IL-12依赖于TRAIL途径增强NK细胞对Jurkat细胞的杀伤功能

目的:探讨IL-12增强正常人NK细胞对Jurkat细胞杀伤功能的相关机制。方法:纯化正常人NK细胞,分为加或不加IL-12两个刺激组,通过基因芯片筛选差异基因,并通过流式细胞术在单个细胞水平检测相关杀伤分子的表达。结果:基因芯片系统结果显示IL-12刺激组和未刺激组相比,17种基因具有显著性差异(fold change≥10),其中5种基因上调,12种基因下调。在IL-12的刺激作用下,TRAIL的表达在CD56+CD16+和CD56-CD16+NK细胞上显著增加。同时,Jurkat细胞亦高表达TRAIL受体TRAIL-R2(DR5)。TRAIL中和抗体RIK-2可以阻断IL-12诱导的NK细胞对Jurkat细胞的杀伤功能。结论:TRAIL是IL-12增强正常人NK细胞对Jurkat细胞杀伤功能的主要途径之一。     

TLR7/8配体R-848体外诱导小鼠脾细胞产生IgG2a的研究

目的检测TLR7/8配体R-848是否能够诱导naive小鼠的脾B细胞和纯化B细胞活化、增殖及产生抗体。方法磁珠分选出纯化的B细胞,将制备好的脾细胞或纯化B细胞,与R-848共培养,流式细胞术检测R-848对B细胞活化和增殖的影响;ELISA检测R-848是否能够诱导脾细胞产生IgG2a和IgG1,并检测与抗体产生相关的细胞因子的产生。结果 R-848上调B细胞表面活化分子CD25和CD80的表达,促进B细胞活化和分裂;R-848以剂量依赖方式诱导小鼠脾细胞产生IgG2a和IgG1;同时诱导脾细胞产生IFN-γ、IL-12P40、IL-10和IL-6。结论 R-848直接作用于B细胞促进其活化和分裂;R-848促进脾细胞产生大量的IgG2a,可能与其增强IFN-γ、IL-6和IL-10的产生有关。     

NKG2D配体在鼻咽癌细胞的表达及其对NK细胞杀伤活性的影响

NK细胞对靶细胞的杀伤活性与其细胞表面的受体和靶细胞表面的配体密切相关，NKG2D为NK细胞活化性受体，表达于所有的NK细胞表面，是介导NK细胞识别和溶解肿瘤细胞的主要活化性受体。NKG2D配体为MHCⅠ类链相关基因产物（MICA、MICB）及ULBPS（人巨细胞病毒UL16蛋白的结合蛋白ULBP1、ULBP2、ULBP3），NKG2D的配体在多种肿瘤细胞表达，其在鼻咽癌细胞的表达尚未见报道。本文通过流式细胞仪技术探讨其在鼻咽癌细胞CNE2的表达情况，并进一步分析其在NK细胞杀伤CNE2细胞中的作用。     

Toll样配体(R-848)与IL-12对人NK细胞亚群IFN-γ产生的作用

目的:研究Toll样配体(R-848)与IL-12对人NK细胞IFN-γ产生的作用和细胞亚群分析。方法:分离人外周血PBMC和纯化的NK细胞,分别与R-848、IL-12或R-848和IL-12共同培养。利用ELISA法检测培养上清中IFN-γ的水平,再利用流式检测并分析产生IFN-γ的NK细胞亚群。结果:正常人PBMC分别与不同浓度的Toll样配体R-848、LPS、CpG培养后,均以剂量依赖的方式诱导IFN-γ的产生,但以R-848的效果最佳。细胞亚群分析的结果表明,R-848对CD4 T和CD8 T细胞IFN-γ的表达无明显作用,但显著地促进CD56 细胞表达IFN-γ。同样地,在IL-12刺激之下,CD56bright和CD56dimNK细胞表达IFN-γ。当R-848和IL-12与PBMC和纯化NK细胞孵育后,对CD56bright和CD56dimNK细胞IFN-γ的表达具有协同作用。结论:Toll样配体与NK细胞Toll样受体结合后,促进CD56brightNK细胞亚群IFN-γ的产生,而且Toll样配体与IL-12具有协同作用,提示Toll样受体与细胞因子在调控NK细胞的生物活性中发挥着十分重要的作用。     

