In Vitro Immune Effects Of Human Peripheral Blood Mononuclear Cells In HBV Infection

Objective:To observe the changes of immune effects after stimulating normal human peripheral blood mononuclear cells(PBMCs)with serum from patients who were immunotolerant hepatitis B virus(HBV)infected persons.Methods:PBMCs which came from healthy individuals were infected with serum from patients who were in the immune tolerance period of chronic HBV infection and cotaining with HBV DNA of 2×10 ~7 IU/mL.The stimulated PBMCs were used as experimental group.The total time of stimulation is 24 days,and PBMCs alone were used as control.The CCK8 assay was performed to measure the number of PBMCs in the experimental group and control group.A sandwich ELISA was used to detect concentrations of interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)in the supernatant of both groups.The cell surface expression of programmed death receptor-1(PD-1)on CD8~+T cells was investigated by flow cytometric analysis.Results:When the infection time was 8 days,the number of PBMC in the experimental group was higher than that in the control group,on the other hand,the experimental group[(1646.89±149.92)pg/ml]showed significant increase in TNF-αlevels compared to the control group[(1061.20±151.29)pg/ml,t=4.76,P=0.009],and the concentration of IFN-γin the experimental group[(134.00±15.10)pg/ml]was higher than that of the control group[(24.26±8.22)pg/ml,t=11.06,P=0.000].On the 24th day of stimulation,the experimental group[(395.67±25.17)pg/ml]showed decrease in TNF-αlevels compared to the control group[(576.33±15.18)pg/ml,t=-10.65,P=0.000],The concentration of IFN-γin the experimental group[(14.05±1.65)pg/ml]was lower than that of the control group[(20.75±2.47)pg/ml,t=-3.91,P=0.017].When the stimulation is not started,the positive rates of PD-1 on the CD8~+T cells were both(12.23±1.72)%in the experimental group and the control group.After 24days of continuous stimulation,the expression levels of PD-1 in the experimental group and the control group were(16.70±1.65)%and(13.17±2.31)%.The expression of PD-1 in the experimental group was higher than that in the unstimulated group(P=0.032)after 24 days of stimulation.There was no significant difference in the expression level of PD-1 in the control group compared with the day when stimulation was not initiated(P=0.605).Conclusion:1.In the early stage of HBV stimulation,the immune cells could proliferate.2.The concentrations of TNF-αand IFN-γwere correlated with the time of HBV infection.3.During the process of HBV stimulation of PBMC,the concentrations of TNF-αand IFN-γhad the basically consistent trend,and both of them increased first and then decreased.4.When HBV is continuously infected,the expression of PD-1 on the surface of CD8~+T cells was upregulated.