Tfh细胞在病毒性心肌炎小鼠中的变化及辅助抗ANT抗体产生的机制研究

Excessive activated immune response, especially mediated by CD4+ T lymphocytes (including autoimmune reaction), is the important reason of myocardial damage in viral myocarditis (VMC). Our initial research shows that, Th17 cells and its effect cytokine IL-17 involved in the pathogenesis of VMC, but IL-17 antibody could not compeletly block the inflammation of VMC. Tfh cells is a new group of CD4+T cells, whose differentiation, distribution or effect molecular abnormalities would cause the disorder of immune system and trigger autoimmune disease. Until now, the role of Tfh cells in VMC has not been reported. This project is to establish the change of Tfh cells during different periods of VMC (murine model, induced by CVB3 peritoneal injection), to explore the relationship between Tfh cells and adenine nucleotide translocator (ANT) antibody. Using IL-21 antibody and IL-21 -/- mice, we aim to observe the pathological changes of myocardiums, the B cell proliferation and the influence of the ANT antibodies. What more, this study aim to elucidate the mechanism by which Tfh cells assist the B cells to produce ANT antibody. This project will further provide new clues for clarifing the pathogenesis of VMC and therapeutic targets of VMC from a new perspective of Tfh cells.

过度激活的免疫反应尤其是由CD4+T淋巴细胞介导的免疫反应（包括自身免疫反应）是病毒性心肌炎（VMC）心肌细胞损伤的重要原因。我们前期的研究显示，Th17细胞及其效应分子IL-17参与了VMC的发病，但IL-17抗体并不能完全阻断VMC的炎症过程。Tfh细胞是一个新的CD4+T细胞亚群,其分化、分布或效应分子异常,均可造成免疫系统紊乱,引发自身免疫疾病，但Tfh细胞在VMC中的作用尚未见报道。本项目应用CVB3建立VMC小鼠模型，探讨Tfh细胞在小鼠VMC不同时期的变化规律及其与抗腺嘌呤核苷酸转位子(ANT)抗体的相关性；通过IL-21抗体和IL-21-/-小鼠，观察干预后VMC小鼠心肌病理改变、Tfh细胞、B细胞增殖及抗ANT抗体的影响；探讨CXCL13介导Tfh细胞辅助B细胞产生抗ANT抗体的机制。本项目从Tfh细胞这个新角度，为探索VMC的发病机制、寻求VMC治疗靶点提供新线索。

病毒性心肌炎（VMC）表现为一种T细胞介导的自身免疫性疾病，激活的CD4+T淋巴细胞能促进B细胞克隆增殖和产生自身抗体, 抗ANT抗体与CVB3诱发的心肌炎进展相关。滤泡辅助性性T细胞(Tfh)的主要功能是在GC反应时辅助B细胞及在体液免疫中产生自身抗体。本项目采用CVB3腹腔注射法制备小鼠VMC模型，检测Tfh细胞、IL-21、抗ANT抗体的变化。结果显示：与对照组比较，VMC小鼠脾中Tfh细胞数1-6周显著升高，2周时达峰值。VMC小鼠心肌中IL-21表达及血清中IL-21蛋白水平、抗ANT抗体1-6周显著升高也明显升高。相关分析提示Tfh细胞数与抗ANT抗体呈正相关，相关系数R=0.758。IL-21水平与抗ANT抗体也呈正相关，相关系数R= 0.88。为进一步确定IL-21在VMC中的作用及其对Tfh细胞的影响，用抗IL-21抗体拮抗VMC小鼠体内的IL-21，结果显示，抗IL-21抗体可减轻VMC小鼠的心肌炎症，降低VMC小鼠脾Tfh细胞数及血清抗ANT抗体浓度。应用IL-21R-/-小鼠观察IL-21R途径缺陷对Tfh细胞、抗ANT抗体的影响。结果显示：与VMC小鼠比较，IL-21R-/-小鼠腹腔注射CVB3后，生存率改善、心肌炎症减轻，脾中Tfh细胞比例及血清中抗ANT抗体水平降低。为明确IL-21是否对B细胞有增殖作用，分离VMC小鼠及IL-21R-/-小鼠脾单个核细胞，应用CFSE标记后进行体外培养，培养液中加入IL-21，测定CD19+B 细胞的增殖情况及培养上清液中抗ANT抗体浓度。结果显示IL-21可增加VMC小鼠B细胞增殖比例，IL-21R信号途径缺陷下，额外给予IL-21不增加B细胞增殖。VMC小鼠心肌组织CXCLl3mRNA及蛋白表达均升高，分离VMC小鼠脾CD4+CXCR5+T细胞与正常小鼠B细胞进行体外分室共培养，下室培养液中加入CXCL13可增加培养上清液中抗ANT抗体浓度，而额外加入抗CD40L抗体或抗ICOSL抗体不增加上清液抗ANT抗体浓度。本研究结果从Tfh细胞这个新视角，为VMC的发病机制及防治提供新思路与新靶点。