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Title: [Effect of tenofovir disoproxil fumarate antiviral therapy on virus-specific CD8+T Cells function in patients with chronic hepatitis B].
Author: Duan SP, Zhu LH, Hou LJ, Wang HW, Zhu XW, Hao J.
Journal: Zhonghua Gan Zang Bing Za Zhi; 2021 May 20; 29(5):421-426. PubMed ID: 34107578.
Abstract:
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-γ in 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-γ was 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-γ and 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-γ secretion 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. 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。.

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