Herceptin与卡铂联合应用对宫颈癌细胞的抑制及其机制

目的:研究自然杀伤(natural killer,NK)细胞表面杀伤细胞免疫球蛋白样受体(killer immunoglobulin-like recep-tor,KIR)和HLA-Cw配体不相合的异基因NK细胞对乳腺癌细胞的体外杀伤作用;分析杀伤活化受体NKG2D及其MICA配体表达水平与NK细胞对乳腺癌细胞杀伤敏感性之间的关系。方法:取10例健康人及5例乳腺癌患者外周血10ml,采用MACS系统NK细胞免疫磁珠分选试剂盒负向分选高纯度NK细胞。取乳腺癌细胞(MCF-7、MDA-MB-435s和SK-Br3)和NK细胞各1×105个,碱裂解法抽提DNA,PCR-SSP方法分别检测HLA-Cw位点、KIR位点表达。MTT法检测KIR相合与不相合组的NK细胞对乳腺癌细胞的杀伤率。流式细胞术检测NK细胞NKG2D表达水平以及RT-PCR方法检测乳腺癌细胞MICA表达。结果:MACS系统分选出的NK细胞,经流式细胞术检测,其纯度在90%以上;KIR与HLA-Cw相合组的NK细胞对乳腺癌细胞株的杀伤率明显高于不相合组,G1组和G2组NK细胞对MDA-MB-435s(G1组)杀伤率分别为(73.2±14.5)%和(34.2±7.6)%,对SK-Br3(G1组)杀伤率为(67.3±12.5)%和(36.5±7.7)%,而对MCF-7(G2组)杀伤率分别为(36.7±8.5)%和(76.5±11.7)%。结果还显示,3株乳腺癌细胞均表达MICA分子;NK细胞与MCF-7细胞共培养,可上调NK细胞表面NKG2D的表达。结论:NK细胞对乳腺癌细胞的杀伤并非由KIR的不相合机制介导;乳腺癌细胞表面表达的MICA分子可上调NK细胞表面NKG2D的表达,激发NK细胞对乳腺癌细胞的细胞毒效应。     

自然杀伤细胞与肿瘤细胞之间的免疫编辑

自然杀伤细胞(nature killer cell, NK)是机体天然免疫的主要承担者，NK细胞表面表达的抑制性杀伤细胞免疫球蛋白样受体(inhibitory killer cell immunoglobulinlike receptor，KIR)和活化性受体NKG2D是调节NK细胞杀伤活性的主要受体。NK细胞通过其细胞表面的活化性受体和肿     

MHCI类样分子(MICA)在部分肿瘤组织和肿瘤细胞系中的表达及临床意义

目的:探讨MHC Ⅰ类样分子(MICA)在肿瘤组织和肿瘤细胞系中的表达及其临床意义.方法:分别采用半定量RT-PCR和免疫组织化学技术检测肿瘤组织和肿瘤细胞系MICA表达,MTT法测定NK细胞细胞毒活性.结果:人红白血病细胞K562、喉癌细胞系Hep2、宫颈癌细胞系Hela、肝癌细胞系HepG2和结直肠癌细胞系HT29均有MICA mRNA和蛋白表达,而人Burkitt淋巴瘤Raji和高转移肺癌PG不表达MICA.MICA表达阳性的肿瘤细胞对NK杀伤敏感性明显高于MICA阴性者.多数肿瘤组织存在MICA mRNA和蛋白表达,但并非在所有肿瘤均有表达,癌旁组织均无MICA表达.结论:肿瘤细胞表达MICA可激发NK细胞对肿瘤的杀伤,NKG2D及其配体的相互作用在肿瘤免疫监视中起着非常重要的作用,肿瘤细胞分泌sMICA分子为肿瘤发生免疫逃逸的机制之一.     

