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     

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。     

结核杆菌耐热抗原对人外周血淋巴细胞NKG2A/NKG2D的影响

目的：以结核杆菌耐热抗原为刺激剂，观察人外岗血淋巴细胞NKG2A受体和NKG2D受体表达量的变化情况。方法：用结核杆菌耐热抗原刺激人外周血淋巴细胞，用流式细胞技术检测NKG2A受体和NKG2D受体的变化。使用RT-PCR和ELISA检测PBMCs中IFN-γ的表达情况。结果：在IL-2和抗原联合刺激下，NKG2A受体的表达量在第12天大幅度上升，NKG2D受体的表达量始终变化不大。NKG2A受体的表达在T细胞、NK细胞、αβT细胞和γδT细胞表面都有不同程度的增加。结论：NKG2A/NKG2D比例的上升有可能会对免疫系统造成一些影响，其中IFN-γ起着不可忽视的作用。     

HIV原发感染者自然杀伤样T细胞NKG2A/NKG2D变化的研究

目的深入了解人类免疫缺陷病毒(human immunodeficiency virus,HIV)原发感染者(primary HIV infection,PHI)NKT样细胞表面NKG2A/NKG2D受体表达的变化。方法选取25例未经高效抗逆转录病毒治疗的HIV原发感染者和27例HIV抗体阴性健康对照,用流式细胞仪检测研究对象外周血NKT样细胞表面NKG2D和NKG2A的表达。结果 HIV原发感染者NKT样细胞绝对数和百分率显著低于健康对照(P<0.01)。HIV原发感染者NKT样细胞表面NKG2A、NKG2D受体表达与健康对照并无显著差异。HIV原发感染者病毒调定点低组NKG2A+NKT样细胞、NKG2A+NKG2D-NKT样细胞以及NKG2A+NKG2D+NKT样细胞百分率均显著低于病毒调定点高组(P<0.05);NKT细胞绝对数和百分率、NKG2D+NKT样细胞、NKG2D+NKG2A-NKT样细胞百分率在两组间相似,没有显著性差异。NKG2A+NKT细胞的百分比与病毒载量正相关(R=0.430,P=0.032)。结论 NKT样细胞数量以及其表面NKG2A受体的表达可作为HIV疾病进程的预测指标之一。     

NKG2A在银屑病患者外周血NK细胞中的表达及调控

目的检测寻常型银屑病患者外周血中NK细胞受体NKG2A的表达及其调控,并分析其与疾病进展的相关性。方法采用流式细胞术检测寻常型银屑病患者外周血中NK细胞表面受体NKG2A的表达水平,并探讨血浆中炎症因子对NKG2A表达的影响,最后分析其与患者PASI评分的相关性。结果与正常对照外周血相比,寻常型银屑病患者外周血中NK细胞的比例无显著统计学改变(P0.05),但NK细胞表达的NKG2A水平则显著下降(P0.05);进一步体外刺激实验发现患者血浆中的TNF-α可诱导NK细胞下调表达NKG2A;相关性分析显示NKG2A的表达水平与患者的PASI评分成负相关(P0.05)。结论寻常型银屑病患者外周血中NK细胞低表达抑制型受体NKG2A并与疾病的进展密切相关,监测其外周血NK细胞表达的NKG2A水平有助于对病程的判断。     

