循环Tfh细胞促进B细胞活化在多发性硬化病理过程中的作用机制研究

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) in which the exact pathogenesis is unclear. It has been generally accepted that myelin-reactive T cell-induced immune responses cause the onset of MS, but the recent evidence has supported a critical role of B cells. However,the regulatory mechanism of the B-cell activation is not fully understood..T follicular helper (Tfh) cells, located in the lymphoid follicles, are the effector cells of B-cell activation,and play a central role in the activation of B cells,the formation of germinal center (GC) and the production of high-affinity antibodies. It has been demonstrated that CD4+CXCR5+ T cells in the peripheral blood posses a similar phenotype and function with GC-Tfh cells. In our recent study, a significant increase of circulating CD4+CXCR5+ T cells has been observed compared with healthy controls, but there is no studies concerning the potential role of these cells in the MS..In this study, we are planning to collect the circulating Tfh cells during relapsing or remitting period of RRMS patients by utilizing immunomagnetic beads and flow cytometry (FACS), evaluate the expression of the specific surface molecules, and explore the correlation between circulating Tfh cells and the disease load. We are trying to test the potential role of circulating Tfh cells in inducing B-cell activation. Moreover, we are still trying to investigate whether the transferred circulating Tfh cells from the mice with experimental autoimmune encephalomyelitis (EAE) affect the onset of EAE and the severity of disease in the normal mice immunized with MOG35-55. This would provide a novel therapeutic strategy for MS.

多发性硬化（MS）作为中枢神经系统自身免疫性疾病，确切发病机制尚不明了。传统观点认为髓鞘反应性T细胞免疫介导了MS发病，新近证据支持B细胞免疫在MS中具有重要作用，但B细胞活化的调控机制尚不完全清楚。.辅助B细胞活化的主要效应细胞是滤泡辅助性T细胞（Tfh），定位于淋巴滤泡，在B细胞活化、生发中心(GC)形成及高亲和力抗体产生中发挥核心作用。研究表明外周血CD4+CXCR5+T细胞具有与Tfh相似的表型和功能，称为循环Tfh，可间接反映Tfh的功能状态。我们前期研究发现MS患者复发期外周血CD4+CXCR5+T细胞比例明显高于健康对照组，但该群细胞在MS发病中的作用如何尚无报道。.本项目采用免疫磁珠+流式细胞分选MS不同病程阶段的循环Tfh，分析其表面分子表达情况及与疾病负荷的相关性；体外细胞培养验证其对B细胞的活化作用；观察过继转移循环Tfh对EAE发病的影响，为MS治疗提供新的作用靶点

本研究以实验性自身免疫性脑脊髓炎（EAE）小鼠模型和MS患者为对象，采用激光共聚焦、流式细胞仪、免疫磁珠分选、体外细胞共培养等手段发现：（1）MS患者外周血Tfh细胞比率在复发期升高，而在缓解期与健康对照组比较无明显差异。尽管循环Tfh细胞比率与病情活动度有关联，但其与EDSS评分和颅内Gd增强病灶数之间并无明确相关性；（2）MS患者外周血B细胞比率在复发期明显升高，而在缓解期与健康对照组比较无统计学差异，与EDSS评分和颅内Gd增强病灶数亦无明确相关性；（3）MOG35-55肽段免疫诱导激活了C57BL/6小鼠体内的免疫反应，导致脾脏生发中心的各类细胞发生了结构重排，其中Tfh细胞与B细胞紧密接触，进一步确定该群Tfh细胞的表型为CD4+CXCR5+ICOS+和CD4+CXCR5+PD-1+；（4）EAE小鼠的中枢神经系统（CNS）出现了异位的淋巴滤泡样结构，其中Tfh细胞与B细胞密切接触，为两者相互作用提供了形态学证据；（5）EAE小鼠脾脏、淋巴结及CNS中Tfh细胞和B细胞亚群比率、血清IL-21水平和抗MOG35-55肽段抗体滴度随病情变化而发生波动，IL-21和抗MOG35-55肽段抗体与EAE评分均呈正相关；（6）EAE小鼠脾脏中Tfh细胞相关因子CXCR5、Bcl-6和IL-21蛋白表达随病情而发生变化，以高峰期升高为著，与Tfh细胞的变化趋势相同，支持了Tfh细胞参与EAE疾病的观点；（7）过继转移EAE小鼠Tfh细胞和B细胞入正常C57BL/6小鼠体内，经MOG35-55肽段免疫诱导后，EAE发病时间有提前，症状评分较正常小鼠诱导组更高。