Roles Of CD4~+ CD25~+ Regulatory T Cells In The Host Immune Tolerance To Hbv And Its Mechanisms

Objective: To study the role of CD4~+CD25~+ regulatory T cells in the host immune tolerance to HBV and its mechanisms; To search the approach of breaking the host immune tolerance to HBV and preventing chronic hepatitis B infection by studying the frequency and function of CD4~+CD25~+ regulatory T cells in chronic hepatitis B patients, asymptomatic HBV carries and HBV transgenic mice.Method:1. To detect the frequencies of peripheral CD3~+CD4~+T cells, CD3~+CD8~+T cells and CD4~+CD25~+ regulatory T cells from chronic hepatitis B patients, asymptomatic HBV carries and HBV transgenic mice by Flow cytometry.2. The expression of hepar Forkhead/winged helix sranscription factor(Foxp3)protein was detected by immunohistochemistry.3. To analyze the percentages of peripheral IFN-γ-producing CD3~+CD4~+T cells and CD3~+CD8~+T cells in chronic hepatitis B patients, asymptomatic HBV carries and HBV transgenic mice after in vitro activation with PMA , ionomycin or IL-2 by flow cytometry. 4. CD4~+CD25~+ regulatory T cells and CD4~+CD25- T cells were isolated from HBV transgenic mice splenocytes by negative and positive selection using a CD4~+CD25~+ regulatory T cells isolation kit.5. To analyze the percentage of TGF-β-producing CD4~+CD25~+T cells in spleen tissue from HBV transgenic mice after in vitro activation with PMA and ionomycin by flow cytometry .6. Purified CD4~+CD25-T cells were cultured alone or co-cultured with CD4~+CD25~+T self-cells in the vitro activation with HBsAg or ConA for 72 hour. The proliferation capability of purified CD4~+CD25-T cells was determined by [3H]-thymidine incorporation (cpm); The Concentrations of IFN-γand IL-4 produced by purified CD4~+CD25-T cells were measured by ELISA method.7. The correlation of CD4~+CD25~+T cells with ALT or HBV-DNA in chronic hepatitis B patients was analyzed by correlation analysis method.Result:1. The frequency of CD3~+CD4~+T cells or CD3~+CD4~+CD25~+ regulatory T cells in chronic hepatitis B patients was significantly higher than that in asymptomatic HBV carries and healthy controls(P<0.01) . However , The frequency of CD3~+CD8~+T cells in chronic hepatitis B patients was significantly lower than that in asymptomatic HBV carries and healthy controls(P<0.01) . There was no significant difference in the frequencies of CD3~+CD4~+T cells, CD3~+CD8~+T cells or CD3~+CD4~+CD25~+ regulatory T cells between asymptomatic HBV carries and healthy controls(P>0.05).2. There was no significant difference in the frequencies of CD3~+CD4~+T cells, CD3~+CD8~+T cells or CD3~+CD4~+CD25~+ regulatory T cells between HBV transgenic mice and healthy mice(P>0.05).3. There was no significant difference in the expression of Foxp3 protein between HBV transgenic mice and healthy mice(P>0.05).4. The percentage of peripheral IFN-γ-producing CD3~+CD4~+T cells or CD3~+CD8~+T cells in chronic hepatitis B patients was significantly lower than that in asymptomatic HBV carries and healthy controls(P<0.01). There was no significant difference in the percentage of peripheral IFN-γ-producing CD3~+CD4~+T cells or CD3~+CD8~+T cells between asymptomatic HBV carries and healthy controls(P>0.05).5. There was no significant difference in the percentage of peripheral IFN-γ-producing CD3~+CD8~+T cells between HBV transgenic mice and healthy mice(P>0.05).6. There was no significant difference in the percentage of TGF-β-producing CD4~+CD25~+T cells in spleen tissue between HBV transgenic mice and healthy mice(P>0.05).7. Purified CD4~+CD25-T cells were co-cultured with CD4~+CD25~+T self-cells in the presence of HBsAg or ConA. In the non-HBsAg stimulation group, there was no significant difference in the proliferation ability of CD4~+CD25-T cells and the Concentrations of IFN-γand IL-4 produced by purified CD4~+CD25-T cells between HBV transgenic mice and healty mice(P>0.05); However, In the HBsAg stimulation group, the proliferation ability of CD4~+CD25-T cells and the Concentrations of IFN-γand IL-4 produced by purified CD4~+CD25-T cells in HBV transgenic mice were significantly lower than that in healty mice(P<0.05, P<0.01).8. The proliferation ability of CD4~+CD25-T cells and the Concentrations of IFN-γand IL-4 produced by purified CD4~+CD25-T cells in the vitro activation with HBsAg or ConA for 72 hour from HBV transgenic mice and healty mice significantly decreased when CD4~+CD25-T cells were co-cultured with CD4~+CD25~+T self-cells(P<0.01).9. we observed a significant positive correlation between the frequency of CD4~+CD25~+ regulatory T cells and serum ALT level in chronic hepatitis B patients(P<0.05),and no significant correlation between the frequency of CD4~+CD25~+ regulatory T cells and HBV loads in chronic hepatitis B patients was found (P>0.05).Conclusion:1. In chronic hepatitis B patients, the frequency of CD4~+CD25~+T regulatory T cells increases, while the frequencies of CD4~+T and CD8~+T cells decrease with impaired functions. It is probably the increased frequency of CD4~+CD25~+ regulatory T cells that leads to the immune tolerance in chronic hepatitis B patients.2. The frequency and function of CD4+CD25+ regulatory T cells in HBV transgenic mice were comparable to that of healty mice. Specific immune tolerance to HBV in HBV transgenic mice was at the level of T cells.3. CD4+CD25+ regulatory T cells can non-specifically inhibit the function of auto-activated CD4+ and CD8+ T cells.In this research, we studied, for the first time, the frequency and function of CD4+CD25+ regulatory T cells in HBV transgenic mice and, further, in chronic hepatitis B patients, so as to study the role of CD4+CD25+ regulatory cells in the host immune tolerance to HBV and probe into approache of breaking immune tolerance induced by chronic hepatitis B infection. Our study provided experimental basis for these aspects.