抗NKG2D多克隆抗体抑制NK和LAK细胞细胞毒效应的研究

目的 :分析抗NKG2D多克隆抗体 ( pAb)对NK和LAK细胞毒作用的影响。方法 :应用密度梯度离心法分离外周血单个核细胞 (PBMC) ,经 10mg/LPHA和 1× 10 6U/LrhIL 2诱导LAK细胞产生 ,再应用流式细胞术 (FCM)分选NK细胞并进行表型检测。加入抗NKG2DpAb封闭NK和LAK细胞表面的NKG2D分子后 ,用MTT比色法检测其细胞毒效应。结果 :经FCM分析证实 ,获得高纯度、高活性的NK细胞。抗NKG2DpAb能显著抑制NK和LAK细胞对K5 6 2、HepG2细胞的细胞毒效应。NK细胞对两种靶细胞的细胞毒效应分别下降了 82 .9%和 75 .6 % ;LAK细胞对两种靶细胞的细胞毒效应分别下降了 5 2 .8%和 5 0 .2 %。但抗NKG2DpAb不能显著抑制两种效应细胞对人鼻咽癌细胞系CNE的细胞毒效应。结论 :抗NKG2DpAb可通过封闭NK和LAK细胞表面的NKG2D分子 ,抑制其对肿瘤细胞的细胞毒效应     

微小RNA-384靶向NK细胞表面受体NKG2A及STAT3蛋白对胃癌细胞生物学行为的作用机制北大核心CSCD

目的:探究微小RNA-384靶向NK细胞表面受体NKG2A及STAT3表达对胃癌细胞生物学行为的作用机制。方法:选取滕州市中心人民医院2017年4月至2020年3月已确诊的46例胃癌患者癌组织与癌旁组织标本进行研究。胃癌细胞分为胃癌组(胃癌细胞组)、对照组(转染NC组)、转染组(转染miRNA-384组)。PT-PCR检测胃癌细胞miR-384、NKG2A mRNA水平。MTT检测细胞增殖。流式细胞仪检测细胞凋亡。Western blot检测STAT3、Survivin蛋白水平。双荧光酶报告检测miR-384、NKG2A靶向关系。结果:与癌旁组织相比,胃癌组织中miR-384表达降低、NKG2A表达升高(P<0.05)。与转染miR-384-NC组相比,转染miR-384组NK细胞增殖率随着时间延长而升高,CD16+、CD56+阳性率显著升高(P<0.05),提示转染miR-384后,可促进NK细胞增殖。胃癌组与对照组相比各指标差异均无统计学意义(P>0.05)。与对照组相比,转染组miR-384、凋亡率升高,NKG2A表达、增殖率、STAT3、Survivin表达降低(P<0.05)。NKG2A与STAT3表达呈正相关(r=0.327,P=0.003);NKG2A与Survivin表达呈正相关(r=0.415,P<0.001);STAT3与Survivin呈正相关(r=0.351,P=0.001)。转染miR-384后野生型胃癌细胞中NKG2A活性及STAT3表达降低(P<0.05),突变型胃癌细胞中NKG2A及STAT3表达无明显变化(P>0.05),表明NKG2A及STAT3是miR-384的靶基因。结论:miR-384通过靶向NK细胞表面受体NKG2A及STAT3表达,降低Survivin活性,促进胃癌细胞凋亡并抑制其增殖。     

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。     

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.     

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。     

Tumor-derived soluble MICs impair CD3(+)CD56(+) NKT-like cell cytotoxicity in cancer patients

Upon ligation with its ligands, the activating receptor NKG2D stimulates or costimulates CD8(+) T cells or NK cells. The inducible gene MHC class I chain-related molecules (MICs), which belong to the NKG2D ligand family and usually initiate the process of lymphocyte activation, have been found to be broadly expressed on epithelial tumor cells. Sustained localized expression or release of soluble forms of MICs (sMICs) by tumor cells play key roles in tumor evasion via the impairment of T cell and NK cell functions. NKG2D is also expressed on the surface of CD3(+)CD56(+) NKT-like cells, which participate in tumor rejection via direct cytolysis. We speculated whether sMICs have the same impact on NKT-like cells. In this study, we demonstrated that in vitro killing by freshly isolated NKT-like cells was principally mediated by NKG2D, and the cytotoxic function of NKT-like cells isolated from cancer patients was obviously compromised. We found a significant correlation between elevated tumor-derived sMICs and down-modulation of NKG2D expression on NKT-like cell surfaces in human ovarian cancer and prostate cancer patients. We determined that elevated serum sMIC impairs the lytic activity via downregulation of the NKG2D receptor because incubation of NKT-like cells with sera obtained from cancer patients down-modulated surface NKG2D expression, whereas the addition of neutralizing anti-MIC mAbs restored surface NKG2D expression. We suggest that tumors shedding MICs may promote immune evasion by impairing NKT-like cell cytotoxicity.     

