移植物NK和T细胞上抑制型受体表达对异基因造血干细胞移植后影响的观察

目的:探讨异基因造血干细胞移植(allo-HSCT)中移植物和移植后1个月患者外周血NK细胞和T细胞上4种抑制型受体(CD158a、CD158b、NKB1及CD94/NKG2A)的表达对造血重建及急性移植物抗宿主病(aGVHD)的影响.方法:对29例接受异基因造血干细胞移植的患者进行研究,采用流式细胞术检测移植物和移植后1个月患者外周血中NK细胞及T细胞上4种抑制型受体的表达,分析其对造血重建及aGVHD的影响.结果:移植物中NK细胞上CD158a、NKB1、CD94/NKG2A的表达在Ⅱ～Ⅳ度aGVHD组分别为(4.36±4.54)％、(1.37土3.24)％和(9.56±8.98)％,明显高于0～Ⅰ度aGVHD组的(0.52±0.51)％、(0.56±0.97)％和(1.85±1.71)％,P值均＜0.05；而CD8+T细胞上CD94/NKG2A的表达在0～Ⅰ度aGVHD组明显高于Ⅱ～Ⅳ度aGVHD组,分别为(25.67±14.66)％和(11.80±8.09)％,P＜0.05.结论:Allo-HSCT移植物中NK细胞高表达CD158a、NKB1和CD94/NKG2A时可能增加Ⅱ～Ⅳ度aGVHD的发生率,而CD8+T细胞上高表达CD94/NKG2A可能降低Ⅱ～Ⅳ度aGVHD的发生率.     

苦参碱诱导自然杀伤细胞对白血病K562细胞杀伤的实验研究

目的 探讨中药苦参碱对人自然杀伤（NK）细胞体外杀伤白血病细胞的作用及可能的分子机制.方法 以人慢性粒细胞白血病K562细胞为靶细胞,采用CFSE/PI双染色法流式细胞术检测不同质量浓度（02、0.5、0.8 mg/ml）苦参碱处理后,人NK细胞在不同效靶比下对K562细胞的体外杀伤活性.流式细胞术分析不同浓度苦参碱处理24 h对NK细胞主要活化性受体NKG2D和抑制性受体CD158a、CD158b表达的影响及K562细胞膜上NKG2D配体MICA/B、ULBP1、ULBP 2、ULBP 3表达的改变.结果 效靶比为5∶1时,NK细胞对0.2、0.5和0.8 mg/ml苦参碱处理后的K562细胞杀伤率分别为32.8％、38.1％和40.5％,较处理前均有不同程度增高（29.2％）;但进一步增加效靶比（10：1）后,NK细胞杀伤活性改变差异无统计学意义（P＞0.05）.苦参碱处理24 h,NK细胞抑制性受体CD158a、CD158b的表达均较处理前降低,而活化受体NKG2D的表达则增高.K562细胞表面NKG2D配体ULBP1和ULBP2分子的表达也较处理前增高（平均荧光强度分别为174.33±39.93比275.67±32.88,517.6±47.97比1368.6±49.43,P＜0.05）.结论 苦参碱可增强NK细胞对白血病K562细胞的体外杀伤活性,其机制可能与NK细胞受体及配体表达调节作用有关.     

乳腺癌患者可溶性MICA对自然杀伤细胞受体的影响

目的观察乳腺癌患者外周血自然杀伤（NK）细胞杀伤活性及受体的变化,探讨可溶性MICA（sMICA）对NK细胞受体及杀伤活性的影响。方法ELISA法检测外周血血清sMICA的含量。流式细胞术（FCM）检测NK细胞百分比、NK细胞活化性受体NKG2D、抑制性受体KIR（CD158b）表达。MTT法检测NK细胞对乳腺癌细胞株MCF-7的杀伤活性。结果与健康人比较,乳腺癌患者中81．6％表达sMICA,含量为（205．36±71．27）ng／L,且sMICA含量与TNM分期呈正相关。乳腺癌患者外周血NK细胞所占百分比无明显差异,但血清sMICA阳性的乳腺癌患者中NK细胞杀伤活性明显降低,NKG2D表达下降,CD158b表达增高。当NK细胞培养体系中加入sMICA阳性的乳腺癌血清时,其杀瘤活性明显降低【（76．2±6．7）％与（48．4±4．1）％】,NKG2D的表达明显下调[（92．5±7．1）％与（62．5±6．4）％],而CD158b的表达明显上升【（10．6±3．2）％与（43．6±3．4）％】。sMICA阳性的乳腺癌患者NK细胞与细胞因子IL-15共培养,NK细胞的杀瘤活性、NKG2D的表达明显升高,KIR（CD158b）的表达明显下降。结论乳腺癌外周血血清中sMICA可通过下调NK细胞NKG2D表达以及上调KIR表达,降低NK细胞杀瘤活性。IL-15可逆转sMICA对NK细胞的免疫下调作用。     