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。     

NKG2D配体在13种肿瘤细胞系中的表达及意义

背景与目的:NKG2D及其配体的相互作用在肿瘤免疫监视中起着非常重要的作用。本研究探讨NKG2D配体在13种肿瘤细胞系中的表达及其意义。方法:采用半定量RT-PCR法检测肿瘤细胞系中NKG2D配体的mRNA表达。应用MTT法检测人NK细胞对肿瘤细胞的杀伤活性,免疫组织化学技术和Western blot法检测肿瘤细胞中MHCⅠ类相关蛋白A(MHC classⅠchain-related A)蛋白表达情况。结果:13种肿瘤细胞系表达不同水平的NKG2D配体mRNA。其中Hep2细胞中MICA强阳性表达,HeLa、HepG2、MDA231、HT29、SGC7901、M21、K562、Jurkat细胞MICA表达阳性,而Caski、PG、HL-60和Raji细胞中MICA mRNA阴性。MICA和MICB的表达水平与NK细胞杀伤活性具有高度相关性(r=0.851,P<0.001;r=0.652,P<0.05)。除ULBP3外,其余ULBP成员的表达水平与NK细胞杀伤均无相关性。结论:在6种人NKG2D配体中,MICA的表达水平与肿瘤细胞对NK细胞杀伤的敏感性关系最为密切,肿瘤细胞MICA的表达水平可能决定着机体NK细胞抗肿瘤免疫应答的强弱。     

重组可溶性MICA蛋白负调控NK细胞对白血病细胞的杀伤活性

目的:体外研究重组可溶性MHCⅠ类分子相关A(soluble MHC classⅠchain-related gene A,sMICA)蛋白对自然杀伤细胞(natural killer cell,NK cell)表面活化性受体NKG2D(natural killer group 2 member D)的表达、杀伤白血病细胞的活性和分泌IFN-γ的影响。方法:免疫磁珠分选健康人外周血NK细胞,分为空白对照组、重组sMICA组(200、500、800μg/L三亚组),相互作用24 h后,流式细胞术检测NK细胞NKG2D表达水平,LDH释放法检测NK细胞对白血病细胞K562的杀伤活性,ELISA法检测培养上清中IFN-γ的水平。结果:200、500、800μg/L sMICA作用组与对照组相比,NK细胞表面NKG2D的表达均下调[(68.79±6.87)%,(55.75±9.31)%、(45.14±5.70)%vs(92.75±6.91)%,P<0.05或P<0.01],均抑制NK细胞杀伤白血病细胞K562的活性[(18.67±2.35)%、(15.01±2.25)%、(7.33±2.52)%vs(36.33±2.51)%,P<0.05或P<0.01],均抑制IFN-γ的分泌[(164.48±22.48)、(112.71±10.89)、(70.23±9.64)vs(313.72±16.06)pg/ml,P<0.05,P<0.01]。结论:急性白血病细胞表面脱落的sMICA可下调NK细胞NKG2D的表达和IFN-γ的分泌,抑制了NK细胞对白血病细胞的杀伤活性。     

MICA蛋白在肿瘤生物治疗中的作用

近年,NK细胞的活化性受体NKG2D已成为研究热点,作为NKG2D配体的MICA(人类MHC-Ⅰ类分子链相关基因A)蛋白越来越受关注.机体内MICA以膜型和分泌型两种形式存在.膜型MICA与NKG2D相互作用在肿瘤免疫监视中起着非常重要的作用;与此相反,分泌型MICA不仅下调NKG2D受体的表达,而且下调其细胞毒活性,对免疫细胞抗肿瘤起阻碍作用,可能是肿瘤免疫逃逸的一种新的机制.因此,可以利用这些特点发挥MICA蛋白在肿瘤生物治疗中的应用价值.     

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.     

