摘要
人类γδT细胞是T淋巴细胞中的一个特殊群体,在机体抗肿瘤及抗感染免疫应答中发挥着重要作用。然而,由于已发现和鉴定出的抗原(或配体)数量有限,且缺乏相应的反应性亚克隆T细胞抗原识别受体(T cell receptor, TCR)的结构信息,γδT细胞与肿瘤细胞或病毒感染细胞之间相互作用的分子机制尚有待于进一步阐明。
人类MutS同源蛋白2(Human MutS homologue 2, hMSH2)是DNA错配修复蛋白家族的一个重要成员,通常定位于细胞核并与hMSH3或hMSH6形成异源二聚体,参与修复DNA合成中的碱基错配,以保证基因组的忠实性。遗传性或获得性的hMSH2缺陷与诸多癌症的发生、发展及转归密切相关。在前期的研究工作中,本实验室采用基于TCR互补决定区36链(Complementary determining region 3δchain, CDR3δ)肽结合特性的免疫亲和筛选技术策略,用OT3肽探针从卵巢癌细胞系SKOV3的细胞提取物中钓取了三个可能被Vδ2 TCR识别的配体,hMSH2是其中之一。初步的研究结果表明,hMSH2在SKOV3的细胞表面有异位表达,γδT细胞对SKOV3细胞的杀伤作用亦可为抗hMSH2抗体(Antibody, Ab)部分封闭。鉴于此,我们推测hMSH2是一个能为γδT细胞所识别的、肿瘤相关的内源性抗原(或配体)分子。本研究旨在对此进行验证,并进一步探讨γδT细胞识别hMSH2的分子机制及hMSH2在γ6 T细胞介导的天然免疫监视应答中的作用和意义。
本文的第一项研究内容旨在探索hMSH2的膜异位表达在上皮肿瘤细胞中是否具有普遍意义。针对此问题,分析了宫颈癌、胃癌、肺癌、结直肠癌及卵巢癌肿瘤细胞系HeLa、803、NCI-H520、HR8348、SKOV3、HO8910及ES-2细胞中hMSH2的基因序列、mRNA水平及蛋白质表达情况,并对这五种肿瘤病人癌组织标本中hMSH2的分布进行了分析。流式细胞术检测结果表明,hMSH2在受测的七种上皮肿瘤细胞表面均存在异位表达(阳性率14.2～68.2%),而在正常对照细胞HK-2、成纤维细胞及γδT细胞表面则几乎没有表达(阳性率<2.2%),提示hMSH2的膜异位表达在上皮肿瘤细胞中可能具有普遍意义。激光共聚焦扫描显微镜术和免疫组织化学染色分析的结果也显示,hMSH2在上皮肿瘤细胞中出现异常的亚细胞分布,表现为广泛的胞膜、胞质异位表达,胞核分布则多减弱或消失。基因测序结果表明,hMSH2的基因序列在大多数上皮肿瘤细胞系中并无突变,其mRNA表达水平在不同上皮肿瘤细胞系间也存在较大差异。
hMSH2在上皮肿瘤细胞表面不寻常的异位表达为其作为γδT细胞识别的配体提供了可能。因此,本文的第二项研究内容重点验证了γδT细胞对肿瘤细胞膜异位表达的hMSH2的识别作用。首先,运用抗体杀伤封闭实验,在HeLa、803及NCI-H520细胞中初步证实了hMSH2的膜异位表达与γδT细胞对上皮肿瘤细胞的细胞毒作用间存在联系。其次,运用小RNA干扰(Small interference RNA, siRNA)技术靶向沉默HeLa、803及NCI-H520细胞中hMSH2的表达,并比较γδT细胞对干扰组及Mock组杀伤效率的差异。结果表明,异位表达的hMSH2的确能被γδT细胞识别,并参与γδT细胞对上皮细胞肿瘤的免疫监视作用。随后,通过杀伤实验及重组hMSH2 (Recombinant hMSH2, rhMSH2)体外刺激实验进一步证实Vδ2 T细胞是hMSH2的主要反应性γδT细胞亚群。最后,通过对HeLa、803及NCI-H520细胞膜蛋白的分离纯化Western blot分析,鉴定出上皮肿瘤细胞膜异位表达的hMSH2为分子量(Molecular weight, MW)约105千道尔顿(Kilodalton, kDa)的全长蛋白质。
由于Vδ2 T细胞组成性地表达NKG2D受体,且有研究表明γδT细胞的某些配体分子如MHCⅠ类分子链相关抗原A和B(MHC classⅠ-related chains A and B, MICA/B)、UL16结合蛋白4(UL16-binding protein 4, ULBP4)等存在TCR及NKG2D双重识别机制,本文的第三项研究内容着重探讨了γδT细胞对hMSH2的识别是否同样存在TCRγδ及NKG2D双重途径。首先,用抗体封闭与siRNA干扰相结合的方法,研究TCRγδ和/或NKG2D封闭是否能够抑制膜异位表达hMSH2所介导的γδT细胞(或NK-92细胞)对肿瘤细胞的识别与杀伤作用；其次,将rhMSH2蛋白在体外与γδT细胞预孵育,经流式细胞术分析rhMSH2对TCRγδ及NKG2D的竞争性结合作用；此外,运用表面离子共振技术(Surface plasmon resonance, SPR)对NKG2D与rhMSH2的直接结合作用及亲和力进行了进一步分析。结果显示,hMSH2介导的γδT细胞(或NK-92细胞)对肿瘤细胞的识别与杀伤能被anti-TCRγδ和/或anti-NKG2D Ab封闭所抑制；rhMSH2能竞争性地与TCRγδ及NKG2D结合;NKG2D能直接与rhMSH2结合,Kd值为132 nM。这些结果说明γδT细胞对hMSH2的识别存在TCRγδ及NKG2D双重途径。
