S P Duan1, L H Zhu2, L J Hou1, H W Wang1, X W Zhu1, J Hao3. 1. Department of Infectious Diseases, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China. 2. Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China. 3. Medical Section, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China.
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.
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 CHBpatients 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.