High expression of NKG2A/CD94 and low expression of granzyme B are associated with reduced cord blood NK cell activity

Human umbilical cord blood （CB） has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease （GVHD） and graft-versus-leukemia （GVL）. It was reported that the activity of CB NK cells was lower than that of adult peripheral blood （PB） NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-γ, TNF-α and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.     

Requirements for control of B‐cell lymphoma by NK cells

The role of NK cells in the control of endogenously arising tumors is still unclear. We monitored activation and effector functions of NK cells in a c‐myc‐transgenic mouse model of spontaneously arising lymphoma. At early stages, tumors demonstrated reduced MHC class I expression and increased expression of natural killer group 2D ligands (NKG2D‐L). NK cells in these tumors showed an activated phenotype that correlated with the loss of tumor MHC class I. With increasing tumor load however, NK‐cell effector functions became progressively paralyzed or exhausted. In later stages of disease, tumors re‐expressed MHC class I and lost NKG2D‐L, suggesting a role of these two signals for NK cell‐mediated tumor control. Testing a panel of lymphoma cell lines expressing various MHC class I and NKG2D‐L levels suggested that NK cell‐dependent tumor control required a priming and a triggering signal that were provided by MHC class I down‐regulation and by NKG2D‐L, respectively. Deleting either of the “two signals” resulted in tumor escape. At early disease stages, immune stimulation through TLR‐ligands in vivo efficiently delayed lymphoma growth in a strictly NK cell‐dependent manner. Thus, NK‐receptor coengagement is crucial for NK‐cell functions in vivo and especially for NK cell‐mediated tumor surveillance.     

Suppressive Effect of a Standardized Mistletoe Extract on the Expression of Activatory NK Receptors and Function of Human NK Cells

Despite long-term use of mistletoe extracts for cancer treatment, their mode of action remains elusive. In this study, it was studied in vitro if mistletoe extract is able to modulate the expression of natural cytotoxic receptors (NCRs) and NKG2D receptor, which stimulate natural killer cell-mediated cytotoxicity. Unexpectedly, a mistletoe extract, ABNOBA viscum Fraxini, inhibited the expression level of NKp46 and NKG2D receptors in dose- and time-dependent manners. The levels of NKp30 and NKG2D receptors were remarkably induced and NKp44 was slightly induced after 48 h treatment with IL-2 and IL-15 in both mRNA and surface expression. The activatory NK receptors were not induced significantly after treatment with IL-12, IL-18, and IL-21 for 48 h. Induction of activatory NK receptors by IL-2 and IL-15 was suppressed almost to the untreated levels by treatment with mistletoe extract, which appeared to induce apoptosis of NK cells in a dose-dependent manner. However, the treatment with IL-2 and IL-15 did not prevent the mistletoe-induced NK-cell death. Mistletoe extract inhibited significantly the cytotoxic activity of resting and IL-2- or IL-15-stimulated NK cells. These results suggest that inhibition of survival and function of NK cells by mistletoe extract may curtail in part the therapeutic effects of mistletoe. Soo Jung Lee, Young-Ok Son, and Hyunjin Kim contributed equally to this article     

c‐Cbl regulates MICA‐ but not ULBP2‐induced NKG2D down‐modulation in human NK cells