NKG2D介导NK 细胞对鼻咽癌细胞杀伤作用的体外研究

 目的 探讨鼻咽癌CNE2细胞表面HLA-Ⅰ类分子表型和NKG2D配体的表达情况，进一步了解其对同种异体NK细胞杀伤活性的影响。方法 流式细胞仪检测NKG2D的配体MICA、MICB、ULBP1、ULBP2、ULBP3在K562、CNE2细胞的表达情况。PCR-SSP法分析CNE2细胞HLA-A、B、Cw分型和NK细胞KIR分型。LDH释放法测定5例健康者NK细胞在不同效靶比时对K562、CNE2细胞的杀伤活性，效靶比20：1时观察抗NKG2D配体的单抗对NK细胞杀伤K562、CNE2细胞活性的影响。结果 CNE2细胞表达MICA、MICB、ULBP2，不表达ULBP1、ULBP3。K562细胞表面表达MICA、MICB、ULBP1、ULBP2、ULBP3。5例健康者NK细胞抑制性KIR与CNE2细胞表面的HLA-Ⅰ类分子之间存在错配。效靶比5：1、10：1、20：1、30：1时NK细胞对K562、CNE2细胞的杀伤活性分别为（29．02±0．45）％、（10．50±2．17）％；（44．43±1．36）％、（27．68±1．47）％；（57．82±1．35）％、（36．99±3．13）％：（71．24±2．36）％、（55．00±2．20）％，在各效靶比时NK细胞对K562细胞的杀伤活性较CNE2细胞明显增强（P=0．000）；在效靶比20：1时anti-MICA、anti-MICB、anti-ULBP1、anti—ULBP2、anti-ULBP3可明显抑制NK细胞对K562细胞的杀伤活性，与阻断前相比有显著性差异（P=0．000）；anti—MICA、anti—MICB、anti—ULBP2可明显抑制NK细胞对CNE2细胞的杀伤活性，与阻断前相比有显著性差异（P〈0．01），但anti—ULBP1、anti—ULBP3不能阻断NK细胞对CNE2细胞的杀伤活性。结论 NKG2D配体影响NK细胞对靶细胞的杀伤活性，提高NKG2D配体的表达有可能提高NK细胞的抗肿瘤活性。     

NKG2D及其配体与肿瘤免疫治疗研究新进展

NKG2D为自然杀伤(NK)细胞表面的C型凝集素样活化性受体,NKG2D与肿瘤细胞表面配体结合,杀伤肿瘤细胞,但在肿瘤患者和患肿瘤小鼠体内存在着依赖NKG2D的免疫逃避机制.近年来利用各种分子生物技术调节受体和配体的表达来避免免疫逃避.     

Direct and natural killer cell-mediated antitumor effects of low-dose bortezomib in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) displays particular resistance to conventional cytostatic agents. Alternative treatment strategies focus on novel substances exhibiting antineoplastic and/or immunomodulatory activity enhancing for example natural killer (NK) cell antitumor reactivity. However, tumor-associated ligands engaging activating NK cell receptors are largely unknown. Exceptions are NKG2D ligands (NKG2DL) of the MHC class I-related chain and UL16-binding protein families, which potently stimulate NK cell responses. We studied the consequences of proteasome inhibition with regard to direct and NK cell-mediated effects against HCC. EXPERIMENTAL DESIGN: Primary human hepatocytes (PHH) from different donors, hepatoma cell lines, and NK cells were exposed to Bortezomib. Growth and viability of the different cells, and immunomodulatory effects including alterations of NKG2DL expression on hepatoma cells, specific induction of NK cell cytotoxicity and IFN-gamma production were investigated. RESULTS: Bortezomib treatment inhibited hepatoma cell growth with IC(50) values between 2.4 and 7.7 nmol/L. These low doses increased MICA/B mRNA levels, resulting in an increase of total and cell surface protein expression in hepatoma cells, thus stimulating cytotoxicity and IFN-gamma production of cocultured NK cells. Importantly, although NK cell IFN-gamma production was concentration-dependently reduced, low-dose Bortezomib neither induced NKG2DL expression or cell death in PHH nor altered NK cell cytotoxicity. CONCLUSIONS: Low-dose Bortezomib mediates a specific dual antitumor effect in HCC by inhibiting tumor cell proliferation and priming hepatoma cells for NK cell antitumor reactivity. Our data suggest that patients with HCC may benefit from Bortezomib treatment combined with immunotherapeutic approaches such as adoptive NK cell transfer taking advantage of enhanced NKG2D-mediated antitumor immunity.     

