Characteristics And Immune Function Of CXCR5+CD8+T Cells In Chronic HBV Infection

BackgroundChronic hepatitis B infection is an immune-mediated liver injury disease.Hosts’immune function play important roles in the control of hepatitis B virus(HBV)infection and determine the clinical outcome of the disease.CD8+T cell responses are crucial and the exhaustional function of it is an dominant cause of chronic HBV infection.However,continuous researches have shown that CD8+T cells are composed of different phenotypes and functional heterogeneous subpopulations.Different subsets of CD8+T cell may play different roles in the control of HBV infection.Therefore,futher exploration of the subpopulation of CD8+T cells may help to find new strategies for chronic hepatitis B cure.Aims:The purpose of this study was to reveal the phenotypic and functional characteristics of CXCR5+CD8+T cells in a horizontal cohort of patients with chronic HBV infection and a longitudinal cohort of chronic HBV patients during antiviral therapy and its role in disease development,and to clarify the relationship between the intrahepatic expression of CXCL13 and the chemotaxis of CXCR5+CD8+T cells and its predictive effect of antiviral therapy.Finally,the immune mechanism of CXCR5+CD8+T cells exerting intrahepatic antiviral effect was clarified by the HBV mouse model.Methods:The phenotype and functional characteristics of CXCR5+CD8+T cells were measured in healthy control(HCs)and patients with chronic HB V infection and clinical treatment cohorts by flow cytometry,ELISA,RNA sequencing and other methods.In vitro co-culture experiments were used to detect the inhibitory effect of CXCR5+CD8+T cells on HBV in HepG2.2.15 cells.Then the characteristics of intrahepatic CXCR5+CD8+T cells were analyzed by using HBV infected liver cancer specimens.In addition,we also examined the serum CXCL13 levels of patients in longitudinal cohorts of antiviral therapy and anti-viral therapy-discontinued cohorts,and their relationship with the prognosis of the disease,to clarify the molecular mechanism of CXCL13 chemotaxis of CXCR5+CD8+T cells exerting antiviral activites in the liver.Then high-pressure tail vein injection was used to apply pAAV1.2 plasmid to eatablish HBV mouse models.And we used IL-21R gene-knockout(IL-21R KO)mice and B cell deficent(μMT)mice to explore the phenotypic and functional differences of CXCR5+CD8+T cells in peripheral blood liver and spleen,and the effects of IL-21R and B cells on the function of CXCR5+CD8+T cells were also detected.Finally,the antiviral effect of CXCR5+CD8+T cells was confirmed through adoptive immunity experiments.Results:1.Characteristics of CXCR5+CD8+T cells in patients with chronic HBVinfection1.1 The frequency of CXCR5+CD8+T cells was significantly higher in HBV patients and HBsAg loss(HBsAg clearance in patients with chronic HBV infection)patients than that in HCs.And the frequency of CXCR5+ CD8+T cells in HBsAg loss was also higher than that in HBV group.1.2 Compared with CXCR5-CD8+T cells,the phenotype profiles of CXCR5+CD8+T cell showed higher PD-1,CTLA-4,TIM-3,CD38,CD69,CCR7,CD45RO,CD62L,and the ability to secrete IFN-y,IL-2,IL-4,IL-17 and IL-21 also increased,but the ability to secrete granzyme B weakened.1.3 By collecting intrahepatic lymphocytes and peripheral blood mononuclear cells(PBMC)from HBV-infected liver cancer patients,we found that compared with cells in the peripheral blood,CXCR5+CD8+T cells in the liver expressed higher PD-1,CTLA-4,TIM-3,CD38,CD69,HLA-DR and chemokine receptors,and the ability of CXCR5+CD8+T cells in the liver secreting IFN-y was stronger than CXCR5-CD8+T cells,and stronger than that of CXCR5+CD8+T cells in peripheral blood.The pentamer staining experiments proved that the proportion of HBV C18-27-specific CD8+T cells in intrahepatic CXCR5+CD8+T cells was significantly increased.2.The frequency of Peripheral blood CXCR5+CD8+T cells and the expression of CXCL13 predict the efficacy of Telbivudine(LdT)antiviral therapy.2.1 The dynamic changes of CXCR5+CD8+T cells in PBMCs of patients with Telbivudine antiviral treatment cohort were tested.As a result,we found that compared with the Treatment-incomplete-response group(NCR group),the