【作者】 殷珊珊；

【导师】 何维；

【作者基本信息】 北京协和医学院 ， 免疫学， 2013， 博士

【摘要】 γδT细胞能够以主要组织相容性复合体(major histocompatibility complex, MHC)非限制性方式识别多种肿瘤相关抗原,有效的杀伤肿瘤细胞,并能分泌干扰素(interferon, IFN)-γ等细胞因子。因此,γδT细胞已成为当前恶性肿瘤免疫治疗中一种很有前景的候选细胞。γδT细胞表面除表达γ和δ链组成的T细胞受体(T cell receptor γδ, TCRγδ)外,还表达自然杀伤(natural killer, NK)细胞的重要功能受体NKG2D。这两种受体分子在γδT细胞对肿瘤细胞的杀伤中发挥重要的作用。目前,大部分文献报导倾向于γδT细胞活化为TCRγδ依赖性的,而NKG2D仅起共刺激作用。然而,为什么单独的TCRγδ刺激即可活化γδT细胞,NKG2D的共刺激作用又是如何发挥的?只有进一步回答这些问题,才能澄清γδT细胞生物学效应(主要为细胞毒效应)的分子机制,尤其是信号转导机制。鉴此,本研究主要针对以下三个科学问题展开：一是γδT细胞杀伤肿瘤细胞的主要效应途径是什么?二是TCRγδ和NKG2D在活化γδT细胞杀伤功能中的具体作用是什么?三是γδT细胞杀伤功能活化的调控机制是什么?本文分以下两部分就上述三个科学问题进行研究。第一部分工作旨在进一步澄清γδT细胞杀伤肿瘤细胞的相关机理。首先,我们比较了穿孔素-颗粒酶和Fas-FasL两条途径在γδ T细胞杀伤肿瘤细胞中的贡献。我们选取了五种不同组织来源的肿瘤细胞作为γδT细胞杀伤途径研究的靶细胞,包括Daudi(人Burkkit淋巴瘤细胞)、G401(人肾癌Wilms细胞)、NCI-H446(人小细胞肺癌细胞)、HR8348(人结直肠癌细胞)和MGC-803(人胃癌细胞)。流式细胞术检测结果表明,这五种不同组织来源的肿瘤细胞表面Fas受体呈现不同程度的表达：Daudi为5.14%、G401为7.24%、NCI-H556为44.10%、HR8348为69.40%以及MGC-803为82.30%。然后,对这五种Fas受体表达水平不同的肿瘤细胞进行穿孔素-颗粒酶途径及Fas-FasL途径的封闭实验。结果表明,封闭穿孔素-颗粒酶途径后,γδT细胞对这五种肿瘤细胞的杀伤能力均显著降低,而封闭Fas-FasL途径对γδT细胞杀伤能力无显著影响。随后的酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)结果显示,穿孔素-颗粒酶途径封闭的γδT细胞与靶细胞共孵育后分泌IFN-γ的能力亦显著降低。而Fas-FasL途径封闭后,γδT细胞分泌IFN-y的能力无显著变化。以上结果表明γδT细胞杀伤肿瘤细胞的主要途径为穿孔素-颗粒酶途径。随后,利用激活抗体包被的P815靶细胞杀伤体系,探讨了TCRγδ和NKG2D在活化γδT细胞杀伤功能中的作用,并分析了二者功能存在差异的原因。P815靶细胞杀伤实验结果表明,单独给予抗TCRγδ抗体刺激即可活化γδT细胞,杀伤抗体包被的P815靶细胞,并分泌IFN-γ;而单独的抗NKG2D抗体刺激却不能活化γδT细胞的杀伤功能,也不能引起IFN-γ的分泌。然而,抗NKG2D抗体可以增强抗TCRγδ抗体刺激所引起的γδT细胞的杀伤功能和分泌IFN-γ的能力。流式细胞术检测结果表明,抗TCRγδ抗体和抗NKG2D抗体均可引起γδT细胞的脱颗粒反应；激光共聚焦检测结果表明,单独的抗TCRγδ抗体刺激即可引起γδT细胞裂解性颗粒的极化,单独的抗NKG2D抗体刺激却不能。尽管如此,抗NKG2D抗体可以在某种程度上增强抗TCRγδ抗体所引起的裂解性颗粒极化。结合杀伤实验结果,提示引起γδT细胞裂解性颗粒极化能力的不同是TCRγδ和NKG2D在活化γδT细胞杀伤功能方面存在功能差异的主要原因。第二部分研究工作旨在明确调控γδT细胞杀伤功能活化的相关信号通路。首先对γδT细胞杀伤功能相关的活化信号通路进行了研究,主要集中在Vavl信号通路、磷酸脂酶C-γ1(Phospholipase C-γ1, PLC-γ1)信号通路、丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)/细胞外信号调节激酶(extracellular signal-regulated kinase, Erk)信号通路、磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase, PI3K)信号通路等四条与αβ T细胞和NK细胞活化相关的信号通路。运用Western blot技术,证实了单独的抗TCRγδ抗体刺激即可显著性活化Vav1信号通路、PLC-γ1信号通路以及Erk信号通路；而单独给予抗NKG2D抗体刺激则无此作用；在联合给予抗NKG2D抗体刺激时,这三条信号通路的活化显著增强。PI3K信号通路在给予抗TCRγδ抗体刺激或抗NKG2D抗体刺激时均明显活化,而联合给予二者刺激时,PI3K信号通路活化亦未见增强。结合杀伤实验结果,提示Vav1信号通路、PLC-γ1信号通路以及Erk信号通路与γδT细胞杀伤功能密切相关。利用siRNA技术敲低γδT细胞内的Vav1分子表达时,发现γδT细胞对靶细胞的杀伤、IFN-γ分泌以及裂解性颗粒极化能力均显著降低；同时,敲低Vavl亦可显著抑制PLC-γ1信号通路以及Erk信号通路的活化。利用信号通路抑制剂抑制γδT细胞内的PLC-γ1信号通路以及Erk信号通路时,发现抑制PLC-γ1信号通路可显著抑制γδT细胞的杀伤、IFN-γ分泌以及裂解性颗粒极化,而抑制Erk信号通路对γδT细胞的杀伤功能及裂解性颗粒极化均无显著影响,但抑制Erk信号通路可抑制γδT细胞IFN-γ的分泌。此外,抑制PLC-γ1信号通路可显著抑制γδT细胞内Erk信号通路的活化。上述结果提示,Vav1-PLC-γ1信号通路位于Erk信号通路上游,并在γδT细胞杀伤功能活化中具有重要的作用。随后的研究工作证实了Cbl-b在γδT细胞杀伤功能活化中的负调控作用。利用siRNA技术敲低γδT细胞内Cbl-b分子表达时,γδT细胞对靶细胞的杀伤能力、杀伤功能相关的Vav1-PLC-γ1信号通路活化以及裂解性颗粒极化均显著提高；特别值得一提的是,单独的抗NKG2D抗体刺激即可活化与γδT细胞的杀伤功能相关的Vav1-PLC-γ1信号通路,并引起裂解性颗粒极化,提示Cbl-b是单独使用抗NKG2D抗体不能活化γδT细胞杀伤功能的主要负调控因素,亦提示γδT细胞杀伤功能活化需要一个较强的活化信号来克服活化抑制信号；Vav1过表达实验进一步证实了这一点。