结核病患者抗原特异性CD4+T细胞分化及功能异常的分子机制研究

Eomesodermin and T-bet are important transcription factors that regulate differentiation and functions of CD4+ effector and memory T cells, however, their role in anti-tuberculosis (TB) immunity has not been studied so far. In our previous studies, we found that expression of CD27 on antigen-specific CD4+ T cells was significantly reduced in patients with active TB, accompanied by decreased T cell proliferation and IFN-gamma production, suggesting malfunction of CD4+ T cell. Based on these observation, we postulated that Eomesodermin and T-bet might play an important role in regulation of TB antigen-specific CD4+ T cell differentiation and functions in active TB patients. In this proposal, we plan to study the expression profile of Eomesodermin and T-bet in T cells of patients with TB, individuals with latent infection , in order to understand their relationship with disease severity of TB; to study the role of Eomesodermin and T-bet in differentiation of antigen-specific CD4+ effector and memory T cell and in production of effector molecules such as IFN-gamma, TNF-alpha. The study has important implications in TB prevention and treatment.

转录因子Eomesodermin和T-bet在CD4+效应和记忆T细胞分化及功能中起关键调控作用，但其在结核病患者抗结核细胞免疫中的作用尚未见报道。我们的前期研究发现，活动性结核病患者结核抗原特异性CD4+T细胞CD27表达显著降低，IFN-gamma产生及T细胞增殖能力显著下降，提示结核病患者抗原特异性CD4+T细胞分化及功能存在异常。我们推测，Eomesodermin和T-bet可能在结核病患者抗原特异性CD4+T细胞分化及功能异常中起了重要的作用。本申请计划研究结核病患者与潜伏感染者结核抗原特异性CD4+T细胞Eomesodermin和T-bet的表达特点及其与结核病严重程度的关系；研究Eomesodermin和T-bet在结核抗原特异性CD4+效应及记忆T细胞分化及效应分子IFN-gamma、TNF-alpha等产生中的作用。该研究对探索结核病防治新途径有重要意义。

本课题研究了活动性结核病患者结核抗原特异性CD4+ T细胞的特征及调控机制，发现活动性结核病患者结核抗原特异性CD4+ T细胞内T-bet和Eomesdermin表达显著高于潜伏感染者。活动性结核病患者与潜伏感染者总CD4+ T细胞中TCM和TEM的比例无显著差异，但在结核抗原特异性CD4+ T细胞中TCM和TEM的比例存在显著差异，活动性结核病患者TCM比例显著降低。在CD4+CD154+CD45RO+细胞中分析T-bet的表达与TCM/TEM比例的关系，显示T-bet在TEM表达显著性升高，但在TCM显著性降低，提示T-bet促进TEM的分化。结核抗原特异性CD4+ T细胞表面PD-1的表达水平显著高于潜伏感染者，T-bet+细胞表面PD-1表达显著高于T-bet-细胞，提示T-bet促进PD-1表达。粘膜相关恒定T细胞（MAIT细胞）是一类新的天然免疫样T细胞，我们发现活动性结核病患者外周血MAIT细胞比例显著低于正常对照者（p＜0.0001）和潜伏感染者（p=0.0105）；结核性胸膜炎患者胸腔积液中MAIT细胞与肺结核患者外周血MAIT细胞比例相似，而结核性腹膜炎患者腹水中MAIT细胞比例显著高于胸腔积液（p=0.0059）。提示MAIT细胞可能在抗结核免疫中起了重要作用。活动性结核病患者和正常健康对照者MAIT细胞经结核分枝杆菌H37Rv裂解液及BCG刺激后CD69表达显著增高，BCG刺激后IFN-gamma和TNF-alpha产生也增高，提示MAIT细胞可以被结核特异性抗原激活产生效应分子。结核病患者和正常健康对照者MAIT在PMA/ionomycin刺激后IFN-gamma和TNF-alpha产生无显著差异；采用E.coli刺激，结核病患者MAIT细胞IFN-gamma和TNF-alpha产生显著低于正常对照者（p=0.0007和p=0.0032），提示存在功能缺陷。发现PD-1在活动性结核病患者MAIT细胞表达显著高于正常健康对照者（p=0.0015）。采用PD-1受体特异性单克隆抗体阻断PD-1信号通路后，结核病患者MAIT细胞IFN-gamma表达显著增高（p=0.0178），提示MAIT细胞功能受PD-1调节。该研究对了解抗结核免疫保护机制有重要意义。