可溶性MICA在乳腺癌免疫逃逸中的作用

目的: 观察乳腺癌患者血清中可溶性MICA(sMICA)表达情况, 探讨sMICA在乳腺癌免疫逃逸中的作用.方法: ELISA方法检测乳腺肿瘤患者外周血sMICA的分泌.流式细胞术(FCM)检测sMICA及白细胞介素15(IL-15)对NK细胞表面NKG2D表达的影响.MTT法检测NK细胞对乳腺癌细胞的杀伤活性.结果: ELISA结果显示, 血清sMICA含量在健康成年人血清中为阴性, 在乳腺良性肿瘤患者血清中含量为(76.8±22.3)ng/L, 在恶性肿瘤患者血清中含量为(205.36±71.27)ng/L, 乳腺癌患者中血清sMICA含量高低与TNM分期呈正相关.应用含sMICA的血清与NK细胞共培养可明显下调NK细胞的杀瘤活性[(76.2±6.7)% vs (48.4±4.1)%], NK细胞表面NKG2D表达和上清中IFN-λ分泌量也下降.IL-15明显上调NK细胞表面NKG2D表达, 增加培养上清IFN-λ分泌量和增强NK对MCF-7的杀瘤活性.结论: sMICA表达与乳腺癌TNM分期正相关, sMICA通过下调NK细胞NKG2D表达水平而降低NK细胞杀瘤活性, 在肿瘤免疫逃逸中起重要作用.IL-15可以上调NK细胞NKG2D的表达并增强NK细胞杀瘤活性.     

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。     

Soluble MICA as an independent prognostic factor for the overall survival and progression-free survival of multiple myeloma patients

Major histocompatibility complex class I-related chain A (MICA) molecules are frequently expressed in lymphoproliferative malignancies including multiple myeloma (MM). MICA activates NK cells and co-stimulates T cells by interaction with its immunoreceptor NKG2D. In contrast, soluble MICA (sMICA) molecules impair the functions of NKG2D(+) T and NK cells, which may facilitate tumor cell escape from immunosurveillance. Here, we analyzed the clinical relevance of sMICA in 97 MM patients. sMICA (mean+/-SEM pg/ml) was significantly increased (p<0.0001) in patients (429+/-20) compared to controls (230+/-20; N=43). Serial determination showed a strong correlation between increments of sMICA and paraprotein levels (r=0.543, p<0.0001, N=49). sMICA levels >305 pg/ml are associated with a poor overall (p=0.004) and progression-free survival (p=0.002). Multivariate analysis revealed sMICA as an independent predictive factor for overall (p=0.007) and progression-free survival (p=0.002). Thus, our results suggest sMICA as a potent prognostic marker in MM, which may be useful to identify risk patients.     

膜型/可溶型MHC Ⅰ类链相关分子A及其受体NKG2D在骨肉瘤中的表达及其意义

目的探讨膜型／可溶型MHCI类链相关分子A（MICA）及其受体NKG2D在骨肉瘤中的表达及其意义。方法采用免疫组织化学（LSAB法）检测膜型MICA（mMICA）在43例骨肉瘤组织中的表达;用流式细胞仪检测上述病例中的16例患者外周血淋巴细胞NKG2D的表达;用ELISA法检测患者血清中可溶型MICA（sMICA）的水平。结果骨肉瘤细胞广泛表达mMICA（37／43）,外周血淋巴细胞NKG2D的表达普遍下降,两者的表达水平均与骨肉瘤的分化呈正相关（P〈0．05）,与临床分期呈负相关（P〈0．05）。检测16例骨肉瘤患者血清中sMICA含量,其中5例升高,且与骨肉瘤的远处转移及临床分期呈正相关（P〈0．05）。结论（1）骨肉瘤细胞表达mMICA,外周血淋巴细胞NKG2D的表达以及血清中sMICA的含量与骨肉瘤的分化及临床分期相关,因此可望作为骨肉瘤病理诊断和估计预后的生物学指标。（2）MICA-NKG2D介导的免疫监视障碍可能促进了骨肉瘤的进展。     