γδT细胞在抗病毒感染免疫中发挥着重要作用。γδT细胞识别的某些内源性抗原(或配体)分子,如热休克蛋白(Heat shock protein, HSP) 90、HSP60及ULBPs等,在病毒感染细胞中出现表达上调。因此,本文的第四项研究内容通过建立Epstein-barr病毒(Epstein-barr virus, EBV)转化细胞模型,观察hMSH2在EBV转化类淋巴母细胞中的表达变化及这种变化对γδT细胞清除病毒感染细胞作用的影响,进一步研究了hMSH2在γδT细胞介导的病毒感染免疫监视中的作用。结果表明,EBV感染细胞hMSH2的转录水平及膜异位表达均明显增高；膜异位表达的hMSH2亦介导了γδT细胞对EBV感染细胞的识别,并显著增强了γδT细胞的靶向杀伤作用。这提示hMSH2可能如γδT细胞识别的大多数内源性抗原(或配体)一样具有应激分子的特性,且在γδT细胞介导的抗EBV感染免疫应答中发挥重要作用。
综上所述,本文得出的主要结论如下：(1)hMSH2的膜异位表达在上皮肿瘤细胞中具有一定的普遍性；(2)异位表达的hMSH2能被γδT细胞的抗原识别受体TCRγδ和NKG2D双重识别,并主要参与Vδ2 T细胞介导的对肿瘤细胞的杀伤作用；(3)上皮肿瘤细胞膜异位表达的hMSH2为MW约105 kDa的全长蛋白；(4)hMSH2的异位表达能被EBV感染所诱导,并显著增强γδT细胞对病毒感染细胞的清除作用。
本研究的创新点体现在：一是初步验证了hMSH2的膜异位表达在上皮肿瘤细胞中具有一定的普遍性,提示hMSH2可能是一个新发现的上皮肿瘤标志分子；二是首次证明hMSH2作为一个肿瘤相关的内源性抗原分子,能通过TCRγδ及NKG2D双重识别途径被γδT细胞识别,为γδT细胞识别的抗原(或配体)谱增添了新的分子,揭示了γδT细胞识别、杀伤上皮来源肿瘤细胞的新的分子机制；三是首次报道了hMSH2分子在EBV感染细胞中的诱导表达,并阐明了该分子在γδT细胞介导的抗EBV感染免疫中的作用,揭示了γδT细胞识别和清除EBV病毒感染细胞的新的分子机制,丰富了目前对γδT细胞在病毒感染免疫中重要作用的认识。
总之,本文通过对上皮来源肿瘤细胞及EBV转化B细胞表面hMSH2的异位表达及γδT细胞对其识别机制的深入研究,进一步揭示了γδT细胞免疫监视的新型作用方式,为全面阐明γδT细胞的生物学功能提供了新的线索,亦为建立新的肿瘤及病毒感染免疫治疗策略提供了可能的靶标分子。
Human y8 T cells account for a minor fraction of human peripheral T lymphocytes. The innate-like feature and unique major histocompatibility complex (MHC)-independent antigen recognition pattern define these cells a special status in tumor surveillance and pathogen defense. However, the molecular mechanisms underlying tumor/infected cells recognition byγδT cells are poorly understood, due to the limited number of antigens/ligands recognized byγδT cells that have been identified so far.
Human MutS homologue 2 (hMSH2) is a critical element of the highly conserved DNA mismatch repair system, normally located in the nucleus and dimerized with hMSH3 or hMSH6 to form complexes essential for the maintenance of genome integrity. Inherited and acquired defects in hMSH2 are closely related to the pathogenesis of hereditary nonpolyposis colon cancer as well as various sporadic cancers. Previously, with a CDR3δpeptide-based affinity screening system, we identified hMSH2 as a putative tumor-associated protein ligand for Vδ2 TCRs. In addition, we observed an unusual surface expression of hMSH2 on SKOV3 cells that was seemingly correlated with an enhanced cytotoxicity ofγδT cells.