frequency of peripheral blood CXCR5+CD8+T cells in patients with Treatment-complete-response group(CR group)increased at baseline and at week 12,and the ability of this cell subset secreting IFN-y also increased significantly at baseline and week 24.2.2 In the treatment cohort,HBV DNA levels at week 52 were positively correlated with the frequency of CXCR5+CD8+T cells in peripheral blood at week 12.2.3 Serum testing of Telbivudine antiviral therapy in the longitudinal cohort found that the serum CXCL13 levels in CR patients at baseline,week 12 and week 52 of antiviral therapy,were significantly higher than those in NCR patients.After antiviral treatment withdrawed,serum CXCL13 levels in patients who did not relapse were significantly higher than those in the relapse group.Using the liver biopsy specimens from patients with HBV infection,results shown that the expression of intrahepatic CXCL13 mRNA was positively correlated with the level of CXCL13 in serum.3.CXCR5+CD8+T cells produced IFN-y and helped the antibody production of B cells to control HBV infection.3.1 Sorting peripheral blood CXCR5+CD8+T cells and CXCR5-CD8+T cells were stimulated in vitro with anti-CD3 and anti-CD28.We found that compared with CXCR5-CD8+T cells,the ability of CXCR5+CD8+T cells to secrete IFN-γ is enhanced while the ability to secrete granzyme B is weakened.After culturing with HepG2.2.15,the expression of HBsAg and HBeAg in the CXCR5+CD8+T cells group decreased more significantly,but the killing effect on HepG2.2.15 cells was weaker.3.2 After sorting CXCR5+CD8+T cells and CXCR5-CD8+T cells to be co-cultured with B cells,results shown that the frequency of HBcAb secreting B cells increased in the co-cultured system of CXCR5+CD8+T cells.4.Characteristics and antiviral effects of CXCR5+CD8+T cells in HBV mouse models4.1 By injecting pAAV-HBV1.2 plasmid to establish HBV mouse model,we found that the frequency of CXCR5+CD8+T cells in the spleen and liver of HBV mouse model was higher than that in the peripheral blood.Compared with CXCR5-CD8+T cells,the expression of PD-1,CCR7,CX62L and ICOS can be observed higher in CXCR5+CD8+T cell subsets.4.2 Lymphocytes in liver and spleen were isolated and stimulated with anti-CD3 and anti-CD28.Results shown that the levels of IFN-y and IL-21 scecrected by spleen or liver CXCR5+CD8+T cells were significantly higher than CXCR5-CD8+T cells,and intrahepatic CXCR5+CD8+T cells were also stronger than spleen-derived CXCR5+CD8+T cells.4.3 After adoptive transfer of CXCR5+CD8+ T cells and CXCR5-CD8+T cells,intrahepatic CXCR5+CD8+T cells can be found at a higher frequency within 24 hours and 48 hours.Compared with mice which received adoptive transfer of CXCR5"CD8+T cells,serum HBsAg levels in mice that received CXCR5+CD8+T cells were significantly reduced,and relatively higher levels of HBcAb could be detected.5.Molecular mechanism that affect the antiviral effects of CXCR5+CD8+T cells5.1 The expression of IL-21 R in intrahepatic and blood CXCR5+CD8+T cells of HBV-related HCC patients was higher than that of CXCR5-CD8+T cells,and the expression of IL-21 R in intrahepatic CXCR5+CD8+T cells was also significantly higher than in peripheral blood.5.2 Increasing of IFN-γ production of CXCR5+CD8+T cells could be observed in HBV patients both after PD-1 and TIM-3 blockade.And IL-21 stimulation alone or IL-21 combined with immune checkpoint inhibitors both could enhance the ability of CXCR5+CD8+T cells to secrete IFN-γ.5.3 The frequency of CXCR5+CD8+T cells in the liver,spleen or peripheral blood of IL-21R-KO mouse model was not different from that of WT mice,but the production of IFN-γ decreased.Conclusion:The frequency and IFN-y production of CXCR5+CD8+T cells are related to the response to LdT in patients with chronic HBV infection.CXCR5+CD8+T cells exert antiviral effects through producing IFN-y and helping the antibody production of B cell.CXCL13 is highly expressed in the liver of patients with hepatitis,which help to promote the recruitment of CXCR5+CD8+T cells towards the liver and thus control HBV infection.Although CXCR5+CD8+T cells are partly exhaustion,in vitro and animal experiments have shown that IL-21 can significantly enhance its antiviral function.