The Research Of HBV-specific T Cells In Chronic Hepatitis B Patient After Antiviral Therapy And Stopping Treatment With Relapse

Objective:To explore the levels and immune function of specific CD8+ T cells in the peripheral blood of the immune tolerance, antiviral therapy or relapse patients with chronic hepatitis B(CHB), and to explore the potential reason why the recurrent patients with drug withdrawal showed severe liver damage within a short time.Methods:1.Research objects:Collect 111 cases of chronic hepatitis B patients. According to HLA genotype by flow cytometry technology, 49 cases HLA-A2+ patients were divided into three groups: Immune tolerance group including 15 cases HLA-A2+patients, of which 11 cases male and 4 female, average 28.8±8.3 years old, all of them were under immune-tolerant phase(with normal ALT, HBe Ag positive, HBV DNA persistently high level, and there is no or only slight inflammatory in liver tissue);Antiviral therapy group including 20 cases HLA-A2+patients, of which 14 cases male and 6 female, average 41.0±14.3 years old, and HBV DNA turned negative more than 6 moths after antiviral treatment; and the remaining 14 cases HLA-A2+patients served as relapse group including 10 cases male and 4 female, average44.9±11.2 years, all of which were relapsed after stop treatment when HBV DNA turned negative at lest more than 6 moths. All these patients were inpatients and outpatients from the Infectious Diseases Department of the first affiliated hospital of Nanchang university from May.2014 to Dec.2014. All these patients according with diagnostic standard in 2010 chronic hepatitis B prevention guideline. Exclude combined with other liver damage caused by drugs, poisons, alcohol and other viruses, and genetic metabolic diseases,et al. 25 cases of HLA-A2+ healthy as negative control(5 cases males and 3 cases female, average 35.9±9.2 years old) with markers as HAV, HBV, HCV, HDV,HEV and HIV negative.2.Methods:Collect some important clinical indicators of the subjects,such as HBV-DNA, HBe Agand alanine aminotransferase(ALT). The levels of HBV-DNA were detected using real-time fluorescent quantitative PCR(FQ-PCR); Serum HBe Ag was tested by chemoluminescence of Axsym auto-immune assay system; Serum ALT was tested using Hitachi 7600 automated biochemistry analyzer. Screening for the HLA-A2 haplotype was performed by flow cytometry; The MHC-pentamers flow cytometry was used to detect the frequencies of HBc Ag18-27-specific CD8+ T cells among HLA-A2-positive patients. Separate peripheral blood mononuclear cells(PBMC), the ability of HBV-specific CTLs to secrete cytokine, such as IFN-γ and TNF-α, was detected by ELISPOT. Compare the differences of the secretion function and quantities of HBV-specific CD8+ T cells among groups, analyze the correlations among the number and secretion function of T cell、HBV-DNA and HBe Ag.Results:(1) Immune tolerance patients had less frequencies of the HBV-specific CD8+T cells and lower immune function than the antiviral therapy patients, the differences were statistically significant(P < 0.01).(2) In the immune tolerance group, significant negative correlations were found between the frequencies of the HBV-specific CD8+ T cells and the serum HBV-DNA titers and HBe Ag(r=-0.882,P<0.001;r=-0.755,P=0.001), significant negative correlations were found between the function of cytokine secretion and the serum HBV-DNA titers(IFN- γ : r=-0.837, P<0.001; TNF- α :r=-0.685, P=0.005) and HBe Ag(IFN-γ: r=-0.787, P<0.001; TNF-α: r=-0.671, P=0.006).(3)In the antiviral therapy group, with longer of HBV DNA turned negative and an decline of the level of serum HBe Ag, the frequency of HBV-specific CTL increased, peak HBV-specific CTL occurred at the first year after HBV DNA turned negative, started to decline after one year later; HBV-specific CTL declined to the lowest level after thirty-one months later and stayed at a low level.(4)The frequencies of the HBV-specific CD8+ T cells and immune function in relapse patients were significantly higher than those from antiviral therapy patients(P< 0.05) and immune tolerance patients(P < 0.01).(5)The frequency of HBV-specific CD8+ T cells and secretion function in healthy controls were significantly lower than observed in immune tolerance patients、antiviral therapy patients and relapse patients(P < 0.01).Conclusions:The HBV-specific CD8+ T-cell in chronic HBV infection patients with few quantity and impaired immune function, may associate with HBV-specific CD8+ T cells are persistently exposure to a large number of viral antigen environment lead to T cell exhaustion. Reduction of viral antigen expression by antiviral treatment can overcome CD8+ T-cell quantity and hyporesponsiveness in patients with chronic HBV infection. But blindly antiviral therapy may be detrimental to the maintenance of specific antiviral immunity. Patients appeared liver injury soon after stopping antiviral treatment, and liver damage is often heavier.One possible reason may be HBV-specific T cell functional recovery and protective memory T-cell produced a lot of effective T cells within a short time when stimulated by rebounded HBV, which lead to strong specific cellular immune response and most hepatocyte was injured.