白细胞介素27诱导肿瘤微环境中IL-10产生与免疫调节分子机制研究

The inflammatory response in tumor microenvironment is closely related to tumor immunity. Cytokines, including interleukins, play a major role in the anti-tumor, induction and limitation of inflammation. Interleukin (IL)-10 has been recognized as a major anti-inflammatory cytokine serving as a negative regulator of the response in both innate and adaptive immune cells, especially in persistent infectious and autoimmune diseases, often acting beneficially to modulate excessive immune response mediated injury. IL-27 is a novel cytokine that has broad effects on the differentiation of T helper type 1 (Th1), Th2, Th17 subsets as well as regulatory T (Treg) cells throuth regulating the production of the anti-inflammatory cytokines, like IL-10. For its pro-inflammatory and anti-inflammatory properties, IL-27 is a pleiotropic immunological mediator that functions paradoxically like a "double edged sword" in inflammatory response. Previous studies of our research on IL-27 with tumor models provide evidences that IL-27 could suppress several important inflammatory cytokines, such as IFN-γ, TNF-α and IL-1β during its anti-tumor immunity. More recently, IL-27 has been demonstrated to be served as a regulatory cytokine to limit host damage during persistent bacterial and parasitic infection. Given the important role of IL-10 in limiting inflammation and the characteristics of IL-27, we suppose that IL-27 may induce IL-10 production in tumor micro- environment. However, the cellular and molecular mechanisms of IL-27 in controlling IL-10 production by T cells, and particularly by effector T cells in the process of anti-tumor effects are imperfectly understood. Based on the above hypothesis, our study undertake to disclose the fuction of IL-27 on effector T cells in tumor microenvironment. We will analyze the relation between IL-27 induced IL-10 in effector T cells and several important T cell subsets in the tumor-enviroment. We will also uncover the relation between IL-27 induced IL-10 and IFN-γ as well as other cytokines in tumor models. Furthermore, by in vivo and in vitro experiments, our aim is to investigate the mechanisms of IL-27 promoting IL-10 production by the cytotoxicity T lymphocytes (CTL), and to explore whether IL-2, IL-21 pathways, signal transducer and activator of transcription (STAT)1, STAT3, Blimp-1, T-bet transcription factor attend in IL-27 mediated anti-tumor and anti-inflammatory effects. In this regard, we not only deepen the understanding of IL-27 biological function that orchestrate the anti-tumor and anti-inflammatory effects, but also provide feasible scientific basis for the clinical application of IL-27.

肿瘤微环境中的炎症反应与肿瘤免疫反应密切相关。细胞因子在抗肿瘤和诱导与控制炎症中起重要作用。IL-10因负性调节炎症反应可防止宿主免疫损伤，IL-27通过IL-10调控Th1、Th2、Th17和Treg分化，在炎症反应中起"双刃剑"作用。本项目前期研究发现IL-27发挥抗肿瘤效应同时有抑制炎症因子产生和宿主损伤的现象，那么，IL-27在抗肿瘤免疫中是否诱导抑炎因子IL-10产生，其作用机制和相关信号通路如何。本项目以IL-27抗肿瘤作用为基础，分析其诱导肿瘤微环境产生IL-10与T细胞亚群及IFN-γ等细胞因子的关系，通过体内外实验研究IL-27促进效应T细胞产生IL-10的可能作用机制，探索IL-2、IL-21等细胞因子和STAT1/3、Blimp-1、T-bet等转录因子在传递相关信号中的作用。明确IL-27抗肿瘤与抗炎作用间的关系，加深其生物学功能认识，为临床应用提供可行性科学依据。

细胞因子的抗肿瘤机制是肿瘤免疫的重要研究内容，抗肿瘤免疫反应与肿瘤微环境中炎症反应密切相关。IL-10是重要的免疫调节性炎症因子能够防止宿主过度损伤，IL-27通过IL-10调控Th1、Th2、Th17和Treg分化，在炎症反应中起“双刃剑”作用。本项目前期研究发现IL-27发挥抗肿瘤效应同时有抑制炎症因子产生的现象。本课题主要探讨IL-27在抗肿瘤免疫中是否诱导效应T细胞产生抑炎因子IL-10及其作用机制。课题组成员在主研人带领下，首先证明IL-27 在体内外具有抗肿瘤活性，诱导CTL杀伤活性、提高IFN-水平，并发现小鼠移植瘤组织微环境中T细胞亚群分布即CD8+ T细胞、CD4+ T细胞升高而Th17、Treg亚型降低，影响炎症因子IFN-、TNF-α和IL-10、IL-4的表达水平。为探讨IL-27影响肿瘤微环境炎症反应特别是IL-10产生的机制，通过给与CD4+ T单克隆抗体建立去除CD4+ T细胞小鼠模型，发现IL-27诱导CTL产生IL-10依赖于IL-27和IFN-；分离IL-27基因转染的荷瘤小鼠脾细胞，以磁珠分选的方法分别获取CD8+ T细胞、CD4+ T细胞和DC，荧光素CFSE标记体外培养的CD8+ T细胞，制备肿瘤抗原负载DC，以DC-T细胞培养体系获得CD8+ 细胞毒性T淋巴细胞即CTL和CD4+ T细胞，检测CTL单独或与CD4+ T细胞共培养时IFN-、TNF-α及IL-10的分泌，证实IL-27诱导CTL产生IL-10与IFN-、TNF-α等因子和CD4+ T细胞亚群有相关性，并用蛋白印迹法证实IL-27诱导CTL产生IL-10依赖于Blimp-1和磷酸化STAT1。肿瘤微环境免疫逃逸是影响肿瘤进展的重要因素，在研究中还发现，由于IFN-和IL-27均可诱导IDO产生，IDO通过抑制T淋巴细胞增殖促进Treg产生参与肿瘤免疫逃逸。我们研究发现IDO表达水平与肿瘤微环境中CD3+T、CD4+T、CD8+ T、CD4+CD25+Treg等淋巴细胞亚群失衡相关，与肿瘤患者生存期负相关。因此，有必要在未来的工作中深入探讨炎症相关因子在肿瘤微环境免疫逃逸中的作用。本研究结果将促进对肿瘤微环境中炎症反应的认识，将推动肿瘤微环境免疫编辑特别是免疫逃逸的深入研究，打破免疫逃逸与免疫耐受将对肿瘤免疫治疗效果具有重要意义。