此外,RNAi实验也证实了Cbl-b是通过抑制Vavl磷酸化而发挥其负调控功能的。综上所述,本研究得出以下主要结论：1、γδT细胞杀伤靶细胞主要通过穿孔素-颗粒酶途径；2、γδT细胞杀伤功能为TCRγδ依赖性的,NKG2D可增强TCRγδ依赖性的γδT细胞杀伤功能；3、引起裂解性颗粒极化能力的不同是TCRγδ和NKG2D在活化γδT细胞杀伤功能方面存在差别的原因；4、Vav1-PLC-γ1信号通路与γδT细胞杀伤功能密切相关；5、Cbl-b负调控γδT细胞杀伤功能；6、γδT细胞杀伤功能的活化需要一个较强的活化信号来克服活化抑制信号,最终使γδT细胞发挥杀伤能力。本研究深入揭示了TCR依赖性γδT细胞杀伤活性的分子机制,发现γδT细胞杀伤功能的实现需要经Vav1-PLC-γ1信号通路的活化来消除E3泛素连接酶Cbl-b的抑制作用,为深入阐明γδT细胞生物学效应的作用机制提供了研究资料。更多还原

【Abstract】 y8T cells are able to target a broad spectrum of tumors because of their unique properties, including major histocompatibility complex (MHC)-independent recognition, potent cytotoxicity, and cytokine (like IFN-γ) secretion. Therefore, γδ T cells have recently become attractive candidate effector cells for tumor immunotherapy.Besides T cell antigen receptor y8(TCRyδ), y8T cells also highly express natural killer group2, member D (NKG2D). TCRyS and NKG2D are considered as two important receptors of y8T cells that play important roles in the recognition of tumor cells. Evidences of TCRγδ-dependent y8T cells activation have been well documented, whereas NKG2D is generally accepted to act as a costimulatory receptor for γδ T cell cytotoxicity. However, why TCRγδ alone can activate γδ T cell cytotoxicity, and how does NKG2D costimulatory effect take place? Only when these questions are answered, the molecular mechanisms, especially the signal transduction underlying the biological effects of γδ T cells (mainly cytotoxic effect) could be well clarified.In view of these facts, our current study focused on the following three scientific questions. First, what is the principal pathway related to γδ T cell mediated tumor killing? Second, what are the specific functions of TCRγδ and NKG2D in the activation of γδ T cell cytotoxicity? Third, what is the regulative mechanism of y8T cell cytotoxicity?The first part of this work aimed at further clarifying y8T cell killing mechanism. First of all, the individual contribution of perforin-granzyme pathway and Fas-FasL pathway to y8T cell mediated tumor killing was compared. Five tumor cell lines were selected as the target cells, including Daudi (Human Burkkit lymphoma cells), G401(Human renal cancer Wilms cells), NCI-H446(Human small cell lung cancer cells), HR8348(Human colorectal cancer cells), and MGC-803(Human gastric cancer cells). Flow cytometry (FCM) analysis showed that these five tumor cell lines have different levels of Fas expression:Daudi5.14%, G4017.24%, NCI-H55644.10%, HR834869.40%, and MGC-80382.30%. Cytotoxicity assay following perforin-granzyme pathway and Fas-FasL pathway blockade showed that Fas-FasL signaling blockade with supplemented