MICA triggering signal for NK cell tumor lysis is counteracted by HLA-G1-mediated inhibitory signal

MICA, a highly glycosylated membrane-anchored cell-surface MHC Class I-related chain, has recently been reported to activate NK cell cytolytic responses in epithelial tumors. Tumor cells may escape from NK lysis by counteracting NK cytotoxicity activating signals with inhibitory ones. Among the molecules that mediate an NK inhibitory signal, HLA-G1, a non-classical MHC Class I antigen, is of particular interest. HLA-G1 is ectopically expressed in various tumors, including melanoma and constitutes the major NK inhibitory ligand in the M8 melanoma cell line when coexpressed with HLA-A, -B, -C and -E molecules. We have evaluated the balance between 2 powerful signals that affect NK cell tumor lysis, one inhibitory and the other one activating, respectively HLA-G1 and MICA. For this purpose, we transfected the M8 melanoma cell line, which spontaneously expresses MICA, with HLA-G1 cDNA, using it as a target for the NKL effector. We carried out cytotoxicity assays, using antibodies that disrupt interactions between the MICA and HLA-G1 ligands and their respective NK effector counterparts, the NKG2D activating and ILT2 inhibitory receptors. Results showed that 1) MICA expressed in the M8 melanoma cell line triggered NK cell tumor lysis and 2) HLA-G1 coexpression mediated the inhibition of NK cytotoxicity by mitigating the MICA activating signal. HLA-G1 expression in a tumor cell line in which MICA is switched on would therefore appear to be a powerful way to turn off NK cells, supporting the emerging idea that the balance between positive and negative NK cytolysis signals critically influences tumor progression.     

Interferon‐γ down‐regulates NKG2D ligand expression and impairs the NKG2D‐mediated cytolysis of MHC class I‐deficient melanoma by natural killer cells

NKG2D operates as an activating receptor on natural killer (NK) cells and costimulates the effector function of αβ CD8⁺ T cells. Ligands of NKG2D, the MHC class I chain‐related (MIC) and UL16 binding protein (ULBP) molecules, are expressed on a variety of human tumors, including melanoma. Recent studies in mice demonstrated that NKG2D mediates tumor immune surveillance, suggesting that antitumor immunity in humans could be enhanced by therapeutic manipulation of NKG2D ligand (NKG2DL) expression. However, signals and mechanisms regulating NKG2DL expression still need to be elucidated. Here, we asked whether the proinflammatory cytokine Interferon‐γ (IFN‐γ) affects NKG2DL expression in melanoma. Cell lines, established from MHC class I‐negative and ‐positive melanoma metastases, predominantly expressed MICA and ULBP2 molecules on their surface. Upon IFN‐γ treatment, expression of MICA, in some cases, also of ULBP2 decreased. Besides melanoma, this observation was made also for glioma cells. Down‐regulation of NKG2DL surface expression was dependent on the cytokine dose and the duration of treatment, but was neither due to an intracellular retention of the molecules nor to an increased shedding of ligands from the tumor cell surface. Instead, quantitative RT‐PCR revealed a decrease of MICA‐specific mRNA levels upon IFN‐γ treatment and siRNA experiments pointed to an involvement of STAT‐1 in this process. Importantly, IFN‐γ‐treated MHC class I‐negative melanoma cells were less susceptible to NKG2D‐mediated NK cell cytotoxicity. Our study suggests that IFN‐γ, by down‐regulating ligand expression, might facilitate escape of MHC class I‐negative melanoma cells from NKG2D‐mediated killing by NK cells. © 2008 Wiley‐Liss, Inc.     