体外扩增对食管癌患者NK细胞表面受体表达及其抑瘤活性的影响

目的： 探讨食管癌患者NK细胞扩增前后的受体表达及其对肿瘤细胞的杀伤。 方法： 收集福建省肿瘤医院食管癌患者外周血20例,健康供者（对照组）外周血10例。NK细胞培养采用细胞因子（IL-2+IL-12+IL-15+IL-18）组合,流式细胞术检测NK细胞免疫表型及其受体（CD56+、CD69+、NKG2D、NKp30、NKp44、NKp46、CD158b、CD159a）的表达,LDH法检测不同效靶比时NK细胞对多种肿瘤细胞株（K562、Raji、Eca-109、TE-1）的杀伤作用。 结果： 与对照组相比,食管癌患者外周血CD3+、CD4+细胞比例以及CD4+/CD8+T细胞比值明显降低（P<0.05）,NK细胞（CD3-CD56+）及调节性T细胞（Treg）比例明显升高（P<0.05）。经细胞因子IL-2+IL-12+IL-15+IL-18组合定向扩增20 d后,食管癌患者NK细胞比例高达90%以上,NK细胞数扩增达1 000倍以上（P<0.01）;而CD3+T细胞、CD4+、CD8+T细胞、CD19+B细胞、Treg细胞及单核巨噬细胞（CD14+）比例均显著降低（P<0.01）,且食管癌患者与对照组之间无统计学差异（P>0.05）。经细胞因子体外培养20 d后,NK细胞表面CD69及活化性受体（NKG2D、NKp30、NKp44、NKp46）均明显上调,而抑制性受体（CD158b、CD159a）均明显下调（P<0.05）。培养20 d后,食管癌患者NK细胞对肿瘤细胞K562、Raji、Eca-109、TE-1的杀伤能力均显著高于培养前\[（69.2±5.1）% vs （42.3±3.0）%,（44.6±3.2）% vs （21.1±2.0）%,（69.7±3.9）% vs （50.3±35）%,（67.1±4.5）% vs （41.2±3.3）%;均P<0.01\]。 结论: 细胞因子IL-2+IL-12+IL-15+IL-18组合能有效扩增外周血NK细胞并上调其活并性受体的表达、下调抑制性受体的表达,其数量及功能均能满足临床治疗需要。     

肺癌患者外周血CD+8 自然杀伤T细胞表面受体NKG2D和NKG2A表达的临床意义

 【摘要】　目的　检测肺癌患者外周血CD+8 自然杀伤（NK）T细胞表面受体NKG2D和NKG2A的表达,探讨二者表达失衡与肺癌免疫逃逸的关系。方法　选择95例原发未治疗的肺癌患者,采用流式细胞术检测CD+8 NKT细胞表面受体NKG2D和NKG2A的表达,以50名健康人为对照。结果　肺癌组CD+8 NKT细胞NKG2D+表达率[（77.07±5.77）%]明显低于对照组[（84.13±4.49）%],差异有统计学意义（t＝8.14,P＜0.05）;在TNM分期中,Ⅰ～ⅢA、ⅢB、Ⅳ期患者CD+8 NKT细胞NKG2D+表达率依次为（81.07±5.02）%、（76.95±4.70）%、（72.80±5.16）%,差异有统计学意义（F＝18.74,P＜0.05）。肺癌组CD+8 NKT细胞NKG2A+表达率[（33.58±8.82）%]明显高于对照组[（25.31±8.38）%],差异有统计学意义（t＝-5.46,P＜0.05）;在TNM分期中,Ⅰ～ⅢA、ⅢB、Ⅳ期患者CD+8 NKT细胞NKG2A+的表达率依次为（25.10±6.93）%、（33.24±3.76）%、（43.64±6.10）%,差异有统计学意义（F＝75.73,P＜0.05）。结论　肺癌患者CD+8 NKT细胞表面NKG2A和NKG2D表达失衡可能抑制该细胞的杀伤功能,而这可能是肿瘤免疫逃逸的机制之一。     

