DcR3与配体相互作用在肝细胞癌免疫逃逸中分子机制的研究

DcR3 is an important protein in cell apoptosis signaling pathways. The latest study found that DcR3 is also an immune suppressor, and is highly expressed in many tumor tissues. DcR3 can interact with its ligand involving in tumor immune escape, of which the specific molecular mechanism has not been fully elucidated. During study, the applicant is committed to the tumor immune escape mechanism and has found that: high expression of DcR3 in liver cancer cells may be associated with the immune escape of hepatocellular carcinoma (HCC), but the specific molecular mechanism has not yet in-depth discussion. In this study, we will first find the T cell subtype for DcR3 combining with by proteomics approach. Then, we will detect DcR3 ligands in T cell subtypes using immune coprecipitation, immunofluorescence localization and GST pull-down technology, and explore DcR3 signaling pathways in regulating T cells functions by high-throughput sequencing and chromatin immune co-precipitation technique. Finally, we will assess the immune regulation of DcR3 on HCCs though in vivo and in vitro experiments. Collectively, our study aims to illuminate the molecular mechanism of DcR3 regulating immune escape of HCCs and provide a possible strategy for HCC immunotherapy.

DcR3是细胞凋亡信号通路中的重要蛋白，最新的研究发现其具有免疫抑制的作用，在多种肿瘤组织中呈高表达，并与其配体相互作用参与肿瘤的免疫逃逸，但是其具体分子机制尚不清楚。申请人在前期研究肿瘤发生分子机制时发现：肝癌细胞高表达的DcR3可帮助癌细胞逃避免疫系统的监视，但是对其具体分子机制尚未进行深入探讨。本研究为了进一步深入研究DcR3在肝癌免疫逃逸中的分子机制，拟利用蛋白质组学的方法寻找DcR3所结合T细胞的亚型，并利用免疫共沉淀、免疫荧光共定位及GST pull-down等技术寻找DcR3在该亚型上的配体，再通过高通量测序与染色质免疫共沉淀技术探究DcR3调控T细胞所利用的信号通路，最后利用体内外实验验证DcR3通过结合T细胞配体对肝癌细胞免疫调控作用的影响。以期为肝细胞癌的免疫治疗提供新靶点及新思路。

前期研究发现DcR3在多种肿瘤组织中呈高表达，并提示其与配体相互作用可能参与了肿瘤的免疫逃逸，但关于DcR3在肝细胞癌免疫逃逸中的相关分子机制尚不清楚。因此，本研究中我们首先通过一系列分子生物学技术检测了DcR3在肝细胞癌组织中的表达情况，发现DcR3在肝细胞癌中高表达，并与肿瘤的演进密切相关；并通过体外细胞共培养模型，体内小鼠肝癌模型以及流式细胞术等技术证明了肝癌细胞来源的分泌因子DcR3与机体的免疫抑制活动密切相关；同时利用细胞因子芯片、免疫共沉淀、免疫荧光共定位等实验技术证明了肝癌细胞来源的DcR3可以和CD4+T淋巴细胞表面的配体LIGHT相结合，进而调控T淋巴细胞的免疫功能；最后通过生物信息学分析、western blotting技术、流式细胞术、以及Elisa技术等明确了肝癌细胞来源的DcR3与CD4+T淋巴细胞相结合后，通过激活T淋巴细胞内的NF-κB信号通路，进而调控淋巴细胞的功能。综上所述，我们的研究证实了DcR3可以通过与CD4+T细胞表面的LIGHT配体结合，通过激活T细胞内的NF-κB信号通路，进而抑制CD4+T细胞的免疫调控功能，促进肝癌细胞免疫抑制微环境的形成。