m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection.

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.     

Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gammadelta T cells

Few studies have demonstrated that innate lymphocytes play a major role in preventing spontaneous tumor formation. We evaluated the development of spontaneous tumors in mice lacking beta-2 microglobulin (beta2m; and thus MHC class I, CD1d, and CD16) and/or perforin, since these tumor cells would be expected to activate innate effector cells. Approximately half the cohort of perforin gene-targeted mice succumbed to spontaneous disseminated B cell lymphomas and in mice that also lacked beta2m, the lymphomas developed earlier (by more than 100 d) and with greater incidence (84%). B cell lymphomas from perforin/beta2m gene-targeted mice effectively primed cell-mediated cytotoxicity and perforin, but not IFN-gamma, IL-12, or IL-18, was absolutely essential for tumor rejection. Activated NK1.1+ and gammadeltaTCR+ T cells were abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and gammadeltaTCR+ T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor recognition.     

分娩发动前后体内NK细胞、NKT细胞表型分析

目的研究分娩发动前后蜕膜及外周血中NK细胞、NKT细胞表面NKG2A和NKG2D受体的表达情况,探讨其在产程发动中的作用。方法选取正常孕足月孕妇分为分娩发动经阴道分娩组（以下简称分娩组20例）和分娩未发动行选择性剖宫产术组（以下简称剖宫产组20例）,分离蜕膜及外周血中单个核细胞后采用流式细胞学技术分别检测CD56＋细胞、NK细胞、NKT细胞表面NKG2A和NKG2D受体的表达情况。结果外周血单个核细胞中分娩组CD56＋NKG2A＋细胞构成下降、CD56＋NKG2D＋细胞构成增加,NK细胞、NKT细胞表面NKG2D表达上升。蜕膜单个核细胞中分娩组CD56＋NKG2A＋和CD56＋NKG2D＋细胞构成及NK细胞表面NKG2A和NKG2D表达均下降,NKT细胞表面NKG2A和NKG2D表达无显著差异。结论临产前体内NK细胞、NKT细胞表面NKG2A和NKG2D受体表达的变化可能在在产程的发动中起着重要作用。     

In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.

Natural killer (NK) receptor signaling can lead to reduced cytotoxicity by NK cells and cytolytic T lymphocytes (CTLs) in vitro. Whether T cells are inhibited in vivo remains unknown, since peptide antigen-specific CD8(+) T cells have so far not been found to express NK receptors in vivo. Here we demonstrate that melanoma patients may bear tumor-specific CTLs expressing NK receptors. The lysis of melanoma cells by patient-derived CTLs was inhibited by the NK receptor CD94/NKG2A. Thus, tumor-specific CTL activity may be decreased through NK receptor triggering in vivo.     

自然杀伤T淋巴细胞对黑色素瘤细胞的免疫调节及杀伤功能实验研究

目的探讨自然杀伤T淋巴细胞(NKT细胞)在细胞免疫调节及杀伤功能中的作用。方法建立混合淋巴细胞培养(MLC)体系,B16F10-luc-G5细胞作为靶细胞,以总淋巴细胞为效应细胞。(1)调节效应实验以NKT细胞或CD4~+CD25~+T淋巴细胞为调节细胞,分为3组:NKT组、CD4~+CD25~+T组、靶细胞空白对照组;另设有1640空白对照组(RPMI1640溶液)。(2)杀伤效应实验以NKT或自然杀伤(NK)细胞为效应细胞,分为3组:NKT组、NK组、靶细胞空白对照组。混合培养24、48、72h后,通过活体成像系统检测该培养系统的靶细胞生物发光,以监测NKT细胞的调节及杀伤效应。结果 (1)调节效应实验:NKT组及CD4~+CD25~+T组测定光子数分别与两空白对照组比较,以及NKT组内培养24h与72h时测得光子数比较,差异均有统计学意义(P0.05)。(2)杀伤效应实验:NKT组及NK组与靶细胞空白对照组所测光子数比较,差异均有统计学意义(P0.05);且培养24、72h时NKT组与NK组光子数比较,差异均有统计学意义(P0.05)。结论 NKT细胞具有抑制总淋巴细胞对靶细胞的杀伤效应,且抑制效应具有时间性,其调节作用较CD4~+CD25~+T淋巴细胞强。NKT细胞具有对靶细胞的杀伤效应,但较NK细胞弱。     

乙型肝炎病毒相关性肝硬化患者外周血NK和NKT细胞功能特点及意义

目的 探讨乙型肝炎病毒相关性肝硬化(HBV-LC)患者外周血自然杀伤细胞(NK)和自然杀伤T细胞(NKT)功能相关分子表达特点及意义.方法 收集代偿期HBV-LC患者(HBV-CLC)、失代偿期HBV-LC患者(HBV-DLC)和同期体检健康者(HC)各25例作为研究对象.流式细胞术检测各组外周血NK和NKT细胞比例、...     

Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells

Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.     

自然杀伤T细胞在幽门螺旋杆菌阳性消化性溃疡患者中的水平与临床意义

目的探讨幽门螺旋杆菌(Hp)阳性消化性溃疡患者(PU)外周血自然杀伤T细胞(NKT)的表达与临床意义。方法选择83例Hp阳性PU患者(A组),以79例Hp阴性PU患者作为阳性对照(B组),以60例健康人作为阴性对照(C组);采用流式细胞术检测A、B及C三组外周血NKT及其胞内细胞因子γ-干扰素(IFN-γ)及白介素4(IL-4)水平。将A组患者随机分为D及E组,两组均给予雷贝拉唑及甲硝唑治疗,D组在此基础上给予克拉霉素治疗,E组给予阿莫西林治疗,两周后进行NKT、IFN-γ及IL-4的水平比较,并将3种指标与治疗前Hp感染的程度进行相关性分析。结果 B与C组间的NKT及IFN-γ差异均无统计学意义,但A组NKT及IFN-γ水平低于B及C组,差异有统计学意义(P0.05)。A组IL-4低于B组,且B组低于C组,差异有统计学意义(P0.05)。治疗前D及E组间的NKT、IFN-γ及IL-4差异均无统计学意义(P0.05),治疗后D组3种指标均显著高于E组(P0.05)。Spearman分析显示NKT、IFN-γ及IL-4均与Hp感染程度呈负相关。结论 NKT、IFN-γ及IL-4水平下降导致杀伤力降低可能原因为Hp感染致PU的免疫机制破坏,且感染越重三者水平越低;克拉霉素在上调NKT、IFN-γ及IL-4的表达效果优于阿莫西林。     

A defect in interleukin 12-induced activation and interferon gamma secretion of peripheral natural killer T cells in nonobese diabetic mice suggests new pathogenic mechanisms for insulin-dependent diabetes mellitus.

The function of natural killer T (NKT) cells in the immune system has yet to be determined. There is some evidence that their defect is associated with autoimmunity, but it is still unclear how they play a role in regulating the pathogenesis of T cell-mediated autoimmune diseases. It was originally proposed that NKT cells could control autoimmunity by shifting the cytokine profile of autoimmune T cells toward a protective T helper 2 cell (Th2) type. However, it is now clear that the major function of NKT cells in the immune system is not related to their interleukin (IL)-4 secretion. In fact, NKT cells mainly secrete interferon (IFN)-gamma and, activated in the presence of IL-12, acquire a strong inflammatory phenotype and cytotoxic function.     

CD4+CD25+Foxp3+调节性T细胞在CIK细胞诱导中的表达及其功能

目的探讨肿瘤患者自体细胞因子诱导的杀伤细胞(CIK)诱导过程中CD4~+CD25~+Foxp3~+调节性T细胞的变化及其功能。方法应用血细胞分离机采集22例肿瘤患者外周血单个核细胞(PBMC),诱导培养CIK细胞,用流式细胞仪动态监测其CD4~+CD25~+Foxp3~+表型,并分析Tregs细胞负性调控分子转化生长因子-β1(TGF-β1)、细胞毒性T淋巴细胞相关抗原4(CTLA-4)和IL-10的表达水平,采用细胞增殖抑制试验测定Tregs细胞免疫学功能。结果诱导的CIK细胞中存在CD4~+CD25~+Foxp3~+Tregs,其表达量分别为第1天(0.30±0.15)%、第3天(4.48±1.72)%、第5天(3.83±2.12)%、第9天(2.37±1.17)%、第11天(1.65±0.99)%、第14天(1.04±0.76)%。诱导第14天时的Tregs细胞免疫调控负性分子TGF-β1的表达水平为(97.2±2.1)%、CTLA-4为(96.2±3.5)%、IL-10为(4.2±2.3)%。细胞增殖抑制试验中空白对照组、条件对照组以及实验组的增殖细胞表达量分别为(8.55±2.38)%、(42.66±7.32)%、(57.04±7.49)%。结论 CD4~+CD25~+Foxp3~+Tregs细胞可作为潜在的CIK细胞质量评价指标。     