γ干扰素对人NK细胞识别功能的负调节作用

目的 探讨γ干扰素(IFN-γ)对人NK细胞识别功能的负调节作用。方法 用MTT法测定人NK细胞系(NK92,NKL)的细胞毒活性及细胞增殖能力;用RT-PCR检测NK细胞受体(NKG2D、NKG2A／B、KIR2DLI、KIR2DSI)及NKG2D的识别配体主要组织相容性复合体Ⅰ类链相关分子A(MICA)的表达。结果 NK细胞系(NK92、NKL)对MICA表达阳性的肿瘤细胞杀伤活性明显高于对MICA表达阴性者;IFN-γ 1000U／ml以上可明显抑制NK细胞对MICA表达阳性肿瘤细胞的细胞毒活性,并轻度抑制NK细胞的增殖,而对MICA表达阴性肿瘤细胞的杀伤活性无明显抑制作用;IFN-γ可抑制NK细胞系活化受体NKG2D的表达,增强抑制性受体NKG2A／B和KIR2DLI的表达。结论 IFN-γ可能通过下调NK细胞活化受体的表达,上调抑制性受体的表达,使NK识别的信号平衡向抑制性方向倾斜,从而对NK细胞功能发挥负调节作用,这种作用可能是NK细胞自我调节功能的表现。     

乳腺癌患者外周血NKG2A、NKG2D检测的临床意义

背景与目的:机体自身免疫功能与肿瘤的发生、发展有密切的关系.自然杀伤(natural killer,NK)细胞在天然免疫和获得性免疫中均发挥重要作用.目前已发现多种NK细胞表面受体,根据功能可分为抑制性受体和活化性受体二大类.抑制性受体NKG2A和活化性受体NKG2D对NK细胞的杀瘤功能发挥着相反的调节作用,在发生肿瘤的情况下,二者是如何表达以及与肿瘤免疫逃逸的关系尚不明确.本研究旨在探讨乳腺癌与乳腺良性疾病患者外周血NK细胞表面受体NKG2A与NKG2D的平衡状态,分析宿主NK细胞受体与肿瘤免疫逃逸的关系.方法:应用流式细胞术对37例乳腺癌患者和30例乳腺良性疾病患者血样标本行NKG2A、NKG2D检测,同时检测患者细胞免疫功能.结果:乳腺癌患者与乳腺良性疾病患者相比,NKG2A明显上调,NKG2D表达降低;CD3+、CD4+、CD56+细胞百分比更低(p＜0.05).在乳腺癌患者中,细胞免疫功能低下组及腋淋巴结转移数≥4枚组中,NKG2D表达更低(P＜0.05).Ⅲ+Ⅳ期及C-erbB2高表达乳腺癌患者,NKG2A表达更高(P＜0.05).NKG2A、NKG2D表达率在乳腺癌不同病理类型及组织学分级之间的差异均无统计学意义(P＞0.05).结论:外周血NKG2A、NKG2D的测定对于了解肿瘤患者机体免疫功能的状态、估计病情及判断预后具有一定的临床价值.     