Based on our previous study, we hypothesized that hMSH2 may be a tumor-associated ligand recognized by Vδ2 TCRs. To test this possibility, we first examined the surface expression of hMSH2 on HeLa. SKOV3, HO8910, ES-2,803, HR8348 and NCI-H520 cell lines. Flow cytometric analysis with specific anti-hMSH2 antibody (H-300) revealed that 10-70% of the tested tumor lines expressed hMSH2, whereas normal control cells rarely expressed hMSH2 on their surface. Confocal microscopy and immunohistochemistry analyses suggested an aberrant subcellular distribution of hMSH2 in carcinoma cells and tissues as well. The broad cell-surface expression of hMSH2 on carcinoma cells raised the possibility of its being a tumor-associated ligand forγδT cells.
The second issue of this study was to verify the recognition of hMSH2 by yS T cells and to explore the significance of ectopically expressed hMSH2 inγδT cell-mediated anti-tumor immunity. With anti-hMSH2 antibody blocking, we found that the ectopic hMSH2 expression was correlated with an enhancedγδT cell-mediated cytolysis of HeLa,803 and NCI-H520 cells. The hMSH2-mediated specific recognition byγδT cells was further validate using small RNA interference technology to knockdown HMSH2 expression in HeLa,803 and NCI-H520 cell lines. At various E:T ratios, down-regulation of hMSH2 expression with siRNAⅠorⅡinterference resulted in significantly reduced cytolysis of HeLa,803 and NCI-H520 cells byγδT cells. The above results from antibody blocking or siRNAs interference suggest that ectopic hMSH2 mediates the recognition of carcinoma cells by humanγδT cells and contributes toγδT cell-mediated tumor cytolysis. The specific subset that recognizes the surface-expressed hMSH2 on tumor cells was Vδ2 T cells, which could be activated to proliferate and produce IFN-y by rhMSH2 stimulation. Besides, the cell surface-expressed hMSH2 on carcinoma cells was charaterized as a full-length protein with a m.w. of 105 kDa by blotting purified membrane proteins with anti-hMSH2 antibody.
The third issue of this study was to determine whether both TCRγδand NKG2D were involved in the recognition of surface hMSH2. Firstly, the expandedγδT effector cells (or NK-92 cells) were pretreated with anti-TCRγδand/or anti-NKG2D Ab before incubation with siRNA I-treated HeLa cells or mock control cells. The ectopic hMSH2-mediated recognition byγδT cells was significantly inhibited by anti-TCRγδ, anti-NKG2D or both mAbs. Secondly, affinity-purified rhMSH2 was used to confirm the specific binding of hMSH2 by TCRγδor NKG2D receptors with flow cytometry. Decreased fluorescence intensity of TCRγδor NKG2D was observed in rhMSH2-blocking groups, suggesting a competitive binding of hMSH2 to both receptors. Furthermore, surface plasmon resonance analysis suggested a direct binding of rhMSH2 to NKG2D-Fc. These results indicate that both TCRγδand NKG2D participate in the ectopic hMSH2-mediated recognition and cytolysis of carcinoma cells by humanγδT cells.
The final issue of this study was to determine the inducible expression of hMSH2 on EBV-transformed human PBMCs and the role of hMSH2 inγδT cell-mediated anti-viral immunity. We infected PBMCs isolated from healthy donor with EBV and analyzed hMSH2 expression. We found that both mRNA transcription and cell-surface expression of hMSH2 were upregulated by EBV infection, leading to a substantial enhancement ofγδT cell-mediated responses towards EBV-transformed cells. The sensitivity of freshly generated EBV-transformed B cells to autologous or allogenicγδT cell-mediated cytolysis was 2-3 folds greater than that of normal B cells. Blocking with anti-hMSH2 antibody significantly downregulated the specific cytolysis of these EBV-infected cells. The ability of induced hMSH2 to facilitate the killing of EBV-transformed cells byγδT cells was followingly confirmed by specific siRNAs interferencing in lymphoblastoid cell line (LCL) 3D5. Consistent with that observed in carcinoma cells, specific cytolysis of siRNAs-treated 3D5 cells byγδT cells was significantly reduced. These results suggest that the cell surface-expressed hMSH2 is inducible and may mediate the specific clearance of EBV-infected cells by humanγδT cells.
To sum up, we concluded that the ectopic surface expression of hMSH2 is broad on human carcinoma cells. The ectopically expressed hMSH2 interacts with both TCRγδand NKG2D and contributes to Vδ2 T cell mediated cytotoxicity towards hMSH2 bearing tumor cells. Moreover, the surface expression of hMSH2 is induced on EBV-transformed LCLs, which dramatically increases the sensitivity of these LCLs toγδT cell-mediated cytolysis. Our data suggest that hMSH2 functions as a tumor-associated or virus infection-related antigen recognized by both TCRγδand NKG2D, and plays a role in eliciting the immune responses ofγδT cells against tumor and virus-infected cells. The recognition of ectopically surface expressing endogenous antigen by TCRγδand NKG2D may be an important mechanism of innate immune response to carcinogenesis and viral infection.
引文
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