Origin and functions of human natural killer cells

Summary Recent data have substantially modified our view of natural killer cells. Although maturation of natural killer cells occurs in the absence of a functional thymus, we have shown that clonogenic precursors capable of differentiating into mature CD3−16+56+ natural killer cells exist in CD3−4−8−16− populations isolated from human thymus. Analysis of peripheral bloodderived natural killer clones showed that they can lyse normal cells (e.g., phytohemagglutinin-induced blasts) isolated from some individuals. Importantly, natural killer clones isolated from single individuals displayed different patterns of cytolytic activity against a panel of normal allogeneic cells. These data suggested the existence of a natural killer cell repertoire. A number of observations have revealed that the expression of given HLA class I alleles protects target cells from lysis by different groups of natural killer clones. Evidence has been gained by genetic analysis of the determinants responsible for susceptibility/resistance to lysis by natural killer clones together with analysis, as target cells, of HLA-defective variants or HLA transfectants. Thus, natural killer cells were found to express a clonally distributed ability to recognize HLA class I alleles. The selection of new monoclonal antibodies directed against members of a novel family of natural killer specific p58 molecules allowed the identification of the putative natural killer receptors for different MHC class I alleles. Firstly, a correlation was established between the expression of given p58 molecules (e.g., EB6 and GL183) and the class I alleles recognized. Secondly, anti-p58 monoclonal antibodies restored the natural killer-mediated lysis of class I-protected cells. A similar effect was obtained by inducing modulation of p58 surface molecules with anti-p58 monoclonal antibodies. The implications of these receptor/ligand interactions in the physiopathological behavior of natural killer cells are discussed.     

Natural Killer (NK) Cell–mediated Cytotoxicity: Differential Use of ?TRAIL and Fas Ligand by Immature and Mature Primary Human NK Cells

Mature natural killer (NK) cells use Ca²⁺-dependent granule exocytosis and release of cytotoxic proteins, Fas ligand (FasL), and membrane-bound or secreted cytokines (tumor necrosis factor [TNF]-α) to induce target cell death. Fas belongs to the TNF receptor family of molecules, containing a conserved intracytoplasmic “death domain” that indirectly activates the caspase enzymatic cascade and ultimately apoptotic mechanisms in numerous cell types. Two additional members of this family, DR4 and DR5, transduce apoptotic signals upon binding soluble TNF-related apoptosis-inducing ligand (TRAIL) that, like FasL, belongs to the growing TNF family of molecules. Here, we report that TRAIL produced or expressed by different populations of primary human NK cells is functional, and represents a marker of differentiation or activation of these, and possibly other, cytotoxic leukocytes. During differentiation NK cells, sequentially and differentially, use distinct members of the TNF family or granule exocytosis to mediate target cell death. Phenotypically immature CD161⁺/CD56⁻ NK cells mediate TRAIL-dependent but not FasL- or granule release–dependent cytotoxicity, whereas mature CD56⁺ NK cells mediate the latter two.     

连续硬膜外镇痛对非停跳冠状动脉搭桥术患者T淋巴细胞亚群和自然杀伤细胞的影响

目的:探讨连续硬膜外镇痛对非停跳冠状动脉搭桥术患者围术期T淋巴细胞亚群和自然杀伤细胞的影响。方法:选择非停跳冠状动脉搭桥术患者60例,随机分为单纯全身麻醉组(GA组)30例和全身麻醉复合连续硬膜外镇痛组(PCEA)30例。在麻醉诱导前(T0)、术后4 h(T1)、1 d(T2)、3 d(T3)、7 d(T4)5个时间点各抽取外周静脉血2mL,采用流式细胞仪测定T淋巴细胞亚群(CD3、CD4、CD8、CD4/CD8)及自然杀伤细胞(CD16+56)的数量。结果:患者手术经过及术后恢复均顺利。T1~T2时2组T淋巴细胞亚群和自然杀伤细胞较术前显著减少(P<0.05),GA组较PCEA组减少更明显(P<0.05)。T3时GA组T淋巴细胞亚群和自然杀伤细胞仍较术前减少,PCEA组T淋巴细胞亚群基本恢复正常。结论:连续硬膜外镇痛可减轻非停跳冠状动脉搭桥手术对患者围术期的免疫抑制,有利于患者术后免疫功能的恢复。     

Commitment of common T/Natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay

We have established a new clonal assay system that can evenly support the development of T and natural killer (NK) cells. With this system, we show that all T cell progenitors in the earliest CD44(+)CD25(-)FcgammaRII/III(-) fetal thymus (FT) cell population retain NK potential, and that the NK lineage-committed progenitors (p-NK) also exist in this population. T cell lineage-committed progenitors (p-T), which are unable to generate NK cells, first appear at the CD44(+)CD25(-) FcgammaRII/III(+) stage in day 12 FT. The proportion of p-T markedly increases during the transition from the CD44(+)CD25(-) stage to the CD44(+)CD25(+) stage in day 14 FT. On the other hand, p-NK preferentially increase in number at the CD44(+)CD25(-) stage between days 12 and 14 of gestation. The production of p-NK continues up to the CD44(+)CD25(+) stage, but ceases before the rearrangement of T cell receptor beta chain genes. It was further shown that the CD44(+)CD25(-) CD122(+) population of day 14 FT exclusively contains p-NK. These results indicate that the earliest T cell progenitor migrating into the FT is T/NK bipotent, and strongly suggest that the bipotent progenitor continuously produces p-NK and p-T until the CD44(+)CD25(+) stage.     

原发性胆汁性肝硬化患者外周血穿孔素的表达及其临床意义

  目的  研究原发性胆汁性肝硬化(primary biliary cirrhosis, PBC)患者外周血穿孔素(perforin, 又称pore-forming protein, PFP)的表达, 探讨PFP在PBC发病机制中的作用及其临床意义。  方法  应用荧光定量PCR检测86例PBC、56例慢性乙型肝炎(chronic hepatitis type B, CHB)和69名健康对照者的外周血单个核细胞(peripheral bloodmono nuclear cells, PBMCs)中PFP mRNA基因表达的差异; 采用流式细胞术分别检测CD3-CD56+自然杀伤细胞(natu-ral killer cells, NK)、CD3+CD8+细胞毒性T细胞(cytotoxic Tlymphocytes, CTL)和CD3+CD56+自然杀伤性T细胞(natural killer Tlymphocytes, NKT)的PFP蛋白表达量。  结果  PBC组PBMCs中PFP mRNA基因表达较健康对照者升高(P < 0.05);PBC、CHB患者和健康对照者NK、CTL和NKT占PBMCs的比例无明显差别(P>0.05), PBC患者NK、CTL和NKT中表达PFP蛋白的细胞较健康对照者明显升高(P < 0.01)。PFPmRNA基因表达量及NK、CTL和NKT表达PFP蛋白的细胞比例与PBC患者Mayo危险评分呈显著负相关(P < 0.01), PBC患者总胆红素水平与NK、CTL表达PFP蛋白的细胞比例呈负相关(P < 0.05)。  结论  PBC患者PFP的异常表达, 提示PFP可能参与PBC的发病。PBC患者NK、CTL和NKT表达PFP蛋白的细胞比例可做为PBC患者生存预后的有效指标。     

Extracellular vesicle-mediated communication between hepatocytes and natural killer cells promotes hepatocellular tumorigenesis

1. Download : Download high-res image (68KB)
2. Download : Download full-size image

     

SLE患者外周血CD3+CD16+CD56+NK T细胞和CD3+CD4-CD8-DN T细胞的流式细胞分析及意义

目的探讨CD3+CD16+CD56+天然杀伤T(NK T)细胞和CD3+CD4-CD8-双阴性T(DN T)细胞在SLE患者外周血中的变化及意义。方法选取32例SLE活动期患者作为SLE活动组、30例SLE稳定期患者作为SLE稳定组、25例健康人作为健康人对照组,用流式细胞术检测各组对象外周血中NK T细胞和DN T细胞的比率,并比较其与SLE疾病活动性指数(SLEDAI)之间的相关性。结果 SLE活动组外周血NK T细胞比率(3.47±1.49)%显著低于SLE稳定组(5.30±1.83)%及健康人对照组(6.24±2.11)%,P均<0.01,但SLE稳定组与健康人对照组差异无统计学意义(P>0.05);SLE活动组外周血DN T细胞比率(5.16±1.97)%显著高于SLE稳定组(4.00±1.37)%及健康人对照组(3.15±1.06)%,P均<0.01,SLE稳定组亦高于健康人对照组(P<0.05)。SLE患者外周血NK T细胞比率与SLE DAI呈显著负相关(r=-0.439,P<0.01);SLE患者外周血DN T细胞比率与SLE DAI呈显著正相关(r=0.446,P<0.01);SLE患者外周血NK T细胞比率与DN T细胞比率之间无明显相关性(r=-0.215,P>0.05)。结论外周血中NK T细胞及DN T细胞比率改变可能参与了SLE的发病过程,且可能与SLE的病情活动性相关。