活动性肺结核患者LAG-3的表达及其对CD4^(+)T细胞的影响

Expression of LAG⁃3 in CD4^(+)T cells in patients with active pulmonary tuberculosis and the effect of LAG⁃3 on the function of CD4^(+)T cells

目的探讨淋巴细胞活化基因3(LAG-3)在活动性肺结核患者CD4^(+)T细胞中的表达情况及其对CD4^(+)T细胞功能的影响。方法纳入2020年5月-2021年5月就诊于苏州大学附属传染病医院的25例初治活动性肺结核患者作为病例组,20名健康体检者作为对照组,流式细胞术检测外周血和支气管肺泡灌洗液中LAG-3^(+)CD4^(+)T细胞的比例;体外用结核分枝杆菌特异性抗原早期分泌靶抗原6000 Mr与培养滤过蛋白10000 Mr(ESAT-6/CFP10)刺激结核患者的外周血单个核细胞(PBMC),分为ESAT-6/CFP组和不加ESAT-6/CFP蛋白的Control组,探究LAG-3在结核特异性CD4^(+)T细胞上的表达;用可溶性LAG-3(sLAG-3)蛋白抑制LAG-3分子信号通路,作为sLAG-3组,探究LAG-3对CD4^(+)T细胞功能的影响。结果病例组患者PBMC和支气管肺泡灌洗液(BALF)中LAG-3^(+)CD4^(+)T细胞比例升高,治疗后下降,差异均有统计学意义(t=2.291、3.045,P均<0.05)。经过ESAT-6/CFP10刺激后的ESAT-6/CFP10组CD4^(+)T细胞上LAG-3的表达上调,而干扰素-γ(IFN-γ)的表达下降,与Control组相比差异均有统计学意义(t=6.174、3.476,P均<0.05)。抑制LAG-3分子信号后,与ESAT-6/CFP10组相比,sLAG-3组CD4^(+)T细胞上IFN-γ和Granzyme B的表达升高,差异均有统计学意义(t=3.882、3.205,P=0.178、0.033)。结论活动性肺结核患者体内CD4^(+)T细胞上LAG-3的表达升高,抗结核治疗后下降,LAG-3可能可以通过抑制CD4^(+)T细胞IFN-γ的分泌,损害CD4^(+)T细胞抗结核分枝杆菌的能力。

Objective To investigate the expression of lymphocyte activation gene⁃3(LAG⁃3)in CD4^(+)T cells in patients with active pulmonary tuberculosis and the effect of LAG⁃3 on the function of CD4^(+)T cells.Methods 25 patients with active pulmonary tuberculosis and 20 healthy controls were recruited from the Infectious Diseases Hospital Affiliated to Soochow University between May 2020 and May 2021,used as the case group and the control group,respectively.The expression of LAG⁃3 on CD4^(+)T cells in peripheral blood and bronchoalveolar lavage fluid were detected by Flow cytometry.Peripheral blood mononuclear cells(PBMC)of tuberculosis patients was stimulated with Mycobacterium tuberculosis specific antigen early secreted antigenic target 6000 Mr and culture filtrate protein 10000 Mr(ESAT⁃6/CFP10)in vitro to explore the expression of LAG⁃3 on tuberculosis specific CD4^(+)T cells.Divided into the ESAT⁃6/CFP10 group and the Control group without the ESAT⁃6/CFP10 protein.The effects of LAG⁃3 on CD4^(+)T cell functions were investigated by inhibition of solubility LAG⁃3(sLAG⁃3)protein(sLAG⁃3 group).Results The percentage of LAG⁃3^(+)CD4^(+)T cells in PBMC and bronchoalveolar lavage fluid(BALF)increased and decreased after treatment,the differences were statistically significant(t=2.291,3.045;all P<0.05).After stimulated by ESAT⁃6/CFP10,compared with the Control group,the expression of LAG⁃3 in ESAT⁃6/CFP10 group was up⁃regulated on CD4^(+)T cells,while the expression of interferon⁃γ(IFN⁃γ)was down⁃regulated,the differences were statistically significant(t=6.174,3.476;all P<0.05).After inhibition of LAG⁃3 molecular signaling,compared with the ESAT⁃6/CFP10 group,the expression of IFN⁃γand Granzyme B was elevated on CD4^(+)T cells in the sLAG⁃3 group,the differences were statistically significant(t=3.882,3.205,P=0.178,0.033).Conclusions The expression of LAG⁃3 was increased in CD4^(+)T cells of active pulmonary tuberculosis patients and decreased after anti⁃tuberculosis treatment.It might affect the ability of CD4^(+)T cells to secrete IFN⁃γ,to impair the ability of CD4^(+)T cells against Mycobacterium tuberculosis.