Regulation Roles of MICA and NKG2D in Human Renal Cancer Cells

Objective: Our aim was to investigation the roles of MHC class I chain-related gene A(MICA) and naturalkiller cell group 2D(NKG2D) in human renal cancer cells. Materials and Methods: The expression of membraneMICA (mMICA) on renal cells and NKG2D on NK cells were detected by flow cytometry (FCM); the contentof sMICA were detected by enzyme linked immunosorbent assay (ELISA) and the distribution of mMICA onrenal tumor tissues by immunohistochemistry; the interaction between MICA and NKG2D was observed byantibody closed method. Results: Our results showed that the expression of mMICA in renal cancer tissues wassignificantly higher than in controls, where the soluble MICA was not expressed. Cytotoxic activity of NK cellswas significantly reduced after exposure to NKG2D and MICA antibodies (P<0.05), and serum containing sMICAcan obviously lower the function of NKG2D (P<0.05). Conclusions: The interaction of mMICA and NKG2Dplay important roles in mediation of cytotoxicity of NK cells in RCC. On the other hand, sMICA may mediatetumor immune escape through down- regulated NKG2D expression.     

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.     

Valproic Acid Upregulates NKG2D Ligand Expression through an ERK-dependent Mechanism and Potentially Enhances NK Cell-mediated Lysis of Myeloma

Modulation of the antitumor immune response through the engagement of NKG2D receptors with their ligands (L) on targets represents a promising therapeutic approach against cancer. In this study, we tested the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on the expression of NKG2D ligands in myeloma cells. We demonstrated that VPA was able to upregulate both protein and mRNA expression of major histocompatibility complex class I-related chain (MIC) A/B and UL16-binding protein (ULBP) 2 without any significant effect on the expression of ULBP1, ULBP3, and ULBP4 or induction of other natural killer (NK) cell ligands, such as NKp30-L, NKp44-L, and NKp46-L in myeloma cells. A ⁵¹Cr release assay and degranulation assay indicated that the induction of MICA/B and ULBP2 augmented NK cell-mediated lysis of myeloma cells, which was abolished by the addition of a blocking NKG2D antibody. Activation of constitutively phosphorylated extracellular signal-regulated kinase (ERK) by VPA is essential for the up-regulation of MICA/B and ULBP2 expressions. Inhibition of ERK using ERK inhibitor PD98059 decreased both MICA/B and ULBP2 expressions and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in ARK resulted in increased MICA/B and ULBP2 expressions and enhanced NK cell lysis. These data indicate that increased sensitivity of VPA-treated myeloma cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway. Our results provide evidence that targeting ERK signaling pathway may be an additional mechanism supporting the antimyeloma activity of HDAC inhibitors and suggest its possible immunotherapeutic value for myeloma treatment.     

Reduced Expression of Natural Killer Cell-Related Activating Receptors by Peripheral Blood Mononuclear Cells from Patients with Breast Cancer and Their Improvement by Zoledronic Acid

Background/aim: Natural killer (NK) cell receptors affect the NK cell-mediated elimination of malignant cells. In this experimental study the effect of Zoledronic acid (ZOL) was investigated on the expression of NK activating- (NKP46 and NKG2D) and inhibitory (KIR2DL1) receptors by Phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from breast cancer (BC) patients. Materials and Methods: Peripheral blood mononuclear cell-extracted RNA from thirty breast cancer women and twenty-five healthy subjects was analyzed for gene expression of NKP46, NKG2D and KIR2DL1 using real time-PCR. Then, the PBMCs from BC patients were cultured in the presence of PHA with 5 μg/ml, 10 or 20 μg/ml of ZOL for 32 hours and expression of the aforementioned receptors was determined. Results: Expression of NKP46, NKG2D and NKP46/KIR2DL1 ratio in BC women were lower than healthy group (P<0.01, P<0.04 and P<0.05, respectively). NKP46 expression was up-regulated by PHA-stimulated PBMCs treated with 10 μg/ml and 20 μg/ml of ZOL compared with PHA-stimulated cultures (P<0.01 and P<0.05, respectively). NKG2D expression remarkably increased by PHA-stimulated cultures treated with 5 μg/ml, 10 μg/ml and 20 μg/ml of ZOL compared with PHA-stimulated cultures (P<0.05 and P<0.02 and P<0.04, respectively). Conclusion: Expression of NK cell-related activating receptors decreased in BC patients. ZOL can improve the expression of NK activating receptors.