soluble anti-FasL antibody did not impair the specific lysis of tumor target cells or the release of IFN-y by γδ T cells. In contrast, concanamycin A (CMA), a perforin inhibitor that accelerates perforin degradation within lytic granules, dramatically blocked γδ T cell cytotoxicity. Hence, we can conclude that the perforin-granzyme pathway makes the major contribution to γδ T cell cytotoxicity.Later, the function of TCRy8receptor and NKG2D receptor in the activation of γδ T cell cytotoxicity was explored through using TCRγδ antibody and/or NKG2D antibody redirected P815cells as target. The results of P815redirected cytotoxicity showed that anti-TCRy5but not anti-NKG2D activating antibodies initiated γδ T cell specific killing of P815target cells, accompanied by the significant release of IFN-y. Interestingly, NKG2D ligation augmented TCRγδ activation-mediated cytotoxicity and IFN-y production. To compare the contribution of TCRγδ and NKG2D on perforin-granzyme pathway, we first measured cellular degranulation based on cell surface expression of CD107a (LAMP-1). No significant difference was observed in CD107a expression in γδ T cells after TCRγδ or NKG2D activation. Intracellular staining of the perforin-containing granules showed that TCRγδ but not NKG2D engagement induced lytic granule polarization, and NKG2D in combination with TCRγδ activation merely enhanced this effect. These results, taken together, suggest that TCRγδ-induced T cell cytotoxicity mainly depends on lytic granule polarization.The second part of the work was to look for the signal transduction mechanisms responsible for the regulation of γδ T cell cytotoxicity. First, we studied the activation signal pathways related to γδ T cell cytotoxicity. Four important signaling pathways that are associated with αβ T cell and NK cell cytotoxicitywere studied. They are Vavl, PLC-y, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) and PI3K. Western blot analysis showed that stimulation of TCRγδ but not NKG2D induced strong phosphorylation of Vav1, PLC-γ1and Erk, which were enhanced when both TCRy8and NKG2D were engaged. In contrast, phosphorylation of Akt, a downstream target of PI3K activation, showed no enhancement following the coengagement of TCRyS and NKG2D compared to either TCRγδ or NKG2D stimulation alone. These results combined with the cytotoxicity results, suggest that Vav1, PLC-yl and Erk pathways play the major role in y8T cell cytotoxicity. Knockdown of Vavl in γδ T cells by small interference RNA (siRNA) could block γδ T cell-mediated killing, lytic granule polarization and IFN-y release. In the mean while, knockdown of Vavl completely abrogated the phosphorylation of PLC-yl and Erk. Using inhibitor of signaling pathways, we