The NKG2D activating receptor on human NK cells mediates “altered self” recognition, as its ligands (NKG2DLs) are upregulated on target cells in a variety of stress conditions. Evidence collected in the past years shows that, even though expression of NKG2DLs acts as a danger signal that renders tumor cells susceptible to cytotoxicity, chronic exposure to soluble or membrane‐bound NKG2DLs can lead to down‐modulation of receptor expression and impairment of NKG2D‐mediated cell functions. Here, we evaluated whether different cell‐bound NKG2DLs, namely MICA and ULBP2, are equivalently able to induce NKG2D down‐modulation on human NK cells. We found that although both ligands reduce NKG2D surface expression, MICA promotes a stronger receptor down‐modulation than ULBP2, leading to a severe impairment of NKG2D‐dependent NK‐cell cytotoxicity. We also provide evidence that the ubiquitin pathway and c‐Cbl direct MICA‐induced but not ULBP2‐induced NKG2D internalization and degradation, thus identifying a molecular mechanism to explain the differential effects of MICA and ULBP2 on NKG2D expression. A better understanding of the molecular mechanisms employed by the different NKG2DLs to control NKG2D surface expression could be useful for the development of anti‐tumor strategies to restore a normal level of NKG2D receptors on human NK cells.     

Adaptive NK cell response to human cytomegalovirus: Facts and open issues

Human cytomegalovirus (HCMV) infection exerts broad effects on the immune system. These include the differentiation and persistent expansion of a mature NK cell subset which displays a characteristic phenotypic and functional profile hallmarked by expression of the HLA-E-specific CD94/NKG2C activating receptor. Based on our experience and recent advances in the field, we overview the adaptive features of the NKG2C+ NK cell response, discussing observations and open questions on: (a) the mechanisms and influence of viral and host factors; (b) the existence of other NKG2C- NK cell subsets sharing adaptive features; (c) the development and role of adaptive NKG2C+ NK cells in the response to HCMV in hematopoietic and solid organ transplant patients; (d) their relation with other viral infections, mainly HIV-1; and (e) current perspectives for their use in adoptive immunotherapy of cancer.     

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8⁺ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.     

Loss of NKG2D in murine NK cells leads to increased perforin production upon long-term stimulation with IL-2

NK cells are innate lymphocytes responsible for lysis of pathogen-infected and transformed cells. One of the major activating receptors required for target cell recognition is the NK group 2D (NKG2D) receptor. Numerous reports show the necessity of NKG2D for effective tumor immune surveillance. Further studies identified NKG2D as a key element allowing tumor immune escape. We here use a mouse model with restricted deletion of NKG2D in mature NKp46⁺ cells (NKG2D^ΔNK). NKG2D^ΔNK NK cells develop normally, have an unaltered IFN-γ production but kill tumor cell lines expressing NKG2D ligands (NKG2DLs) less efficiently. However, upon long-term stimulation with IL-2, NKG2D-deficient NK cells show increased levels of the lytic molecule perforin. Thus, our findings demonstrate a dual function of NKG2D for NK cell cytotoxicity; while NKG2D is a crucial trigger for cytotoxicity of tumor cells expressing activating ligands it is also capable to limit perforin production in IL-2 activated NK cells.     

Evidence of NK cell dysfunction in SIV-infected rhesus monkeys: impairment of cytokine secretion and NKG2C/C2 expression

Defects in the adaptive immune response have been extensively characterized in human immunodeficiency virus type-1 (HIV-1)-infected individuals; however, much less is known about the function of natural killer (NK) cells during the course of HIV-1 infection. In the present study, we demonstrate that the NK cells from simian immunodeficiency virus (SIV)-infected rhesus monkeys are significantly impaired in their ability to secrete IFN-gamma, TNF-alpha, and IL-2, while NK cell function in SIV-infected long-term non-progressor monkeys is similar to that of normal monkeys. These findings suggest that abnormal NK cell activity may contribute to the global immune dysfunction observed in HIV-1-infected individuals. NK cell function is modulated by several families of cell surface receptors, including the CD94/NKG2 family. We evaluated the messenger RNA levels of these inhibitory and activating NKG2 molecules in SIV-infected rhesus monkeys. These experiments demonstrate that the activating molecules NKG2C and NKG2C2 are significantly down-regulated in peripheral blood mononuclear cells of SIV-infected rhesus monkeys, suggesting that the dysregulation of these molecules may contribute to the abnormal NK cell function observed in the setting of infection.