IL-2促进PBMC来源的NK、NKT、CD8+T细胞高表达NKG2D

目的 观察大剂量IL-2活化的人外周血单个核细胞（PBMC）中，NKG2D在NK细胞、T细胞和NKT细胞表面的表达规律。方法 使用三重免疫荧光标记的流式细胞术检测NKG2D的表达情况。使用sMICA蛋白与人PBMC共同培养，之后使用流式细胞术分析NKG2D在NK细胞中的表达情况。使用半定量RT-PCR方法检测大剂量IL-2活化的人PBMC中NKG2D及其锚定蛋白DAP10 mRNA的表达变化。结果 使用大剂量IL-2活化人PBMC细胞后，NKG2D在NK细胞、CD^＋T细胞和NKT细胞表面的表达均增加，但是在CD4^＋T细胞表面始终不表达。同时IL-2可以拮抗sMICA对NKG2D的下调作用。半定量RT-PCR结果显示，使用大剂量IL-2活化人PBMC之后，NKG2D及其锚定蛋白DAP10的mRNA水平并不发生明显变化。结论 大剂量IL-2培养人PBMC之后，NKG2D在NK细胞、CD8^＋T细胞和NKT细胞表面的表达均增加，可能是PBMC活化并获得广谱抗肿瘤效应的机制之一．     

Sustained localized expression of ligand for the activating NKG2D receptor impairs natural cytotoxicity in vivo and reduces tumor immunosurveillance

Upregulation of the inducible gene products MICA (human) and Rae-1 (mouse) may promote tumor surveillance and autoimmunity by engaging the activating receptor NKG2D on natural killer (NK) cells and T cells. Nevertheless, sustained expression of MICA by tumors can also elicit NKG2D downregulation, perhaps indicating 'immunoevasion'. Investigating this paradox, we report here that constitutive Rae-1epsilon transgene expression in normal epithelium elicited local and systemic NKG2D downregulation, generalized but reversible defects in NK cell-mediated cytotoxicity and mild CD8(+) T cell defects. The extent of NKG2D downregulation correlated well with the incidence and progression of cutaneous carcinogenesis, emphasizing the utility of NKG2D as a marker of tumor resistance. Thus, NKG2D engagement is a natural mediator of immunosurveillance, which can be compromised by locally sustained ligand expression but potentially restored by innate immune activation.     

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.     

5-氟尿嘧啶增强HeLa细胞对自然杀伤细胞敏感性研究

目的 用5-氟尿嘧啶(5-fluorouracil,5-FU)处理HeLa细胞,检测其NKG2D 配体 MICA 的表达及其对NK92细胞杀伤敏感性的变化.方法 不同浓度的 5-FU处理 HeLa细胞,在不同时间点用半定量PCR及流式细胞术检测HeLa细胞表面的NKG2D配体MICA在RNA及蛋白水平的表达变化情况,用MTT法检测NKG2D抗体封闭NK92细胞的NKG2D受体前后, NK92 细胞对HeLa细胞的杀伤作用.结果 不同浓度的5-FU作用于 HeLa 细胞后,半定量RT-PCR结果显示MICA表达随5-FU作用浓度增加逐渐增高.而且40 μg/ml 5-FU作用于HeLa细胞后随着作用时间的延长(0、8、16、24 h)MICA表达增加,流式细胞术检测结果表明,MICA表达的增加主要依赖于未凋亡细胞的 MICA表达.在40μg/ml 5-FU作用24 h,效靶比为2.5∶ 1,5∶ 1,10∶ 1,20∶ 1时都检测到NK92细胞对HeLa细胞的杀伤增强,杀伤作用可部分被NKG2D抗体抑制.结论 5-FU 能够上调HeLa细胞表面NKG2D配体MICA的表达,增强HeLa细胞对NK92细胞的敏感性,提示化疗联合NK细胞免疫治疗宫颈癌可产生协同作用,提高治疗效果.     

NK cell‐mediated anti‐leukemia cytotoxicity is enhanced using a NKG2D ligand MICA and anti‐CD20 scfv chimeric protein

NK cells are important innate cytotoxic lymphocytes that have potential in treatment of leukemia. Engagement of NKG2D receptor on NK cells enhances the target cytotoxicity. Here, we produced a fusion protein consisting of the extracellular domain of the NKG2D ligand MICA and the anti‐CD20 single‐chain variable fragment (scfv). This recombinant protein is capable of binding both NK cells and CD20⁺ tumor cells. Using a human NKG2D reporter cell system we developed, we showed that this fusion protein could decorate CD20⁺ tumor cells with MICA extracellular domain and activate NK through NKG2D. We further demonstrated that this protein could specifically induce the ability of a NK cell line (NKL) and primary NK cells to lyse CD20⁺ leukemia cells. Moreover, we found that downregulation of surface HLA class I expression in the target cells improved NKL‐mediated killing. Our results demonstrated that this recombinant protein specifically lyses leukemia cells by NK cells, which may lead to development of a novel strategy for treating leukemia and other tumors.     

