheptic cholestasis of pregnancy （ICP）, and to investigate the production of interleukin-4 （IL-4）, interferon-γ （IFN-γ） in the culture supernatant of decidual parietalis mononuclear cells （DPMCs）. Compared with controls, the decidua parietalis from ICP were characterized with significant increased percentages of CD3^＋CD56^＋ cells, CD3^＋CD56^＋ cells, CD56^＋CD16＋ cells, CD56^＋CD16^＋ cells, CD56^＋NKG2D＋ ceils, and the significant decreased percentages of CD3^＋ cells, CD3^＋CD4^＋ cells. There were no differences found for the percentage of CD3^＋CD8^＋ cells, CD56^＋NKG2A^＋ cells between control and study group. In addition, the enhanced concentration of IFN-γ was presented in culture supernatant of DPMCs from ICP. It was suggested that the increased NK cells, NKT cells and the decreased T cells in the decidual parietalis and over-secretion of IFN-γ could be correlated with the pathophysiology of ICP patients. Cellular ＆ Molecular Immunology. 2007;4（1）：71-75.     

Clinical Significance of the HLA-E and CD94/NKG2 Interaction

HLA-E belongs to the non-classical HLA (class Ib family) broadly defined by a limited polymorphism and a restricted pattern of cellular expression. So far, only two functional alleles differing at only one amino acid position (non-synonymous mutation) in the α2 heavy chain domain, where an arginine in position 107 in HLA-E*0101 is replaced by a glycine in HLA-E*0103, have been reported. The interaction between non-classical HLA-E molecule and CD94/NKG2A receptor plays a crucial role in the immunological response involving natural killer (NK) cells and cytotoxic T lymphocytes. All proteins forming CD94/NKG2 receptors are encoded by genes situated in the same cluster on chromosome 12, allowing tight control over the order of their expression. The inhibitory members of the NKG2 receptor family are available on the cell surface before activating the members to prevent autoimmune incidents during immune cells’ ontogenesis. In the present review, the potential role of this interaction in viral infection, pregnancy and transplantation of allogeneic hematopoietic stem cells (HSC) is presented and discussed. The review will also include the effect of HLA-E polymorphism on the outcome of HSC transplants in humans.     

脂多糖对蜕膜NK细胞表达NKG2D水平的促进作用

为探讨脂多糖(LPS)及其受体TLR4相互作用并影响蜕膜NK细胞的可能机制,在孕E6.5给BALB/c×C57BL/6孕鼠腹腔注射LPS,而E10.5采用流式细胞术检测小鼠蜕膜NKG2D+TGF-β-NK细胞构成比,计算小鼠胚胎吸收率。此外,采用磁珠亲和细胞分选术纯化E10.5小鼠蜕膜NK细胞并培养,用LPS刺激所培养的细胞,并观察刺激后NK细胞表达NKG2D水平的变化。研究发现,在体内和体外实验中LPS刺激均可显著增高BALB/c×C57BL/6孕鼠蜕膜NK细胞NKG2D的表达水平,其中腹腔注射可显著增高胚胎吸收率。在体外细胞培养实验中,预先在培养基中加入抗TLR4抗体,则LPS刺激后细胞NKG2D表达水平无显著升高。这些结果提示,LPS与TLR4相互作用可增强小鼠蜕膜NK细胞NKG2D的表达,而NKG2D的过高表达不利于同种异基因妊娠成功。     

HLA-E regulates NKG2C+ natural killer cell function through presentation of a restricted peptide repertoire

NK cells interact with the HLA-E molecule via the inhibitory receptor NKG2A and the activating receptor NKG2C. Hence, HLA-E can have a dual role in the immune response. In the present study, we aim to investigate the functional consequences of HLA-E for NKG2A and NKG2C expressing NK cell subsets by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. PBMC derived from healthy subjects were used as targets for isolated NK cells and NK cell activation was examined by analysis of the expression of the degranulation marker CD107a. Peptide induced HLA-E expression inhibited degranulation of NKG2A+ NK cell subsets with almost all peptides, whereas NKG2A− NKG2C+ NK cell responses were enhanced only after incubation with four peptides; 1.3-fold with CMV(I), A80 and B13 and 3.2-fold with HLA-G derived peptide. In addition, the HLA-E:G peptide complex triggered NKG2C receptor internalization, as evidenced by reduction in the percentage of NKG2C+ NK cells when incubated with the peptide, which could be restored by addition of Bafilomycin. In conclusion: in contrast to NKG2A, NKG2C is regulated by HLA-E only when HLA-E is in complex with a restricted peptide repertoire, especially in combination with the HLA-G leader peptide.     

