Effect Of Immune Activated/Tolerant Environment Switching On The Function Of HBV-specific CD8+ T Cells

Objective HBV-specific CD8+ T cell responses play a key role in HBV clearance.However,the immunological characteristic of chronic HBV infection is the functional exhaustion of HBV-specific CD8+ T cells.The aims of this study are as follows: 1)to investigate the kinetics of functional change of HBV-specific CD8+ T cells derived from different stages of acute HBV infection in immune tolerant environment;2)to investigate the effect of switching immune environment on the proliferation,differentiation and antiviral function of exhausted HBV-specific CD8+ T cells.Methods 1.HI was performed to establish acute-resolving(with p SM2 plasmid)or chronic(with p AAV/ HBV1.2 plasmid)HBV replication(AR or CH)in CD45.1 and CD45.2 C57BL/6 mice.Total splenocytes,splenic CD8+ T cells were purified from HBV acute-resolving CD45.1 mice at different time points post virus exposure and were adoptively transferred to chronically HBV replicating mice.The viremia in the recipient mice was monitored.The phenotype and function of donor and recipient immune cells were analyzed by flow cytometry.The effect of breaking HBV tolerance by the immune cells from the spleen of acute-resolving HBV mice was evaluated.2.Chronic HBV replication was established in CD45.2 C57BL/6 mice by hydrodynamically injecting p AAV/HBV1.2 plasmid through the tail veil.Splenic CD8+ T cells were purified from chronically HBV-replicating CD45.2 mice at 21 days post virus exposure and were adoptively transferred to na?ve CD45.1 mice.Meanwhile,non-exhausted memory CD8+T cells were transferrd as positive control.On the first day after cell transfer,in vivo HBV restimulation was performed by injecting p SM2 plasmid to induce immune-activated environment.The viremia in the host mice was monitored.The phenotype and function of exhausted CD45.2+CD8+T cells were analyzed by flow cytometry to study the functional resconstitution of exhausted CD8+ T cells.Results 1.Adoptive transfer of total splenocytes from early(7dpi,days post injection)and late(28dpi)stages of acute-resolving HBV replication had no effect on HBV clearance.Fully viral clearance was achieved in chronically HBV replicating mice by adoptive transfer of 14 dpi total splenocytes.21 dpi total splenocytes led to a better control of viral replication in the HBV persistent mice.2.Adoptive transfer of splenic CD8+T cells from early(7dpi)and late(28dpi)stages of acute HBV replication didn’t promote HBV clearance.Adoptive transfer of 14 dpi purified splenic CD8+T cells partially contributed to HBV clearance.And completely viral clearance was achieved by adoptive transfer of 21 dpi splenic CD8+T cells,which suggested that there was a negative regulation of 21 dpi non-CD8 splenocytes on CD8+T cell response to HBV.3.Further analysis showed that compared to control group,there was an increased infiltration of CD8+T cells,B cells and dendritic cells to the liver in the mice transferred with 14 dpi total splenocytes.And the absolute number of donor-derived HBV-specific CD8+ T cells was significantly increased.CD8+T cells reponse to HBV in mice transferred with 14 dpi splenocytes was significantly higher than that in control.4.And the frequency of intrahepatic infiltrating macrophages in mice transferred with21 dpi splenic CD8+T cells was significantly higher than control.The frequency of donor-derived HBV-specific CD8+T cells and its anti-viral function were significantly higher than that of host CD8+T cells.5.Exhausted CD8+T cells from chronically HBV-replicating CD45.2 mice were adoptively transferred to na?ve CD45.1 mice,followed by in vivo HBV restimulation to induce immune-activated environment.Serum HBs Ag,HBe Ag and HBV DNA clearance were delayed significantly,compared to control.6.Further analysis showed that after transfer into na?ve mice,exhausted CD8+T cells reexpanded in response to acute HBV infection;however,their proliferation intensity was significantly lower than that of non-exhausted memory CD8+ T cells from acute-resolving HBV-replicating mice.Exhausted CD8+T cells maintained less activated phenotype and poor antiviral function,which was demonstrated by lower CD43 expression and an absence of effector cytokine production after HBV restimulation.The differentiation phenotypes of exhausted CD8+T cells driven by acute HBV replication demonstrated lower TEM in the liver and lower TCM in the spleen than that of control.Conclusion 1.The antiviral function varies in different splenocytes as well as at different time points during the course of acute-resolving HBV replication.The achievement of breaking HBV tolerance relies on the activation of both splenic CD8+ T cells and non-CD8 cells.Completely virus clearance was achieved in recipient mice by adoptive transfer total splenocytes(14dpi)and splenic CD8+T cells(21dpi)from AR mice.Adoptive transfer of total splenocytes from AR mice induces endogeneous CD8+T cell response by increased mature DCs to break HBV tolerance.Splenic CD8+T cells from AR mice maintain its anti-viral function after transfer into immune tolerance environment and break HBV tolerance directly.2.The characteristic of immune response during chronic HBV infection was the presence of exhausted CD8+T cells which undergo a stable form of dysfunctional differentiation during chronic HBV replication.Functionally exhausted HBV-specific CD8+T cells of chronic replication retained proliferative ability;it was profoundly impaired compared with that of the non-exhausted memory CD8+T cells from HBV resolvers.These cells showed less effector differentiation and maintained less activated phenotypes,an absence of effector cytokine production and poor antiviral function after exposure to acute HBV infection setting.The reconstitution of antiviral function of exhausted HBV-specific CD8+T cells may request powerful immunological means more than switching immune environment.