Critical role of the NKG2D receptor for NK cell‐mediated control and immune escape of B‐cell lymphoma

Little is known on the control of lymphomas by NK cells. Here, we study the role of the NK group 2D (NKG2D) receptor for the immunosurveillance of lymphoma. By using transplantable tumors as well as a λ‐myc‐transgenic model of endogenously arising lymphoma and NKG2D‐deficient mice, we show that NK cells eliminate tumor cells in vivo after receiving two signals. One step involved the activation of NK cells giving rise to IFN‐γ expression, which was effected by MHCI^low tumor cells or DCs. However, this was necessary but not sufficient to mediate cytotoxicity. Triggering cytotoxicity additionally required a second step, which could be mediated by engagement of the NKG2D receptor. Thus, NKG2D‐deficient NK cells could become activated in vivo, but they were not able to reject transplanted lymphomas or to degranulate in animals bearing autochthonous lymphomas. Tumor growth in NKG2D‐deficient λ‐myc‐transgenic mice was significantly accelerated compared to NKG2D‐competent animals. Whereas the latter developed tumors that lost expression of NKG2D ligands (NKG2D‐L) in late disease stages, this did not occur in NKG2D‐deficient mice. This indicates that NK cells and the NKG2D receptor play a role for control of lymphomas and that selection for NKG2D‐L loss mutants provides a mechanism of tumor escape.     

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.     

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.     

Estrogen upregulates MICA/B expression in human non-small cell lung cancer through the regulation of ADAM17

Estrogen is involved in promoting lung cancer cell division and metastasis. MICA and MICB function as ligands for NKG2D, an important immunoreceptor expressed on natural killer (NK) cells. However, whether estrogen regulates MICA/B expression and affects tumor immune escape remains unknown. In this study, we measured the mRNA levels of MICA, MICB and ADAM17in non-small cell lung cancer (NSCLC) cell lines treated with estrogen. Surface expression of MICA/B on LTEP-a2 and A549 was detected using flow cytometry. We demonstrate that both mRNA and secretory protein levels of MICA/B in lung adenocarcinoma cell lines were upregulated by estradiol. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. This secretion of MICA/B downregulated the NKG2D receptor on the surface of NK92 cells and impaired the cytotoxic activity of NK cells. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. Furthermore, a significant correlation between the concentration of estradiol and the expression of MICA was found in tumor tissues of NSCLC patients. Therefore, we conclude that estrogen can regulate the expression and secretion of MICA/B through ADAM17, which helps lung cancer cells escape NKG2D-mediated immune surveillance.     

Potentiation of NK cell-mediated cytotoxicity in human lung adenocarcinoma: role of NKG2D-dependent pathway

Natural cytotoxicity receptors and NKG2D correspond to major activating receptors involved in triggering of tumor cell lysis by human NK cells. In this report, we investigated the expression of NKG2D ligands (NKG2DLs), MHC class I-related chain (MIC) A, MICB and UL16-binding proteins 1, 2 and 3, on a panel of human non-small-cell lung carcinoma cell lines, and we analyzed their role in tumor cell susceptibility to NK cell lysis. Although adenocarcinoma (ADC) cells expressed heterogeneous levels of NKG2DLs, they were often resistant to NK cell-mediated killing. Resistance of a selected cell line, ADC-Coco, to allogeneic polyclonal NK cells and autologous NK cell clones correlated with shedding of NKG2DLs resulting from a matrix metalloproteinase (MMP) production. Treatment of ADC-Coco cells with a MMP inhibitor (MMPI) combined with IL-15 stimulation of autologous NK cell clones lead to a potentiation of NK cell-mediated cytotoxicity. This lysis is mainly NKG2D mediated, since it is abrogated by anti-NKG2D-neutralizing mAb. These results suggest that MMPIs, in combination with IL-15, may be useful for overcoming tumor cell escape from the innate immune response.     