First trimester pregnancy decidual natural killer cells contain and spontaneously release high quantities of granulysin

Citation Veljkovic Vujaklija D, Gulic T, Sucic S, Nagata K, Ogawa K, Laskarin G, Saito S, Haller H, Rukavina D. First trimester pregnancy decidual natural killer cells contain and spontaneously release high quantities of granulysin. Am J Reprod Immunol 2011; 66: 363–372 Problem Granulysin (GNLY) is a novel cytolytic protein lytic against a variety of tumor cells and microbes. The role of GNLY during pregnancy has not been extensively explored. The aim of this study is to examine GNLY expression and distribution in the first trimester pregnancy peripheral blood (PB) and decidua, the ability of decidual and PB natural killer (NK) cells to secrete GNLY spontaneously, and the role of antigen‐presenting cells (APC) in the regulation of GNLY expression in decidual NK cells. Method of study GNLY expression was analyzed using cell permeabilization method, flow cytometry, and immunohistochemistry. GNLY secretion by purified NK cells was detected by ELISA method. Results GNLY is abundantly expressed at the maternal–fetal interface in the first trimester pregnancy. Decidual T lymphocytes express significantly higher levels of GNLY (58%) then PB T lymphocytes (11%). Over 85% of decidual CD56⁺ cells express GNLY and when cultured spontaneously release high quantities of GNLY. Decidual APC participate in the control of GNLY expression in CD56⁺ cells. Conclusion Abundant expression of GNLY in the decidual immunocompetent cells and the capacity of decidual CD56⁺ cells to spontaneously secrete high quantities of GNLY point to important protective and immunomodulatory role that this molecule could play at the maternal–fetal interface.     

CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients

Natural killer (NK) cells are affected by infection with human cytomegalovirus (HCMV) manifested by increased expression of the HLA-E binding activating receptor NKG2C. We here show that HCMV seropositivity was associated with a profound expansion of NKG2C(+) CD56(dim) NK cells in patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Multi-color flow cytometry revealed that the expanded NKG2C(+) CD56(dim) NK cells displayed a highly differentiated phenotype, expressed high amounts of granzyme B and exhibited polyfunctional responses (CD107a, IFN-γ, and TNF-α) to stimulation with antibody-coated as well as HLA-E expressing target cells but not when stimulated with IL-12/IL-18. More importantly, NKG2C(+) CD56(dim) NK cells had a clonal expression pattern of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self-HLA class I molecules, with predominant usage of KIR2DL2/3. KIR engagement dampened NKG2C-mediated activation suggesting that such biased expression of self-specific KIRs may preserve self-tolerance and limit immune-pathology during viral infection. Together, these findings shed new light on how the human NK-cell compartment adjusts to HCMV infection resulting in clonal expansion and differentiation of educated and polyfunctional NK cells.     

Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirus

The human NKG2D killer lectin-like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3-kinase (PI3 K) and specifically interacts with different stress-inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus-infected cells. This KLR is commonly expressed by human NK cells as well as TCRgammadelta(+) and TCRalphabeta(+)CD8(+) T lymphocytes, but it has been also detected in CD4(+) T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)-specific CD4(+) T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4(+)NKG2D(+) T lymphocytes that coexpressed perforin. NKG2D was detected in CD28(-) and CD28(dull )subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR-dependent activation of CD4(+) T cells, triggering proliferation and cytokine production (i.e. IFN-gamma and TNF-alpha). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV-specific CD4(+) T lymphocytes and thus may have a role in the response against infected HLA class II(+) cells displaying NKG2D ligands.