EB病毒感染所致肝脏损伤患者病毒特异性CD4^(+)T细胞和CD8^(+)T细胞功能分析

Functional analysis of virus-specific CD4^(+)T cells and CD8^(+)T cells in patients with liver injury caused by Epstein-Barr virus infection

目的分析EB病毒(EBV)感染者中发生肝脏损伤和未发生肝脏损伤患者病毒特异性CD4^(+)T细胞和CD8^(+)T细胞功能的差异。方法入组45例EBV感染者,28例发生肝脏损伤,17例未发生肝脏损伤。分离外周血单个核细胞,纯化CD4^(+)T细胞和CD8^(+)T细胞,使用重组EBV核心抗原2(EBNA2)刺激培养96 h。细胞技术试剂盒法检测细胞增殖,流式细胞术检测CD4^(+)T细胞和CD8^(+)T细胞比例。酶联免疫吸附试验检测CD4^(+)T细胞分泌细胞因子和CD8^(+)T细胞分泌毒性分子水平,实时定量PCR法检测CD4^(+)T细胞亚群转录因子mRNA水平和CD8^(+)T细胞中毒性分子mRNA水平,流式细胞术检测CD8^(+)T细胞中免疫检查点分子水平。组间比较采用t检验或Mann-Whitney检验。结果重组EBNA2刺激后外周血单个核细胞增殖、CD4^(+)T细胞和CD8^(+)T细胞比例在EBV感染无肝脏损伤组和EBV感染致肝脏损伤组之间的差异无统计学意义(P>0.05)。重组EBNA2刺激后CD4^(+)T细胞分泌相关细胞因子比例和转录因子mRNA水平在EBV感染无肝脏损伤组和EBV感染致肝脏损伤组之间的差异无统计学意义(P>0.05)。重组EBNA2刺激后EBV感染致肝脏损伤组CD8^(+)T细胞分泌穿孔素[(75.51±23.33)pg/ml对比(58.99±18.39)pg/ml,P=0.017]和颗粒酶B[(117.8±44.55)pg/ml对比(90.22±34.21)pg/ml,P=0.034]的水平高于无肝脏损伤组,EBV感染致肝脏损伤组CD8^(+)T细胞中Fas配体和肿瘤坏死因子相关凋亡诱导配体mRNA水平较无肝脏损伤组分别升高约1.5倍和1.2倍,差异有统计学意义(P<0.001),但CD8^(+)T细胞中程序性死亡分子-1和细胞毒性T淋巴细胞抗原-4表达比例在EBV感染无肝脏损伤组和EBV感染致肝脏损伤组之间的差异无统计学意义(P>0.05)。结论EBV感染致肝脏损伤患者病毒特异性CD8^(+)T细胞毒性作用增强,可能介导EBV诱导的肝脏损伤。

Objective To analyze the functional differences between virus-specific CD4^(+)T cells and CD8^(+)T cells in patients infected with Epstein-Barr virus(EBV)who develop liver injury and those who do not.Methods 45 cases of EBV infections were enrolled,including 28 cases developing liver injuries and 17 that did not.Mononuclear cells from peripheral blood were isolated.CD4^(+)T cells and CD8^(+)T cells were purified and cultured using recombinant EBV core antigen 2(EBNA2)for 96 h with stimulation.The CCK-8 method was used to detect cell proliferation.Flow cytometry was used to detect the proportion of CD4^(+)T cells and CD8^(+)T cells.An enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of CD4^(+)T cells secreting cytokines and CD8^(+)T cells secreting molecular toxicity.Real-time quantitative PCR was used to detect the mRNA levels of transcription factors and molecular toxicity in CD4^(+)T cell subsets.Flow cytometry was used to detect the immune checkpoints at molecular levels in CD8^(+)T cells.The inter-group comparison was performed using a t-test or Mann-Whitney test.Results There was no statistically significant difference(P>0.05)in the proliferation proportion of peripheral blood mononuclear cells,CD4^(+)T cells,and CD8^(+)T cells after stimulation with recombinant EBNA2 between the EBV-infected non-liver injury group and the infected liver injury group(P>0.05).There was no statistically significant difference in the proportion of CD4^(+)T cells secreting related cytokines and the mRNA levels of transcription factors after stimulation with recombinant EBNA2 between the EBV-infected non-liver injury group and the infected liver injury group(P>0.05).The levels of perforin secreted by CD8^(+)T cells and granzyme B after stimulation with recombinant EBNA2 were higher in the EBV infection-induced liver injury group than those in the non-liver injury group[(75.51±23.33)pg/ml vs.(58.99±18.39)pg/ml,P=0.017][(117.8±44.55)pg/ml vs.(90.22±34.21)pg/ml,P=0.034].The mRNA levels of Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand in CD8^(+)T cells in the liver injury group caused by EBV infection were approximately 1.5 and 1.2 times higher than those in the non-liver injury group,respectively,and the difference was statistically significant(P<0.001),but there was no statistically significant difference in the proportional expression of programmed cell death-1 and cytotoxic T lymphocyte-associated antigen-4 in CD8^(+)T cells between the EBV-infected non-liver injury group and infected liver injury group(P>0.05)Conclusion Patients with liver injury caused by EBV infection have strong virus-specific CD8^(+)T cell toxic effects,which may mediate EBV-induced liver injury.