用Percoll法、Ficoll法及常规法制备的效应细胞杀伤功能的比较

本文用Percoll配成的不连续密度梯度(Percoll法)对C57BL、C_(3H)和CBA品系小鼠的脾细胞进行了分离,并用不同密度梯度层分离所得的细胞对~3H-TdR或~(51)Cr标记的YAC-1靶细胞进行杀伤功能检测.发现以50～60％、60～70％二个密度梯度层之间的细胞杀伤活性最高,poly I:C可以增强这种活性.并且在同一效靶比例下,这种效应细胞对YAC-1靶细胞的杀伤活性均高于用Ficoll-Isopague分离的脾细胞(Ficoll法)及常规制备的脾细胞(常规法),前者与后二者所获数据间呈显著性差异(p<0.01)。     

SNAP-23对NK细胞杀伤效应的调控

目的探讨SNAP-23对NK细胞杀伤效应的调控作用。方法通过核酸转染仪转染SNAP-23 shRNA（short hairpin RNA）质粒阻断NK92细胞中SNAP-23蛋白的表达，分别利用乳酸脱氢酶（LDH）释放实验和β-己糖氨酶释放实验检测SNAP-23表达下降对NK细胞杀伤效应和胞吐效应的影响。结果核转染仪可将shRNA质粒成功转入NK92细胞，所设计的3条SNAP-23的shRNA能抑制SNAP-23的蛋白表达，继而导致NK92细胞的杀伤活性和胞吐效应下降。结论SNAP-23通过调控NK细胞的胞吐效应而参与NK细胞杀伤效应的调控。     

同种异体NK细胞对人脐静脉内皮细胞ECV304的杀伤活性差异及分子机制探讨

目的 探讨供者KIR分子表达差异对NK细胞杀伤人脐血内皮细胞系ECV304活性的影响,并观察参与杀伤的活化信号通路.方法 RT-PCR及流式细胞仪检测ECV304细胞NKG2D配体MICA/B、ULBP1-3表达,PCR-SSP法行HLA-A、B、Cw分型.自8例健康供者分离外周血NK细胞,流式细胞仪检测KIR2DL1的表达率,LDH释放法测定NK细胞在效靶比20∶1时对ECV304细胞的杀伤活性及anti-KIR2DL1mAb对NK细胞杀伤活性的影响.结果 ECV30细胞在mRNA水平表达MICA/B、ULBP1-3,但在膜蛋白水平均不表达.HLA分型表明,ECV304表达KIR2DL1的配体,而不表达KIR2DL2/3、KIR3DL1的配体.8例健康供者NK细胞KIR2DL1表达率有较大差异,对ECV304细胞的杀伤活性也有不同,双变量相关分析示个体KIR2DL1表达率与NK细胞对ECV304的杀伤率存在负相关(rS=-0.994,P=0.000).anti-KIR2DL1 mAb明显增强NK细胞对ECV304的杀伤活性(t=-4.860,P=0.002).结论 NK细胞对ECV304细胞的杀伤分子机制主要为HLA-KIR信号系统错配,目前已知的NKG2D配体MICMB、ULBP1-3并不参与,这有助于临床活体器官移植时在遗传指导下选择供体.     

