结直肠癌组织微环境CD4+FOXP3+T-bet+Treg细胞免疫功能及其调控机制研究

CD4 + Foxp3 + Treg is an immunosuppressive regulatory cell subset that plays an important role in maintaining autoimmune tolerance. In many cancers, the degree of Treg infiltration in cancerous tissues is closely related to tumor progression and prognosis. Our preliminary data showed that T-bet expression in Treg cells was significantly up-regulated in colorectal cancer, suggesting that T-bet +FOXP3+ Treg cells may play an important role in the malignant progression of colorectal cancer. In combination with HIF1α inhibitor, T-bet expression up-regulated in Treg cells induced by hypoxia treatment and its immunosuppressive function, which depending on HIF1α. In addition, ubiquitination and nuclear segregation experiments confirmed that hypoxia up-regulated ubiquitinase Usp10 stable T -bet protein . Therefore, it is hypothesized that the hypoxia microenvironment in colorectal cancer activates HIF1α and causes USP10 to nuclear translocation and deubiquinate T-bet, thereby increasing the proportion of T-bet + FOXP3+ Treg cells and their immunosuppressive function, and eventually inhibiting the antitumor immunity of colorectal cancer. This project will investigate the regulation and immune function of T-bet + FOXP3+ Treg cells in colorectal cancer hypoxic microenvironment using FACS and AOM / DSS enteritis model.

CD4+FOXP3+Treg是一种免疫抑制性调节细胞亚型，在维持自身免疫耐受中发挥重要作用。多种肿瘤癌组织中Treg浸润程度与肿瘤进展和预后密切相关。前期FACS检测发现结直肠癌FOXP3+Treg细胞内T-bet表达显著上调，提示T-bet+FOXP3+Treg细胞可能在结直肠癌恶变中扮演重要角色。结合HIF1α抑制剂，缺氧处理体外诱导分化的iTreg上调T-bet表达，其免疫抑制功能依赖于HIF1α。泛素化沉淀和核质分离实验证实缺氧可使泛素化酶USP10稳定T-bet蛋白。因此推测：结直肠癌缺氧微环境稳定HIF1α，引起USP10入核并稳定T-bet，从而上调T-bet+FOXP3+Treg细胞比例及其免疫抑制功能，最终抑制结直肠癌抗肿瘤免疫反应。本项目将利用FACS、AOM/DSS肠炎模型等手段进一步研究结直肠癌缺氧微环境中T-bet+FOXP3+Treg细胞的调节及其作用机理。

我们前期利用AOM/DSS诱导肠炎相关肠癌模型发现相比于对照组，CD4+T中敲除缺氧感受器HIF1a和HIF2a更容易诱发肠炎相关肠癌，临床表现上HIF1a和HIF2a CKO组更早出现便血和体重减轻。待造模结束后流式分析脾脏、外周淋巴结、肠系膜淋巴结和肠道固有层发现肠道固有层中的Τh17比例和活性显著下调，而巨噬细胞比例显著上调，而Treg细胞比例仅在脾脏中明显下调，而Τ-bet+Treg细胞比例无显著性变化，说明缺氧感受器HIF1a和HIF2a在CD4+T细胞向Τh17和Treg细胞极化过程中发挥重要作用，机制分析发现HIF2a可能通过caspase蛋白降解途径抑制FOXP3、RORγt蛋白稳定性，同时代谢组和转录组测序数据表明牛磺酸和次牛磺酸代谢通路在其中发挥作用，而研究表明肠道中次级胆汁酸主要由肠道菌群辅助合成，说明HIF1a和HIF2a可能通过抑制CD4+向Τh17和Treg细胞极化，引起肠组织中浸润的免疫细胞异常导致肠道菌群紊乱，进而引起牛磺酸和次牛磺酸代谢通路异常，正反馈至免疫细胞，最终诱发肠炎相关肠癌发生，因此我们目前正在检测肠道中菌群变化差异，为接下来研究缺氧感受器HIF1a和HIF2a在CD4+T细胞调控肠道菌群稳态介导牛磺酸和次牛磺酸代谢中奠定理论依据，同时为肠炎相关肠癌发生提供新的治疗靶点。此外，我们利用GEO178341单细胞转录组测序数据和课题组前期的单细胞转录组测序数据，提取Treg细胞亚群进行聚类分析，发现相比于正常黏膜组织，腺瘤、癌旁组织和腺癌组织中Tumor Treg、Hsp Treg和Tfh-like cell比例具有显著性差异，且HIF1a主要表达于Tfh-like细胞亚群，同时发育分化轨迹结果说明Tumor Treg、Hsp Treg和Tfh-like在Treg亚群发育分化不同节点，但无明显的T-bet+Treg细胞聚类，综上说明Tumor Treg、Hsp Treg和Tfh-like可能在结直肠癌癌变过程中发挥重要作用，尤其是Tfh-like细胞细胞可能在结直肠癌缺氧微环境中发挥重要作用，为后续开展结直肠癌中Tumor Treg、Hsp Treg和Tfh-like的功能研究奠定理论依据。