外泌体ORM1作为肿瘤免疫微环境中T细胞耗竭(T Cell Exhaustion)生物标志物及其功能研究

T细胞耗竭（T-cell exhaustion）是一种T细胞失能的状态，也是肿瘤免疫治疗的靶点，本项目拟探寻其血清标志物及肿瘤诱导T细胞耗竭的分子机制。前期我们用分离了同种肿瘤不同免疫分型的亚克隆，外泌体测序发现T耗竭型亚克隆产生外泌体ORM1。运用临床标本验证发现在T细胞耗竭型肿瘤患者外周血中高表达外泌体ORM1。体内实验显示外泌体ORM1抑制了肿瘤微环境中CD8+T细胞杀伤和活化。因此，我们推测外泌体ORM1可作为T细胞耗竭型肿瘤的血清学标志物，并抑制T细胞的杀伤功能。本项目拟运用数字PCR技术在T细胞耗竭型肿瘤中验证外泌体ORM1的诊断效能。运用免疫荧光、流式和western-blot，明确外泌体ORM1对T细胞功能的影响。用RNA pull-down和流式细胞术深入探讨ORM1 RNA作用的靶分子和机制。本项目将发掘T细胞耗竭型肿瘤的血清标志物并揭示肿瘤免疫逃逸的新机制。

T-cell exhaustion is a state of T-cell dysfunction and the target of tumor immunotherapy. We aim to identify its serum biomarkers and underlying mechanisms. Previously, we constructed subclones with different immune subtypes in homologous tumor. Exosomes RNA sequencing showed that the T-cell exhaustion subclones produced exosomal ORM1. Clinical specimens showed that exosomal ORM1 was highly expressed in serum of T-cell exhaustion renal cell carcinoma. Our experiments showed that the exosomal ORM1 inhibited the cytotoxic and activation of CD8+T cells in tumor microenvironment in vivo. Therefore, we speculate that the exosomal ORM1 serves as a marker of T-cell exhaustion and inhibits the cytotoxic function of T cells. This project intends to use digital PCR technology to verify the diagnostic role of exosomal ORM1 in T-cell exhaustion tumors. Immunofluorescence, flow cytometry and western blot were used to determine the effect of exosomal ORM1 on T cells. RNA pull-down and immunofluorescence were used to investigate the binding proteins and mechanism of exosomal ORM1 RNA. This project will explore the serum markers of T-cell exhaustion and reveal the new mechanism of tumor immune escape.