食管癌患者外周血自然杀伤T 细胞及其表面受体NKG2A与NKG2D检测的临床意义

 【摘要】　目的　研究外周血中自然杀伤（NK）T细胞及其CD+8 NKT细胞亚群在食管癌患者与健康人中的表达水平及NKT细胞活性改变,探讨NKT细胞受体与临床病理分期的相关性及其临床意义。方法　采用流式细胞术分析53例食管癌患者及39名健康对照者外周血中NKT细胞及CD+8 NKT亚群,检测NKT细胞受体NKG2A和NKG2D的表达并结合临床病理因素作比较分析。结果　与健康对照组相比,食管癌患者外周血NKT细胞表达增加[（4.32±0.73）%,（5.97±1.29）%]（t＝3.562,P＜0.01）,NKT细胞表面NKG2D的表达水平降低[（17.56±5.92）%,（15.12±1.56）%]（t＝3.892,P＜0.05）,而NKG2A的表达水平升高[（4.02±1.41）%,（5.99±4.59）%]（t＝4.015,P＜0.05）,且其变化与食管癌的病情进展有关。结论　NKT细胞及其CD+8 NKT亚群在食管癌患者中表达增加,提示患者机体抗肿瘤效应的免疫反馈增强;NKT细胞表面活化性受体NKG2D表达减少与其表面抑制性受体NKG2A表达增加可能是致使NKT细胞活性降低及食管癌患者免疫逃逸的机制之一,且这种NKT细胞表面受体的变化和食管癌病情的发展有一定的相关性。     

细胞因子组合体外扩增人NK细胞的研究

目的：探讨细胞因子IL-2、IL-12、IL-15和IL-18组合对体外扩增人外周血来源的NK细胞受体表达及杀伤肿瘤细胞能力。方法：NK细胞的培养分为cIK组、IL2＋II,15组和IL-2＋IL-12＋IL-15＋IL-18组;流式细胞术检测培养的细胞表型及NK细胞受体表达;LDH法检测不同效靶比NK细胞对不同肿瘤细胞株的杀伤作用。结果：外周血淋巴细胞经细胞因子IL-2＋IL广12＋IL-15＋IL-18组合作用下,17d后NK细胞（CD3-CD56＋）比例上升至80％以上,NK细胞数扩增〉1000倍,明显高于其他两组,F=37．154,P〈0．001。NK细胞活化标志CD69。‘分子增加（t-21．271,P〈0．001）,NK细胞活化性受体（NKG2D、NKp30、NKp44和NKp46）均有不同程度上调（P值均〈0．05）,而NK细胞抑制性受体CDl58b（t=3．416,P=0．021）和CDl59a（t=4．209,P=0．018）不同程度下调,T淋巴细胞、B淋巴细胞（CD19＋）、单核巨噬细胞（CD14＋）、调节性T细胞（T—reg）等均显著减少,P〈0．001。对扩增的NK细胞杀伤敏感的肿瘤细胞株均高表达MHCI类相关蛋白A（majorhistocompatibility complexclass Ichainrelated moleculesA,MICA）,MICA表达分别为K562（46．2±3．2）％、MCF-7（56．5±4．7）％、HTC-8（52．5±4．1）％和Eca-109（36．5±2．5）％,而对NK细胞抵抗的细胞株均低表达或不表达MICA分子,分别为Raji0、MDA-MB-435s0和HT-29（1．2±0．8）％。结论：细胞因子IL-2＋IL-12＋IL-15＋IL-18组合能有效的扩增外周血来源NK细胞,上调其活化性受体,下调抑制性受体,其数量及功能均满足临床治疗需要。     

NK细胞活化受体NKG2D及sMICA在晚期肺癌免疫监视中的作用

背景与目的 NK细胞活化受体NKG2D及sMICA是近来肿瘤研究领域热点之一.本研究旨在观察晚期肺癌患者外周血中NK细胞受体NKG2D及sMICA表达水平的变化,并探讨它们在晚期肺癌免疫监控中的作用及其临床意义.方法 采用流式细胞术榆测115例肺癌患者外周血NK细胞受体NKG2D、T淋巴细胞哑群及NK细胞百分比,采用酶联免疫吸附反应检测肺癌患者外周血sMICA值,并以50例健康人作为对照.结果 晚期肺癌患者外周血sMICA、CD8+T细胞、NK细胞数量较对照组明显升高,而NK细胞受体NKG2D、CD3+T细胞、CD4+>T细胞、CD4+T/CD8+T值较对照组下降.NK细胞受体NKG2D和sMICA呈负相关(r=-0.319,P<0.05).NK细胞受体NKG2D与CD4+T细胞、CD4+T/CD8+T成正相关(P0.05),与CD8+T细胞成负相关(P<0.05);sMICA与CD4+T细胞、CD4+T/CD8+T成负相关(P<0.05),与CD8+T细胞成正相关(P<0.05);它们与CD3+T、NK细胞均无相关性(P>0.05).结论 外周血sMICA上调介导NK细胞活化受体NKG2D下调机制参与了晚期肺癌以肿瘤为中心抑制免疫网络的形成,它们可作为监视晚期肺癌患者免疫状态的参考指标,也可作为评估肺癌发生、发展的参考依据.     

