TPX2调控肝癌CD8+T细胞耗竭的作用机制研究及其抗肿瘤效应的探索

CD8+T cells are the primary effectors of anti-tumor immunity. However, under persistent stimulation from tumor antigens, the functions of CD8+T cells in tumor microenvironment decreased gradually, a state called "T cell exhaustion", compromising anti-tumor immunity. In our preliminary study, we have separated CD8+T cells from peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TILs) of patients with hepatocellular carcinoma, analyzed their functionality and surface markers (PD1, TIM3) expression. Then, high-throughput RNA sequencing of CD8+T cells from PBMC and TILs respectively was conducted to analyze different expressed genes between the two, followed by real-time PCR and western blot validation. At last, TPX2 was selected for subsequent functional investigation. Based upon these preliminary results, we plan to investigate the effects of TPX2 overexpression (with cDNA) and knockout (with CRISPR-Cas9) on the functionalities, surface markers expression and anti-tumor efficacy of CD8+T cells in cultured cells, three-dimensional cell cultures and in patient-derived xenograft (PDX) models of HCC, respectively. Then, we will investigate the mechanisms underlying the regulatory roles of TPX2 on T cell exhaustion with the aid of transcriptome sequencing, co-immunoprecipitation and mass spectrum analysis. We will also conduct patient follow-up to analyze the possible correlation between TPX2 expression in CD8+T cells and patients’ prognosis, exploring the possibility of TPX2 expression in CD8+T cells to serve as a prognostic marker of HCC. In sum, this program is aimed to explore the possibility of reversing CD8+ T cell exhaustion and enhancing the anti-tumor effects of CD8+ T cells in HCC through intervening TPX2 expression, paving the way for its clinical translation.

CD8+T细胞是抗肿瘤免疫主要效应细胞。然而，肿瘤微环境中CD8+T细胞在持续性肿瘤抗原刺激下功能逐渐降低，出现T细胞耗竭。本课题组前期从慢性乙肝背景的肝癌患者外周血和癌组织中分离出CD8+T细胞，检测了其功能和表面标志分子(PD1、TIM3)表达，并通过RNA测序和临床样本验证筛选出肝癌T细胞耗竭相关基因TPX2，做了初步功能验证。在此基础上，我们将利用cDNA过表达及CRISPR-Cas9基因敲除技术，分别在体外培养CD8+T细胞、3D培养体系以及免疫缺陷鼠肝癌模型中干预TPX2表达，然后检测T细胞功能、标志分子表达及其抗肿瘤效应的变化(体内、体外功能验证)；并利用转录组测序和蛋白免疫共沉淀深入研究TPX2调控T细胞耗竭的分子机制，进行临床随访研究CD8+T细胞中TPX2表达与患者预后的关系。从而探索通过干预TPX2来逆转T细胞耗竭并增强其抗肿瘤效应的可能性，为其临床转化提供依据。

靶向T细胞功能障碍中起主导作用的关键基因是一种有效的癌症免疫治疗策略。本研究旨在研究TPX2在肝细胞癌(HCC) CD8+ T细胞中的抗肿瘤作用。通过流式细胞术检测T细胞表面标志物和细胞因子水平，我们发现TPX2在HCC浸润CD8+ T细胞中表达下调。在HCC荷瘤的CD8 -/-小鼠中，TPX2的缺失抑制了CD8+ T细胞的抗肿瘤活性，而过表达TPX2则增加了CD8+ T细胞的抗肿瘤作用。在肝癌患者来源的异种移植(PDX)小鼠模型中，TPX2过表达改善了CD8+ T细胞的抗肿瘤功能和对抗pd -1治疗的反应。在机制上，TPX2促进P65的磷酸化，从而增加细胞核中p-P65的水平，p-P65结合CXCR5的启动子，激活CXCR5的转录，并以TPX2依赖的方式增加CD8+ T细胞上CXCR5的水平。综上所述，TPX2通过NF-κB信号通路调控 CD8+T 细胞中CXCR5表达，在HCC中维持CD8+ T细胞的抗肿瘤作用。CD8+ T细胞中TPX2表达的增加与抗pd -1治疗具有协同作用，有望成为潜在的肝癌免疫治疗方法。