found that the PLC-y inhibitor U73122can fully abrogate y8T cell cytotoxicity, lytic granule polarization and IFN-y release. However, MEK inhibitor U0126, an upstream activator of Erk, did not abrogate γδ T cell cytotoxicity and lytic granule polarization, even under high concentrations. Intriguingly, IFN-y production was inhibited by U0126. In addition, Erk phosphorylation was inhibited by U73122, indicating that Erk might be a downstream molecule of the PLC-y pathway. These results suggest that, unlike IFN-y production, y8T cell cytotoxicity is an independent event downstream of Vavl-PLC-yl but not Erk.Subsequent research confirmed that Cbl-b played inhibitory role in γδ T cell cytotoxicity activation. Knockdown of Cbl-b in γδ T cells by small interference RNA (siRNA) enhanced γδ T cell cytotoxicity towards redirected P815cells following TCRγδ and/or NKG2D engagement. After Cbl-b knockdown, lytic granule polarization toward target cells, phosphorylated Vavl, PLC-yl and Erk were all upregulated following TCRγδ or NKG2D activation. Interestingly, knockdown of Cbl-b enabled unresponsive γδ T cells to respond to the engagement of NKG2D. NKG2D engagement induced the phosphorylation of Vav1and PLC-γ1, as well as lytic granule polarization when Cbl-b expression was knocked down. Taken together, our data suggest that Cbl-b imposes a requirement of TCRγδ-dependent activation, and the activation of γδ T cell cytotoxicity requires a strong signal to overcome the activation threshold set by the inhibitory effect of Cbl-b. Vavl overexpression experiments further confirmed this conclusion. At the same time, RNAi results showed that Cbl-b might regulate y8T cell cytotoxicity by targeting phosphorylated Vavl.In conclusion, our major findings in this study include (1) Perforin-granzyme pathway mediates γδ T cell cytotoxicity towards tumor cells.(2) γδ T cell cytotoxicity is TCRγδ dependent. NK.G2D ligation augments γδ T cell cytotoxicity mediated by TCRγδ engagement.(3) Difference in lytic granule polarization is the main reason why TCRγδ and NKG2D have different function in the activation of γδ T cell cytotoxicity.(4) Vav1-PLCγ1is required for γδ T cell cytotoxicity.(5) Cbl-b inhibits Vavl-dependent γδ T cell activation signals.(6) The activation of γδ T cell cytotoxicity requires a strong signal to overcome the activation threshold set by the inhibitory effect of Cbl-b. This study further reveals the molecular mechanisms underlying TCRγδ dependent γδT cell cytotoxicity, and verifies that the activation of Vavl-PLCγ1pathway is required to overcome the inhibition by E3ubiquitin ligase Cbl-b in γδT cell cytotoxicity, thereby providing important information regarding the biological effects of γδT cells.更多还原