Research On The Function Of Different PD-1 And CXCR5 Expressing T Lymphocytes In Hepatitis B Virus Infection

Background:Virus clearance after hepatitis B virus?HBV?infection requires the participation of a complex immune network.The interaction between the host and the virus mediated by the immune response affects the complexity and outcome of liver disease after HBV infection.The humoral immune response plays a vital role in suppressing and eliminating HBV.B cell activation status plays an important role in controlling the replication of HBV.Hepatitis B e antigen?HBeAg?/hepatitis B surface antigen?HBsAg?seroconverted spontaneously or through treatment,can permanently control virus replication or completely eliminate the virus,and prevent or delay the occurrence of cirrhosis and hepatocellular carcinoma,which means effectively control the progression of the disease.However,the number of peripheral B cells and the ability to secrete specific antibodies in patients with chronic hepatitis B?CHB?have decreased to a certain extent,and the rate of HBeAg/HBsAg seroconversion obtained by spontaneous or treatment is still low.Therefore,it is important to explore the humoral immune mechanism of HBeAg/HBsAg clearance in CHB patients.Follicular helper T cells?Tfh cells?,as a subset of CD4⁺helper T cells,play an important role in assisting humoral immune responses.Studies have confirmed that circulating PD-1^hiCXCR5⁺CD4⁺Tfh cells can mediate the humoral immune response in patients with chronic schistosomiasis and autoimmune thyroid disease,but the role of PD-1^hiCXCR5⁺CD4⁺Tfh cells in patients with chronic HBV infection remains unknown.Interestingly,recent studies have found a new subpopulation of CD4⁺T cells,which is called peripheral helper T cells?Tph cells?,lacks the expression of CXCR5 but highly expresses PD-1.Studies have shown that PD-1^hiCXCR5^-CD4⁺Tph cells display a similar humoral immune response function as Tfh cells.However,there is also a lack of research on PD-1^hiCXCR5^-CD4⁺Tph cells in CHB patients.Therefore,can PD-1^hiCXCR5⁺CD4⁺Tfh cells and PD-1^hiCXCR5^-CD4⁺Tph cells reduce HBsAg and clean HBV DNA in patients with CHB by regulating humoral immune response?The cytotoxic T lymphocyte?CTL?-mediated immune response also plays an important role in the immune response of CHB.Circulating HBV-specific CD8⁺T cells can directly identify viral antigens and kill HBV infected hepatocytes.At the same time,HBV-specific CD8⁺T cells can also secrete IFN-?and TNF-?,and inhibit the expression and replication of HBV genes in other hepatocytes by a non-cytolytic mechanism.However,in CHB patients,HBV cannot be completely and effectively eliminated.The main cause of persistent HBV infection is the deficiency in the number and function of HBV-specific CD8⁺T cells.Exhausted HBV-specific CD8⁺T cells overexpress negative inhibitory molecules,causing CD8⁺T cells to exhibit lower effector functions and loss of memory.Recent studies have found that there is a unique group of CD8⁺T cells expressing CXCR5,namely follicular cytotoxic T cells?Tfc cells?,whose degree of functional exhaustion is significantly lower than that of CXCR5^-CD8⁺T cells and showed a higher virus suppression function in chronic viral infectious diseases and inflammatory diseases.And at the same time,some of these cells had the same characteristics as Tfh cells and could promote B cells to secrete IgG.HBV-specific CXCR5⁺CD8⁺Tfc cells have not yet been studied in HBV infection.Can HBV-specific CXCR5⁺CD8⁺Tfc cells also play a role in inhibiting virus replication and promoting humoral immune response in HBV infection?Part ?.Role of PD-1^hiCXCR5⁺CD4⁺Tfh cells and PD-1^hiCXCR5^-CD4⁺Tph cells in the process of HBsAg decrease and HBV DNA clearance in CHB patients receiving PEG-IFN-?therapyObjective:Given the B helper function of Tfh cells and Tph cells,we researched the roles of circulating PD-1^hiCXCR5⁺CD4⁺Tfh cells and PD-1^hiCXCR5^-CD4⁺Tph cells in the decrease of HBsAg levels and HBV DNA clearance in CHB patients undergoing pegylated interferon-alpha?PEG-IFN-??therapy.Methods:The frequencies of