Chronic HBV Infection Up-regulated 4-1BB Ligand On B Cells To Regulate T Cell Function

Background and Aims:The mechanism of chronic hepatitis B virus(CHB)infection remains unclear.In the natural history of HBV infection,the process of generating a complex repertoire of virus-specific T and B cells is considered of paramount importance.During chronic infection with HBV,HBV-specific T cells become exhausted,and the antiviral response is weakened.As an important component of immune system,the function of B cells is still obscure.Several limited studies have characterized peripheral B cells in CHB patients,and concluded that the B cells were highly active during the natural history of chronic B infection,while decreased HBsAg-specific B-cell responses were associated with HBV persistence in CHB patients.The dysfunction of B cells may also play an important role in the mechanism of CHB infection.4-1BB(CD 137)is a costimulatory member of the TNFR family expressed on activated T cells.Its ligand,4-1BBL,also known as CD137L,TNLG5A and TNF superfamily member 9(TNFSF9),expressed by activated antigen presenting cells including B cells,dendritic cells and macrophages.The 4-1BBL+ B cells could induce activity in T cells by the 4-1BBL/4-1BB axis.Since cross-linking 4-1BBL was also shown to promote cell activation,differentiation or suppression,it is suggested that the cascade of 4-1BBL signaling is likely to be cell-specific and/or context-dependent.Thus,considering that 4-1BBL+ B cells is the activated cells,meanwhile the hyperactivate state of total B cells was found in CHB patients,it is desirable to investigate the expression of 4-1BBL on B cells in CHB patients.In this study we aimed to characterize 4-1BBL on B cells and investigate the role of 4-1BBL on B cells among chronic hepatitis B patients.Method:A total of 131 subjects,85 CHB patients and 46 healthy individuals,were included in the current study.The frequency of 4-1BBL+ B cell population and 4-1BB expression on CD3+ T cells in peripheral blood was monitored by flow cytometry.The mRNA levels of 4-1BBL and 4-1BB were also quantified by real-time PCR in collected B cells and T cells from both CHB patients and HC subjects.The serum IgG levels from 131 subjects in this study were also measured by the human IgG total ELISA kit.To investigate whether 4-1BBL+ B cells may serve as antigen presenting cells(APCs),the expression of costimulatory molecules,including CD80,CD86,MHC classes Ⅰ and Ⅱon the cell surface,were detected by flow cytometry.In order to explore the mechanism of increased 4-1BBL on B cells,B cells were stimulated by sCD40L,HBsAg or HBcAg,then the level of 4-1BBL on B cells was quantified.The 4-1BBL+ B cells and 4-1BBL-B cells were sorted by FACS and co-cultured with T cells,the supernatants of co-culture of T cells and 4-1BBL+ B cells or 4-1 BBL-B cells,respectively,were collected.The expression of CD69 and 4-1BB on T cells by flow cytometry,and the levels of T cell associated cytokines and chemokines including IFN-y,IL-10,IL-12P40,IL-12P70,IL-17A,IL-2,IL-4,IL-6,IL-8,TNF-a and granzyme B in culture supernatant were also quantitatified.Results:1.Compared with the healthy individuals,the CHB patients had similar ages,gender and came from the same area.The serum total IgG levels in CHB patients were significantly higher than that in healthy subjects(P<0.01).While there were no significant differences in the serum total IgG levels among the immune tolerant phase,immune active phase or the inactive HBV carrier state.2.The CHB patients showed increased expression of 4-1 BBL on CD 19+ B cells compared with healthy control subjects(P<0.05).However,there were no significant differences in the frequency of 4-1BBL+ B cells among immune tolerant phase,immune active phase or inactive HBV carrier state in CHB patients.Of the 4-1BB expression on CD3+ T cells,there was no difference between CHB group and the control group.Higher levels of 4-1BBL mRNA were observed in B cells from CHB patients compared to that from HC,especially in the patients in the immune active phase of chronic HBV infection.However,there was no difference in the levels of 4-1BBL mRNA in PBMCs from either CHB patients and or healthy subjects.Furthermore,there was also no difference in the levels of 4-1BB mRNA in T cells or PBMCs between CHB patients and HC subjects.3.After stimulated with sCD40L,B cells in both CHB patients and HC subjects showed upregulated 4-1 BBL expression compared with the groups stimulated with HBsAg,HBcAg and PBS.Meanwhile,after stimulated with HBcAg,B cells from CHB patients and HC subjects all showed upregulated 4-1 BBL expression compared with PBS groups.Nonetheless,there was no significant change for the expression of 4-1 BBL on B cells from CHB patients and HC subjects after stimulated with HBsAg.While the B cells from CHB patients were less sensitive to 4-1BB/4-1BBL signaling axis,compared to those in HC subjects.4-1BBL+ B cells had higher level of costimulatory molecules,including CD80,CD86,MHC classes I and II than 4-1 BBL-B cells.4.After co-cultured with 4-1BBL+ B cells,the expressions of CD69 and 4-1BB on CD4 T cells were significantly higher than that co-cultured with 4-1 BBL-B cells.Cytokines in the culture supernatant including IL-2,IL-4 and IL-6 were significantly higher in 4-1BBL+ B cells group than 4-1 BBL-B cells group and T cells alone group.However,the levels of IFN-,y and TNF-a in 4-1BBL+ B cells group were significantly lower than that in 4-1 BBL-B cells group and T cells alone group.The total IgG level was significantly higher in co-cultured supernatant of 4-1BBL+ B cells group than that in co-cultured supernatant of 4-1 BBL-B cells group.Conclusion:The 4-1 1BBL expression on B cells was significantly higher in CHB patients than that in healthy subjects,meanwhile the CHB patients had higher serological IgG levels.This provides an additional evidence to support B cells hyperactivation in CHB patients.The 4-1BBL+ B cells may function as APCs demonstrated by high levels of costimulatory molecules on the cells.Hyperactive status of B cells observed in CHB patients could be partially derived from high 4-1BBL expression on B cells triggered by HBcAg.Further,we conjectured and characterized the interaction between B cells and T cells,in which 4-1BBL+ B cells could activate CD4+ T cells and further facilitate IL-2,IL-4 and IL-6 secretion but eliminate IFN-y and TNF-α generation,while the cytokines secreted by T cells could enhance IgG secretion of B cells,which partially explain higher IgG levels observed in serum of CHB patients.Thus,during CHB infection,4-1BB/4-1BBL signaling pathway is involved in B cells activation status,and further enhance CD4+ T cell activation and facilitate IgG secretion via B cell-T cells interaction through modulating cytokine secretion from T cells,which might be critical in B cells dysfunction during CHB infection.