滤泡CD8+T细胞在多发性硬化及其小鼠模型免疫发病中的作用及机制研究

Multiple sclerosis (MS) is a myelin-reactive T cell-mediated autoimmune demyelinating disease characterized by cellular immunity. The exact pathogenesis is still unclear. Follicular CD8+ T cells are a newly discovered group of CD8+ T cells with a phenotype of CD8+CXCR5+, which has a stronger cytotoxic effect and plays an important role in diseases such as viral infection and malignant tumors. However, no research has reported on the role of this group of cells in autoimmune diseases. In the previous study, the applicant found that the ratio of follicular CD8+ T cells was significantly increased in MS patients and their animal models, ie, experimental autoimmune encephalomyelitis (EAE), and the infiltration of follicular CD8+ T cells was found in the spinal cord of EAE mice. Therefore, we conclude that it may be involved in the occurrence of the above diseases. This project will analyze the expression of follicular CD8+ T cells in different course of MS and EAE mice, and explore its correlation with the course of disease. The mouse model will be used to investigate the histological distribution of follicular CD8+ T cells under pathological conditions and the molecular mechanism of this group of cells involved in the disease, aiming to provide new theoretical and experimental basis for the pathogenesis of MS, and to provide new targets for the diagnosis and treatment of MS.

多发性硬化（multiple sclerosis，MS）是一种髓鞘反应性T细胞介导的，以细胞免疫为主的自身免疫性脱髓鞘疾病，其确切的发病机制尚不明确。滤泡CD8+T细胞是新近发现的一群CD8+T细胞亚群，表型为CD8+CXCR5+，具有更强的细胞毒作用，在病毒感染和恶性肿瘤等疾病中发挥着重要作用。但目前尚无研究报道这群细胞在自身免疫性疾病中所起的作用。申请者前期研究发现，在复发期MS患者及其小鼠模型EAE的体内，滤泡CD8+T细胞比率显著升高，并在EAE小鼠脊髓中发现了滤泡CD8+T细胞的浸润，推断其可能参与了上述疾病的发生。本项目通过分析MS患者及EAE小鼠不同病程中滤泡CD8+T细胞的表达情况，探讨其与病程的相关性，并借助小鼠模型研究病理状态下滤泡CD8+T细胞组织学分布情况以及该群细胞参与疾病发生的分子机制，旨在为MS发病机制提供新的理论和实验依据，为MS的诊治提供新靶点。

滤泡CD8+T细胞是新近发现的一群CD8+T细胞亚群，表型为CD8+CXCR5+，在抗病毒感染和恶性肿瘤等疾病中发挥着重要作用。目前尚无研究报道这群细胞在自身免疫性疾病中所起的作用。本研究以实验性自身免疫性脑脊髓炎（EAE）小鼠和多发性硬化（MS）患者为研究对象，运用流式细胞术、免疫荧光染色、免疫磁珠分选、细胞共培养等相关手段进行研究，结果发现：（1）复发期和缓解期MS患者CD8+CXCR5+T细胞比率和数量均较健康人明显升高，但复发期和缓解期患者之间无明显差异。尽管CD8+CXCR5+T细胞与MS疾病活动度可能无关，但这群细胞的比率与患者钆增强病灶数呈正相关；（2）利用MOG35-55肽段构建EAE小鼠，免疫荧光染色发现小鼠中枢神经系统（CNS）中确有CD8+CXCR5+T细胞的浸润，提示这群细胞可能参与了脱髓鞘和轴索损伤的过程；（3）EAE小鼠脾脏和CNS中CD8+CXCR5+T细胞、滤泡辅助性T细胞（Tfh）和滤泡B细胞的比率随病情变化而波动，三者均在疾病高峰期达到最高值，且CD8+CXCR5+T细胞的比率与Tfh细胞和滤泡B细胞均正相关，支持了CD8+CXCR5+T细胞参与EAE疾病的观点，提示了CD8+CXCR5+T细胞可能有促进体液免疫活化的作用；（4）EAE小鼠CD8+CXCR5+T细胞和CD8+CXCR5-T细胞具有不同的转录本表达，CD8+CXCR5+T细胞中与淋巴滤泡、体液免疫等相关的基因表达量显著上调；（5）功能学试验发现CD8+CXCR5+T细胞比CD8+CXCR5-T细胞具有更强的细胞毒性作用，且CD8+CXCR5+T细胞还能促进B细胞分化和分泌特异性抗体，因此CD8+CXCR5+T细胞可能在EAE发病过程中同时参与细胞和体液免疫应答。