Role of NKG2D in cytokine-induced killer cells against multiple myeloma cells

The cytokine-induced killer cells (CIK) have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanisms that CIK cell recognizing MM cells remain unknown. Recent studies indicated that the interaction between NKG2D receptor and NKG2D ligands plays an important role in inducing cytotoxicity against various target cells by natural killer cells (NK). We suspect whether NKG2D receptor and NKG2D ligands interaction is also responsible for the killing of MM cells by CIK as the same way did NK cells. We expanded CIK cells from healthy controls with interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2), and checked expression of NK cell receptors on CIK cells by flow cytometry. About 86% bulk CIK cells expressed NKG2D receptor but not other NK receptors, such as CD158a, CD158b and NCRs. We analyzed NKG2D ligands expression in MM patients by flow cytometry, primary plasma cells from 8 out of 13 (62%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. Interestingly, when stimulated with MM cell line U266 that expressed some levels of MICA/B, only NKG2D expressing CIK cells released IFN-γ. CIK cells showed cytotoxicity against NKG2D ligands expressing U266 and primary MM cells, and the cytotoxicity was partially blocked by treating CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligand interaction may be one of the mechanisms by which CIK cells kill MM cells.     

Natural killer receptors on CD8 T cells and natural killer cells from different HLA-C phenotypes in melanoma patients.

PURPOSE: Because immune mechanisms involved in cutaneous melanoma have not been fully elucidated, efforts have been made to achieve prognosis markers and potential targets for immune therapies, but they have not been entirely fruitful thus far. Therefore, the goal of this study was to investigate the involvement of early changes in CD8 T cells and CD56 natural killer (NK) cells expressing NK receptors in different HLA-C dimorphism groups of melanoma patients. EXPERIMENTAL DESIGN: CD8 T cells and CD56 NK cells were analyzed in 41 patients and 39 sex- and age-matched controls with different HLA-C genotypes by flow cytometry. HLA-C dimorphism at position 80 was tested by PCR sequence-specific primers and PCR sequence-specific oligonucleotide to examine whether it could mediate in the emergence of cells expressing killer cell immunoglobulin-like receptors. RESULTS: Thirty-five of 41 patients had benign sentinel node, and showed an imbalance in the absolute number of CD8(+)DR(+) or CD8(+)CD161(+) peripheral blood T cells according to the CD28 coexpression compared with controls. CD8(+)CD28(-)CD158a(+) T and CD56(+)CD158a(+) NK cells were significantly increased in HLA-C(Lys80) homozygous nonmetastatic patients, whereas only CD56(+)CD158a(+) NK cells increased in heterozygous ones. An up-regulation of the CD158a KIR receptor was also seen on NK cells but not in T cells of patients at advanced disease stages. CONCLUSIONS: This work provides, for the first time, evidence of immune activation in early stages of cutaneous melanoma, together with an increase of cells expressing CD158a in patients bearing the corresponding HLA-C ligand, which may be important to evaluate the disease progression and to use individualized immune therapeutic approaches.     

Analysis of the Expression of Surface Receptors on NK Cells and NKG2D on Immunocytes in Peripheral Blood of Patients with Nasopharyngeal Carcinoma

Background: The aberrant expression of surface receptors on immunocytes may represent potential markers of tumorescape for nasopharyngeal carcinoma (NPC). The aim of this study was to investigate the expression of representativereceptors on natural killer (NK) cells and NK group 2, member D (NKG2D) on immunocytes in the peripheral bloodof patients with NPC. Methods: Patients (n = 64) with NPC prior to initiation of treatment were defined as the studygroup. Healthy volunteers (n = 31) served as the control group. The expression of NK cells and NKT cells; the triggeringreceptors NKp30, NKp44, and NKp46 on NK cells; the activating receptor NKG2D on NK cells, CD4+ T cells, andCD8+ T cells; and the inhibitory receptors CD158b and CD159a on NK cells were analyzed by flow cytometry in thetwo groups. Results: Here, our study showed that no differences were observed in terms of the numbers of NK cells orNKT cells, or the expression of CD158b and CD159a on the surface of NK cells between the two groups. Nevertheless,the expression levels of NKp30 and NKp46 on NK cells in the NPC patients were significantly lower than in the healthyindividuals (P < 0.05). No differences existed in the expression of NKG2D on NK cells, but NKG2D on CD8+ T cellsshowed a markedly lower expression in the study group (P < 0.001). Conclusions: Our findings may reflect a possiblemechanism of immune evasion for NPC. The enhancement of immunotherapy concerning NKp30, NKp46, and NKG2Dmay be an innovative treatment strategy for patients with NPC.     

