IL-35在结直肠癌中表达调控及其介导肿瘤免疫逃逸的机制研究

Colorectal cancer is a common type of malignancy (cancer) with the improvement of living standards，its mechanism of immune escape has not yet been fully elucidated. IL-35 is an novel inhibitors，which plays an important role in the immune balance as the functional executor of Treg. However, the characteristics of IL-35 are still shallow understanding. IL-35 expression in human T cells is plausible, and the mechanisms of its upstream activation and downstream effects in tumor immunity have not yet been reported, which needs our further study. Our results showed that increased expression of Treg and IL-35 secreting by CD4 cells in patients with colorectal cancer. We assume that IL-35 in colorectal cancer by inducing Tconv into Treg and / or IL-35 signaling pathway downstream of ligand binding inhibitory effect of T cell function. To verify this hypothesis, Tconv and Treg cells of CRC will be sorted and co-cultured in vitro using Transwell system to investigate the expression of IL-35 and Treg, and immune suppression mechanism. Transgenic mice will be used to further study the mechanisms in vivo and in vitro, including the reconstitution of Foxp3-EGFP/cre/EBI3fl/fl-YFP and CD4-cre/EBI3fl/fl-YPF mice, the establishment of AOM / DSS-induced colorectal cancer model, the preparation of IL-35 functional blocking antibody to clarify the function of IL-35 in colorectal cancer development, the regulation mechanism of IL-35 step by step. Our aim is to lay the foundation by means of mediating the immune response to enhance the efficiecy of tumor immunology and biological therapy by IL-35 blockage, and introduce a new way to treat other diseases.

CRC是常见恶性肿瘤之一，其免疫逃逸机制迄今仍未完全阐明。新型抑制因子IL-35作为Treg功能执行者在机体的免疫平衡中发挥着重要的作用。但对IL-35的认识尚浅显，IL-35人T细胞中表达的争议及其在疾病中的上游激活以及下游效应机制,在肿瘤免疫中的作用和机制几乎未知。我们以CRC为研究对象，发现疾病中Treg和IL-35表达增。我们假设IL-35在CRC中通过诱导Tconv转化为Treg和/或通过IL-35R下游信号通路抑制效应T细胞发挥功能。为验证这一假设，我们将分选CRC的Treg和Tconv细胞在体外利用Transwell共培养体系对IL-35增加Treg数量和免疫抑制的机制进行研究。利用IL-35条件性敲除小鼠建立CRC模型开展体内和体外机制研究，阐明IL-35在肿瘤中的重要作用。为通过IL-35调控机体的免疫应答，提高肿瘤生物治疗效果，开辟新的治疗思路奠定理论和实践基础。

IL-35是一种新颖的异源二聚体并抑制细胞因子,属于IL-12家族,由白介素-12亚基α（P35）和Epstein-Barr病毒诱导基因3（EBI3）两个亚基构成。 已报道IL-35可由一定细胞类型分泌，特别是调节性T细胞，并通过STATx信号传导途径发挥其免疫抑制作用。本课题中，我们证明了结肠直肠癌患者血清和肿瘤组织中IL-35表达升高。肿瘤旁组织IL-35的表达增加与肿瘤转移显著相关，同时应用免疫荧光技术，发现IL-35主要表达于癌旁组织CD4+T细胞。重组人IL-35能够抑制CD4+ CD25 -T效应细胞在体外的增殖，抑制作用呈现剂量依赖性，并且这种抑制在加入IL-35中和抗体后被逆转。人CD4+T细胞经过IL-35处理后能够活化STAT1和STAT3磷酸化。同时，IL-35通过正反馈，增加IL-35的表达参与肿瘤免疫。研究显示结直肠癌患者来源调节性T细胞能够诱导EBI3和P35的表达能力较健康个体来源的调节性T细胞更强。为了确定IL-35是否直接推动了IL-35自身生产，通过构建EBI3基因启动子区荧光素酶报告质粒，发现用IL-35刺激后，EBI3启动子活性比空质粒高。我们的研究表明，IL-35在结肠直肠癌中高水平表达，通过激活STAT1和STAT3促进IL-35产生，抑制T细胞增殖，并有参与肿瘤免疫。