4-1BB在粘膜相关恒定T细胞抗结核免疫中的调节作用

Mucosal-associated invariant T cells (MAIT cells) from patients with tuberculosis have reduced frequency and exhibit functional defects, which correlate with disease severity of tuberculosis. Previous study found that PD-1 was associated with functional defects; however, blockade of PD-1 signaling only led to partial recovery of MAIT cell function in patients with tuberculosis, suggesting that other mechanisms might play an important role. To understand the mechanisms of MAIT cell functional defects, we identified differentially expressed genes of MAIT cells from patients with tuberculosis by RNA-seq, and found that 4-1BB was one of the genes that were significantly up-regulated. Further analysis with flow cytometry showed that 4-1BB expression was highly correlated with IFN-γ expression. Our hypothesis is that 4-1BB is a critical regulator of MAIT cell functions, and stimulating 4-1BB signaling can restore MAIT cell function and lead to improved control of tuberculosis. We plan to investigate the role of 4-1BB in MAIT cell function, and study the effect and mechanisms of stimulating 4-1BB and blocking PD-1 in improving function of MAIT cells from patients with tuberculosis. The study has important implications in the treatment of tuberculosis.

活动性结核病患者MAIT细胞比例显著降低且存在功能缺陷，与结核病发病和疾病进展密切相关。我们前期研究显示，MAIT细胞功能缺陷与PD-1相关，但阻断PD-1只能部分改善MAIT细胞功能，提示其他机制也发挥重要作用。为了研究MAIT细胞功能缺陷的机制，我们采用RNA-seq研究了MAIT细胞在结核分枝杆菌抗原刺激后的基因表达差异，发现4-1BB是主要的差异表达基因之一；流式细胞术显示4-1BB表达与IFN-γ产生显著相关。我们的假说是: 4-1BB信号通路在MAIT细胞功能中起了关键的正调控作用，激活4-1BB信号通路可逆转活动性结核病患者MAIT细胞功能缺陷，促进结核病的有效控制。本课题计划研究4-1BB信号通路在MAIT细胞抗结核免疫功能中的调节作用及分子机制，研究激活4-1BB、阻断PD-1信号通路在逆转结核病患者MAIT细胞功能缺陷中的作用及机制，为探索结核病治疗新途径奠定基础。

为了研究4-1BB在结核感染过程中对MAIT细胞免疫功能的调节作用及机制，采用流式细胞分选获得4例结核性胸膜炎患者胸腔积液4-1BB+和4-1BB-MAIT细胞，并进行RNA转录组测序，发现众多差异表达基因。4-1BB-对照和Mtb刺激的4-1BB+MAIT细胞相比较，68个基因表达上调，227个基因表达下调。生物信息学分析发现，4-1BB的表达可能主要与MAIT细胞的活化和细胞因子产生有关。KEGG通路分析发现4-1BB与T细胞受体、NF-κB信号通路、细胞因子-因子受体相互作用和蛋白酶体相关。进一步MAIT细胞功能研究发现，结核性胸膜炎患者胸腔积液4-1BB+MAIT细胞中产生IFN-γ的细胞比例显著高于4-1BB-MAIT细胞；相关性分析显示，结核抗原诱导MAIT细胞产生的IFN-γ与其表达的4-1BB密切相关。4-1BB+MAIT细胞产生的IL-17A与IL-17F显著高于4-1BB-MAIT细胞。通过对Ki-67的检测，发现4-1BB+MAIT细胞的增殖能力显著高于4-1BB-MAIT细胞。IL-2阻断抗体可导致MAIT细胞4-1BB表达的显著下降；而加入IL-12阻断抗体后，4-1BB在MAIT细胞上的表达无明显变化。为了进一步明确IL-2的作用，我们在结核抗原刺激PBMCs时添加IL-2，能显著提高MAIT细胞4-1BB的表达；该结果提示MAIT细胞4-1BB表达依赖于IL-2。IL-2的功能依赖于它的三个受体，IL-2Rα,IL-2Rβ和γc。我们将抗IL-2Rα,IL-2Rβ和γc阻断抗体分别添加到MAIT细胞培养液中，结果显示IL-2Rα和IL-2Rβ阻断抗体并不影响MAIT细胞4-1BB的表达，但γc受体的阻断导致MAIT细胞4-1BB表达显著下调，说明γc受体与MAIT细胞4-1BB表达相关。