Up-regulation of inhibitory natural killer receptors CD94/NKG2A with suppressed intracellular perforin expression of tumor-infiltrating CD8+ T lymphocytes in human cervical carcinoma

Inhibitory signals that govern the cytolytic functions of CD8(+) T lymphocytes have been linked to the expression of natural killer cell receptors (NKRs) on CTLs. There is limited knowledge about the induction of inhibitory NKR (iNKR) expression in vivo. Up-regulation of iNKRs has been linked to the modulation of the virus- and/or tumor-specific immune responses in animal models. In the present study, we directly examined the expression of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human cervical cancer. We found that in human cervical cancer, the percentage expression of immunoglobulin-like NKR(+)CD8(+) T lymphocytes were similar in gated CD8(+)-autologous TILs and peripheral blood mononuclear cells. On the contrary, cervical cancer-infiltrating CD8(+) T lymphocytes expressed up-regulated C-type lectin NKRs CD94/NKG2A compared with either peripheral blood CD8(+) T cells or normal cervix-infiltrating CD8(+) T lymphocytes. Dual NKR coexpression analyses showed that CD94 and NKG2A were mainly expressed on CD56(-)CD161(-)CD8(+) TILs within the cancer milieu. Immunohistochemical study showed that cervical cancer cells expressed abundant interleukin 15 (IL-15) and transforming growth factor-beta (TGF-beta). In kinetic coculture assay, cervical cancer cells can promote the expression of CD94/NKG2A on CD8(+) T lymphocytes. The cancer-derived effects can be reversed by addition of rIL-15Ralpha/Fc and anti-TGF-beta antibody. Functional analyses illustrated that intracellular perforin expression of CD8(+) T cells was minimal upon up-regulation of CD94/NKG2A. Kinetic cytotoxicity assays showed that up-regulated expressions of CD94/NKG2A restrain CD8(+) T lymphocyte cytotoxicity. Our study strongly indicated that cervical cancer cells could promote the expression of iNKRs via an IL-15- and possibly TGF-beta-mediated mechanism and abrogate the antitumor cytotoxicity of TILs.     

K562工程细胞联合IL-2扩增方案对人NK细胞扩增和活化的效果

目的：评价新建立的K562工程细胞联合IL-2扩增方案对人NK细胞扩增和活化的效果。 方法：采集健康志愿者和肿瘤患者的外周血PBMC并分离NK细胞,采用前期构建的K562工程细胞(将IL-15、4-1BBL和IL-18在白血病K562细胞上进行跨膜表达获取）联合IL-2培养方案对NK细胞进行扩增和活化,以流式细胞术检测NK细胞的扩增效果和NK细胞表面受体表达水平,CCK-8法检测扩增后NK细胞对肿瘤细胞的杀伤活性和ADCC活性,CCK-8法检测在培养方案扩增末期加入TKD多肽对NK细胞的活化效果。结果：对于健康志愿者的NK细胞,新建立扩增培养方案可使NK细胞在PBMC中的比例提高至（93±3）%;使NK细胞中活化性受体NKG2D、CD94、NKp30、NKp44和NKp46的比例分别提高60%、40%、20%、40%和63%,而抑制性受体的表达变化不大;扩增后NK细胞对白血病细胞K562、肺癌细胞A549、肝癌细胞SMMU-7721和乳腺癌细胞MCF-7的杀伤活性分别提高了19%、29%、26%和28%,其ADCC活性从（33±5.6）%上升至（65±12）%;方案中增加TKD可使NK细胞的杀伤活性从（86±4）%提高至（96±2）%。对于肿瘤患者的NK细胞,新扩增方案使其在PBMC的比例提高至（90.0±8.0）%,其对K562细胞的杀伤活性提高了17%左右。 结论： K562工程细胞联合IL-2扩增方案可高效扩增NK细胞,明显激活其杀伤活性,扩增和活化的NK细胞可满足临床治疗的需要。     

