替诺福韦酯抗病毒治疗对慢性乙型肝炎患者病毒特异性CD8^(+)T细胞功能的影响

Effect of tenofovir disoproxil fumarate antiviral therapy on virus-specific CD8^(+)T Cells function in patients with chronic hepatitis B

目的观察替诺福韦酯(TDF)抗病毒治疗对HBeAg阳性慢性乙型肝炎患者外周血HBV特异性CD8^(+)T细胞功能的影响,评估其与HBeAg血清学阴转的相关性。方法纳入2016年10月至2018年7月就诊的HLA-A02限制性HBeAg阳性慢性乙型肝炎患者63例,予以TDF(300 mg/d)抗病毒治疗,分选基线和治疗48周时外周血CD8^(+)T细胞,流式细胞术检测外周血T细胞计数,酶联免疫斑点试验检测分泌穿孔素、颗粒酶B和γ干扰素(IFN-γ)的HBV特异性CD8^(+)T细胞频数,建立HBV特异性CD8^(+)T细胞与HepG2.2.15细胞直接接触和间接接触共培养系统,检测培养上清液中HBV DNA,通过检测乳酸脱氢酶水平计算靶细胞死亡率,酶联免疫吸附试验检测细胞因子表达,评估病毒特异性CD8^(+)T细胞的细胞杀伤和非细胞杀伤功能。2组计量资料比较采用t检验或配对t检验。结果TDF治疗48周时病毒学应答率为100%,生化学应答率为90.48%(57/63),HBeAg阴转率为25.40%(16/63)。外周血T细胞计数在TDF治疗48周时与基线及对照组比较差异均无统计学意义(P>0.05)。TDF治疗48周时,CHB患者分泌穿孔素、颗粒酶B和IFN-γ的HBV特异性CD8^(+)T细胞频数较基线显著升高(P<0.001),HBeAg阴转的CHB患者病毒特异性CD8^(+)T细胞分泌穿孔素、颗粒酶B和IFN-γ的频数亦显著高于HBeAg未阴转的患者(P<0.05)。在直接接触和间接接触培养系统中,TDF治疗48周时HBV特异性CD8^(+)T细胞均可诱导HepG2.2.15细胞培养上清液中HBV DNA显著下降(P<0.001),分泌IFN-γ和白细胞介素-2的水平显著升高(P<0.05),但仅在直接接触共培养系统中病毒特异性CD8^(+)T细胞诱导HepG2.2.15细胞死亡的比例升高(21.7%±6.18%比16.1%±4.15%,P<0.001)。HBeAg阴转的CHB患者HBV特异性CD8^(+)T细胞较HBeAg未阴转患者诱导HBV DNA下降水平更显著(P<0.001)、IFN-γ分泌水平升高(P<0.05),但靶细胞死亡比例在HBeAg阴转和未阴转患者之间的差异无统计学意义(P>0.05)。结论TDF治疗过程中,伴随病毒载量下降,病毒特异性CD8^(+)T细胞的细胞杀伤和非细胞杀伤功能均显著增强,且与HBeAg阴转密切相关。

Objective To observe the effect of tenofovir disoproxil fumarate(TDF)antiviral therapy on HBV-specific CD8^(+)T cell function in peripheral blood of patients with HBeAg-positive chronic hepatitis B,and to assess its correlation with HBeAg sero-negativeness.Methods Sixty-three cases with HLA-A02 restricted HBeAg-positive chronic hepatitis B who received TDF(300 mg/d)antiviral therapy were enrolled from October 2016 to July 2018.The peripheral blood CD8^(+)T cells were separated at baseline and 48 weeks after treatment.The peripheral blood T cells count were detected by flow cytometry.The frequency of HBV-specific CD8^(+)T cells secreting perforin,granzyme B,and interferon-γ(IFN-γ)were detected by enzyme-linked immunoblotting test.Direct and indirect contact co-culture system was established between HBV-specific CD8^(+)T cells and HepG2.2.15 cells.HBV DNA was detected in the culture supernatant.Target cell mortality was calculated by lactate dehydrogenase level.Cytokines expression was detected by enzyme-linked immunosorbent assay.Virus-specific CD8^(+)T cells cytokilling and non-cytokilling functions were evaluated.Measurement data of the two groups were compared by t-test or paired t-test.Results Viral response,biochemical response,and HBeAg seroconversion rate at 48 weeks of TDF treatment were 100%,90.48%(57/63),and 25.40%(16/63),respectively.There was no statistically significant difference in peripheral blood T cell count when compared with baseline and control group at 48 weeks of TDF treatment(P>0.05).At 48 weeks of TDF treatment,the frequency of HBV-specific CD8^(+)T cells secreting perforin,granzyme B,and IFN-γin CHB patients was significantly higher than baseline(P<0.001).Furthermore,the frequency of HBV-specific CD8^(+)T cells secreting perforin,granzyme B,and IFN-γwas also significantly higher in CHB patients with HBeAg negative than that of non-negative(P<0.05).HBV-specific CD8^(+)T cells had induced significant down-regulation of HBV DNA in the supernatant of HepG2.2.15 cell culture(P<0.001)and remarkable IFN-γand interleukin-2 secretion(P<0.05)at 48 weeks of TDF therapy in direct and indirect contact co-culture system.However,HepG2.2.15 cells death rate induced by virus-specific CD8^(+)T cells was increased only in the direct contact co-culture system(21.7%±6.18%vs.16.1%±4.15%,P<0.001).Compared with HBeAg non-negative patients,HBeAg negative CHB patients with HBV-specific CD8^(+)T cells had induced a strong decrease in HBV DNA(P<0.001)and an increase in IFN-γsecretion level(P<0.05).However,the target cell death proportion difference between HBeAg negative and non-negative patients was not statistically significant(P>0.05).Conclusion During TDF treatment,with the viral load reduction,virus-specific CD8^(+)T cells cytokilling and non-cytokilling functions are significantly enhanced,and are closely related to HBeAg negative.