血管内皮细胞(ECV304)表达的HLA-G1对NK细胞杀伤活性的抑制作用

目的:证实HLA-G1分子能够抑制NK细胞对同种血管内皮细胞系的杀伤作用。方法:采用脂质体介导的DNA转染技术,以构建的真核表达质粒pcDNA3-HLA-G1转染人脐静脉内皮细胞系ECV304,再用免疫荧光法检测表达的HLA-G1分子。并用MTT比色法检测HLA-G1对NK细胞杀伤活性的影响。结果:ECV304细胞上可稳定表达HLA-G1。NK细胞对空质粒pcDNA3转染的ECV304细胞的杀伤率为(50.6±18.1)%;而对pcDNA3-HLA-G1转染的ECV304细胞的杀伤率为(29.7±11.4)%,两者差异具有显著意义(P<0.01)。结论:HLA-G1分子可明显抑制NK细胞对同种血管内皮细胞的杀伤效应。     

NK细胞新概念——趋化因子活化的杀伤细胞(CHAK)

趋化因子是新近发现的一类蛋白质 ,主要参与趋化、激活白细胞亚群 ,调节机体非特异免疫 ,特别是与爱滋病和肿瘤的关系引起了国内外极大关注。国内已发表了不少趋化因子方面的综述 ,但趋化因子与NK细胞的关系却鲜为人知 ,本文重点介绍CHAK的国内外研究进展     

HLA-G分子在体外抑制NK细胞杀伤效应的研究

在获得稳定表达HLA G分子的K5 62细胞克隆的基础上 ,观察 4h及 16h不同效靶比例时NK细胞对靶细胞的杀伤效应 ,结果显示 ,NK细胞对转染HLA G基因的K5 62细胞的杀伤作用较未转染细胞明显降低 (P <0 0 1) ,用W6/ 32及BFL 1单克隆抗体阻断HLA G分子 ,可以恢复NK细胞的杀伤活性。用Y9/Z2 70及XA185 /Z2 70单抗特异性阻断抑制性受体CD94/NKG2A ,未观察到明显影响     

Selective cross-talk among natural cytotoxicity receptors in human natural killer cells

The cytolytic activity of human natural killer cells is induced by several triggering cell surface receptors upon interaction with specific cellular ligands. These receptors include NKp46, NKp30 and NKp44, collectively termed natural cytotoxicity receptors (NCR). Co-operation among NCR has been shown to occur for optimal recognition and killing of most tumor target cells. In this study, we show that the mAb-mediated engagement and clustering of one or another NCR results in the activation of an identical set of tyrosine kinases. These kinases are included in the signaling cascade leading to tyrosine phosphorylation of different receptor-associated signal transducing molecules i.e. CD3 zeta (associated with NKp46 and NKp30) and KARAP/DAP12 (associated with NKp44). In line with the notion that the engagement of inhibitory receptors prevents NCR-mediated responses, we show that the engagement of CD94/NKG2A virtually abrogates the tyrosine phosphorylation of the NCR-associated signaling molecules, i.e. it acts at the very early steps of the signaling cascade. Importantly, the engagement of a single NCR resulted in the activation of the signaling cascades associated with the other NCR. This "cross-talk" is confined to NKp46, NKp30 and NKp44 since neither CD16-nor KIR2DS4-associated signaling polypeptides were phosphorylated following the NCR engagement. These results suggest that a functional cross-talk specifically occurs among different NCR, possibly resulting in the amplification of the activating signals.     

雌激素对天然杀伤细胞的作用

为探讨雌激素对NK细胞的作用,在不同时间把不同剂量的雌激素加入培养体系中,分析雌激素对无和有外来刺激原刺激NK细胞的影响。结果显示培养开始加入生理剂量、低于生理剂量和超生理剂量的雌激素均可明显抑制NK细胞的增殖;培养48 h后加入相同剂量的雌激素,生理剂量和低于生理剂量雌激素仍可明显抑制NK细胞的增殖,超生理剂量雌激素非但不能抑制反而使NK细胞的数量增加。     

T-AK细胞的细胞毒活性研究

探讨抗ＣＤ３单抗和ｒＩＬ－２共同激活诱生的Ｔ－ＡＫ细胞的细胞毒活性本质。方法：采用ＭＴＴ法分别检测Ｔ－ＡＫ细胞杀伤白血病细胞的活性及其构成。结论Ｔ－ＡＫ细胞为异质性细胞群体,其杀伤活性主体为ＣＤ３ＡＫ活性和ＬＡＫ活性。     