Altered CD94/NKG2A and perforin expression reduce the cytotoxic activity in malignant pleural effusions

CD94/NKG2A is an inhibitory receptor expressed by NK cells and cytotoxic lymphocytes and, upon activation by HLA-E, downregulates the cytolytic activities of these cells thus representing a tumour immune escape mechanism.This study was aimed at assessing whether cytotoxic lymphocytes (CD8+) and NK cells from malignant pleural effusions have a deregulated expression of CD94/NKG2A.The expression of membrane CD94/NKG2A and perforin was evaluated by flow-cytometry in CD8+ and NK cells from pleural effusions and autologous peripheral blood of cancer (n = 19) and congestive heart failure (CHF) (n = 11) patients. Intracellular CD94/NKG2A expression was evaluated by flow-cytometry in pleural effusion CD8+ and NK cells from cancer patients (n = 10). Cytotoxic activity against cancer cells exerted by pleural and autologous peripheral blood T lymphocytes from cancer patients was assessed by flow-cytometry assay.Pleural CD8+ from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood and CHF pleural effusions. Reduced numbers of NK cells were present in pleural effusions from both cancer and CHF patients. Pleural NK from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood. Pleural T lymphocytes from cancer patients exhibited a reduced cytotoxic activity against cancer cells when compared to autologous peripheral blood T lymphocytes. The intracellular expression of CD94/NKG2A in CD8+ and NK cells from cancer patients was higher than membrane expression.In conclusion, this study provides compelling evidences of new mechanisms underlying the reduced host defence against cancer within the pleural space.     

The requirement for DNAM-1, NKG2D, and NKp46 in the natural killer cell-mediated killing of myeloma cells

Recent evidence suggests a role for natural killer (NK) cells in the control of multiple myeloma. We show that expression of the NK cell receptor DNAM-1 (CD226) is reduced on CD56(dim) NK cells from myeloma patients with active disease compared with patients in remission and healthy controls. This suggested that this receptor might play a role in NK-myeloma interactions. The DNAM-1 ligands Nectin-2 (CD112) and the poliovirus receptor (PVR; CD155) were expressed by most patient myeloma samples analyzed. NK killing of patient-derived myelomas expressing PVR and/or Nectin-2 was DNAM-1 dependent, revealing a functional role for DNAM-1 in myeloma cell killing. In myeloma cell lines, cell surface expression of PVR was associated with low levels of NKG2D ligands, whereas cells expressing high levels of NKG2D ligands did not express PVR protein or mRNA. Furthermore, NK cell-mediated killing of myeloma cell lines was dependent on either DNAM-1 or NKG2D but not both molecules. In contrast, the natural cytotoxicity receptor NKp46 was required for the killing of all myeloma cell lines analyzed. Thus, DNAM-1 is important in the NK cell-mediated killing of myeloma cells expressing the cognate ligands. The importance of NKp46, NKG2D, and DNAM-1 in myeloma killing mirrors the differential expression of NK cell ligands by myeloma cells, reflecting immune selection during myeloma disease progression.     

成年人脂肪源间充质干细胞治疗急性移植物抗宿主病后CD+8 T细胞亚群的变化

  目的 初步探讨成年人脂肪源间充质干细胞（AMSC）治疗急性移植物抗宿主病（aGVHD）的分子机制。方法 3例行异基因造血干细胞移植术后发生aGVHD的患者,以每1 kg体重2×106个细胞剂量静脉输注AMSC;首先应用尼龙毛柱分离外周血T淋巴细胞,再经CD8磁珠分选出CD+8 T淋巴细胞,应用流式细胞术检测发生aGVHD患者使用AMSC前后外周血CD+8 T细胞亚群的变化。结果 与输注AMSC前相比,输注AMSC后,CD+8 T细胞中的CD+8 CD-28亚群显著上调,同时,患者的aGVHD得以有效控制。结论 AMSC治疗aGVHD的作用机制可能与其上调CD+8 CD-28 T细胞亚群有关,CD+8 T细胞可能是AMSC作用的靶细胞。