circulating PD-1^hiCXCR5⁺CD4⁺Tfh cells and PD-1^hiCXCR5^-CD4⁺Tph cells were measured by flow cytometry in 20 healthy controls?HCs?and 18 PEG-IFN-?treated CHB patients.The absolute numbers of both cell types in per milliliter?mL?of peripheral blood were calculated by the combination of flow cytometry and hematology analysis results.ANOVA of repeated measurements were performed to evaluate the effects of changes of frequencies and absolute numbers of circulating PD-1^hiCXCR5⁺CD4⁺Tfh cells and PD-1^hiCXCR5^-CD4⁺Tph cells on HBsAg decline and HBV DNA clearance after 48 weeks of PEG-IFN-?treatment.Results:1)Higher frequencies of circulating PD-1^hiCXCR5⁺CD4⁺Tfh cells and PD-1^hiCXCR5^-CD4⁺Tph cells were found in patients with CHB compared to HCs.2)In the longitudinal study,there was no significant changes overall in the frequencies and absolute numbers of circulating PD-1^hiCXCR5⁺CD4⁺Tfh cells and PD-1^hiCXCR5^-CD4⁺Tph cells before and after 48 weeks of PEG-IFN-?treatment.3)Serum HBsAg levels decreased rapidly in patients with an increased frequency of circulating PD-1^hiCXCR5⁺CD4⁺Tfh cells after treatment.4)Patients who displayed an increase in the frequency of PD-1^hiCXCR5⁺CD4⁺Tfh cells tended to present a lower level of HBV DNA at the end of treatment?EOT?;however,the difference was not statistically significant in the ANOVA of repeated measurements analysis.5)An increased frequency of PD-1^hiCXCR5⁺CD4⁺Tfh cells exerted a significant positive effect on the HBsAg level decreasing to?2 Log₁₀ IU/mL and?3 Log₁₀ IU/mL at the EOT.6)No significant differences in HBsAg and HBV DNA levels were observed over time,regardless of whether the frequency of PD-1^hiCXCR5^-CD4⁺Tph cells and absolute numbers of both cells were increased or not after treatment.Conclusion:Circulating PD-1^hiCXCR5⁺CD4⁺Tfh cells and PD-1^hiCXCR5^-CD4⁺Tph cells may participated in the immune landscape of chronic HBV infection.Moreover,PD-1^hiCXCR5⁺CD4⁺Tfh cells are associated with decreased HBsAg levels in PEG-IFN-?treated CHB patients.Part ?.The role of HBV-specific CXCR5⁺CD8⁺Tfc cells in HBV infectionObjective:It has been reported that CXCR5⁺CD8⁺Tfc cells have strong cytotoxic function and can assist humoral immune response in other infectious or inflammatory diseases,we want to discuss the immune response mediated by HBV-specific CXCR5⁺CD8⁺Tfc cells after HBV infection in this study.Methods:Either pCDNA3.1-HBV1.3 or pAAV-HBV1.2 plasmid were hydrodynamically transfected into C57BL/6J?B6?mice to establish acute or chronic HBV transfection mice models.Meanwhile,38 CHB patients with different HBeAg states and different alanine aminotransferase?ALT?levels were enrolled.The frequency and phenotypic of HBV-specific CXCR5⁺CD8⁺Tfc cells were detected using flow cytometry in the spleen of mice and peripheral blood of CHB patients.Flow cytometry was also used to detect the secretion levels of IFN-?,TNF-?and IL-21 in the splenic CXCR5⁺CD8⁺Tfc cells of mice after HBc93-100 stimulation.Results:1)HBV-specific CXCR5⁺CD8⁺Tfc cells were detected in the spleen of acute and chronic HBV transfected mice and peripheral blood of CHB patients.2)Compared with the CXCR5^-subset,splenic HBV-specific CXCR5⁺CD8⁺Tfc cells in the acute and chronic HBV transfected mice showed higher PD-1 and Tim-3 expression,but more capable of secreting IFN-?and TNF-?after HBc 93-100 stimulation.3)ICOS and CD40L were expressed on the surface of splenic HBV-specific CXCR5⁺CD8⁺Tfc cells in the acute and chronic HBV transfected mice,and IL-21 is secreted after stimulation by HBc 93-100 antigen peptide.4)Compared with CXCR5^-CD8⁺T cells,CXCR5⁺CD8⁺Tfc cells expressed higher PD-1 and Tim-3 in peripheral blood of CHB patients.And circulating CXCR5⁺CD8⁺Tfc cells expressed ICOS and CD40L in CHB patients.This feature was more pronounced in CHB patients with HBeAg positive and higher ALT levels.Conclusion:HBV-specific CXCR5⁺CD8⁺Tfc cells were partially exhausted but possessed a stronger cytotoxic function than the CXCR5^-subset in HBV infection.In addition,HBV-specific CXCR5⁺CD8⁺Tfc cells may be involved in the humoral immune response against HBV.