Ovarian tumor-associated microRNA-20a decreases natural killer cell cytotoxicity by downregulating MICA/B expression

MicroRNAs （miRNAs） are a class of small non-coding regulatory RNAs, and changes in miRNAs are involved in tumor origin and progression. Studies have shown that miR-20a is overexpressed in human ovarian cancer tissues and that this miRNA enhances long-term cellular proliferation and invasion capabilities. In this study, a positive correlation between serum miR-20a expression and ovarian cancer stage was observed. We found that miR-20a binds directly to the 3＇-untranslated region of MICA/B mRNA, resulting in its degradation and reducing its protein levels on the plasma membrane. Reduction of membrane-bound MICA/B proteins, which are ligands of the natural killer group 2 member D （NKG2D） receptor found on natural killer （NK） cells, y＋ T cells and CD8＋ T cells, allows tumor cells to evade immune-mediated killing. Notably, antagonizing miR-20a action enhanced the NKG2D-mediated killing of tumor cells in both in vitro and in vivo models of tumors. Taken together, our data indicate that increased levels of miR-20a in tumor cells may indirectly suppress NK cell cytotoxicity by downregulating MICA/B expression. These data provide a potential link between metastasis capability and immune escape of tumor cells from NK cells.     

Relationship between soluble MICA and the MICA A5.1 homozygous genotype in patients with oral squamous cell carcinoma

NK and cytotoxic T cells play an important role in the elimination of virus-infected and tumor cells through NKG2D activating receptors, which can promote the lysis of target cells by binding to the major histocompatibility complex class I-related chain A (MICA) proteins. Polymorphisms in MICA may influence its binding to the NKG2D. The soluble form of MICA is released from the surface of tumor cells of epithelial origin. Whereas MICA expressed on the cell surface stimulates the immunoreceptor natural killer group 2, member D (NKG2D), the secreted form down-regulates NKG2D activity, thus allowing the tumor to escape immunosurveillance by NKG2D-expressing cells. In this study, we examined the association between MICA gene microsatellite polymorphisms and serum levels of soluble MICA in patients with oral squamous cell carcinoma (OSCC). We found that patients with OSCC were more likely to have the A5.1 allele when compared to healthy subjects and also more likely to be homozygous for this allele (p = 0.041). Patients with the homozygous A5.1 genotype had higher levels of soluble MICA (p = 0.031) and a lower survival rate (p = 0.026).     

青海高原藏族胃癌患者自然杀伤细胞活化性受体D及其配体MICA表达的研究

目的： 研究高原藏族胃癌患者外周血NK细胞表面活化性受体D(NKG2D)的表达及局部肿瘤组织和癌旁正常组织中相应配体MICA 的表达,探讨宿主NKG2D 在抗胃癌中的作用及其与肿瘤免疫逃逸的关系。方法: 对33例高原藏族胃癌患者及20 例健康人,采用流式细胞术检测外周血NKG2D 的表达状况,RT-PCR 和免疫组织化学技术检测在局部肿瘤组织和癌旁正常组织标本中MICA 的表达。结果: 高原藏族胃癌患者外周血NKG2D 的表达为(13.47 ±5.26)%，明显低于正常组（32.62±10.08)%，2组之间差异显著(P<0．05); 藏族胃癌组织MICA mRNA的表达水平显著高于癌旁正常组织（P<0.05）。胃癌组织MICA蛋白表达在癌旁正常组织中为(21.21%,7/33),胃癌组织阳性率为(78.79%，26/33),高分化组(60.00%,3/5),中分化组(72.73%,8/11),低分化组(88.24%,15/17),组间差异显著(P<0.05),而与肿瘤的大小、性别、年龄、淋巴结转移无关（P>0.05）。 Spearman相关分析显示，MICA表达水平与mRNA的表达水平显著正相关（r=0.903,P<0.01）。结论: 高原藏族胃癌的免疫逃逸可能与NKG2D 表达下调及其配体MICA 的表达升高有关，高原藏族胃癌患者外周血NK细胞活性降低,其NKG2D 表达的下降是NK细胞活性下降的原因之一。NKG2D配体MICA 的基因表达可能与高原胃癌患者的恶性转化有一定的相关性。