Tetrabromobisphenol A has immunosuppressive effects on human natural killer cells

Human natural killer (NK) cells are lymphocytes that destroy tumor cells, virally-infected cells, and antibody-coated cells. Tetrabromobisphenol A (TBBPA) is used both as a reactive and as an additive flame retardant in a variety of materials and appears to contaminate the environment. TBBPA has been found in human blood samples and if it interferes with NK cell function, this could increase the risk of tumor development and/or viral infection. The present study examines the effects of exposure to various concentrations of TBBPA for 24?hr, 48?hr, and 6 days on the lytic function, tumor-target-binding function, and ATP levels of NK cells. These same parameters were also monitored in NK cells that were exposed to TBBPA for 1?h followed by 24?hr, 48?hr, and 6 days in TBBPA-free media. A 24-h exposure of NK cells to 5 μM TBBPA caused a >95% decrease in NK lytic function, a 70% decrease in binding function, and a 34% decrease in ATP levels in NK cells. Exposure to 2.5 μM TBBPA for 24?h decreased lytic function by 76%, binding function by 20%, and had no effect on ATP levels. Exposure of NK cells to 5 μM TBBPA for 1?h followed by 24?h in TBBPA-free media caused a progressive and persistent loss of lytic function (41%) while not affecting either binding ability or ATP levels. The results indicate that TBBPA exposures decrease the lytic function of human NK cells and that an initial brief (1?hr) exposure can cause a progressive loss of function. In addition, the data also indicate that TBBPA-induced loss of NK lytic function can occur at a concentration of TBBPA that does not affect target-binding ability and ATP levels of NK cells.     

CD59 is physically and functionally associated with natural cytotoxicity receptors and activates human NK cell-mediated cytotoxicity

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of several triggering receptors. These include primary receptors such as NKG2D and the natural cytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including 2B4, NTB-A and NKp80, function as co-receptors. As reported in the present study, during an attempt to identify novel NK receptors or co-receptors, we found that CD59 functions as a co-receptor in human NK cell activation; engagement of CD59 by specific mAb delivers triggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar to other NK co-receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)-linked protein, depends on the simultaneous engagement of primary receptors such as NCR. Accordingly, CD59-dependent triggering was virtually restricted to NK cells expressing high surface densities of NKp46, and mAb-mediated modulation of NKp46 resulted in markedly decreased responses to anti-CD59 mAb. Biochemical analysis revealed that CD59 is physically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted in tyrosine phosphorylation of CD3zeta chains associated with these NCR, but not those associated with CD16. Thus, CD59-mediated costimulation of NK cells requires direct physical interaction of this GPI-linked protein with primary triggering NK receptors.     

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A,but lacking inhibitory killer Ig-like receptors

The cognate NK-DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte-derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig-like receptors (KIR). Consistent with these data, the expression of HLA-E (i.e. the cellular ligand of CD94/NKG2A) was down-regulated in iDC. On the other hand, HLA-B and HLA-C down-regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A(+)KIR(-) NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA-E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA-E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.     

Memory responses of natural killer cells

Natural killer (NK) cells have traditionally been classified as a cellular component of the innate immune system, given their ability to rapidly produce effector cytokines and kill infected or transformed cells without prior exposure. More recently, NK cells have been shown to possess features of adaptive immunity such as clonal expansion, longevity, and robust recall responses. NK cell memory can be broadly divided into two categories: antigen-specific and antigen-independent. In the first case, exposure to certain viral or hapten stimuli endows NK cells with antigen-specific immunological memory, similar to T and B cells. In the second case, exposure of NK cells to specific cytokine milieus can imprint long-lasting changes on effector functions, resulting in antigen-independent memory-like NK cells. In this review, we discuss the various conditions that promote generation of these two categories of memory NK cells, and the mechanistic requirements underlying these processes.