| Background:Hepatitis B virus(HBV)inhibits the human immune response after infection,resulting in immune tolerance and develops into chronic HBV infection.Antiviral treatment is the most effective treatment for chronic hepatitis B(CHB),and both the international guidelines and Chinese guideline recommend two broad categories of antiviral drugs,interferon(IFN)and nucleoside/nucleotide analogs.Compared with nucleoside/nucleotide analogs,interferon drugs are more effective in helping patients to obtain hepatitis B virus surface antigen(HBs Ag)seroconversion.Hepatitis B patients who can achieve HBs Ag seroconversion and long-term maintenance will be able to eliminate the virus completely and prevent it from re-invasion.However,the immune regulation mechanism of hepatitis B patients after interferon treatment is not completely clear.As the source of hepatitis b virus surface antibody(HBs Ab),B cells play a crucial role in the process of eliminating HBV.B cell activation and differentiation were regulated by T follicular helper cells(Tfh).Tfh cells are a type of CD4 and CXCR5 double-positive T-lymphocyte subgroup located in the lymphatic follicle.Its main transcription factor is Bcl-6,and characterized by highly expressions of PD-1,ICOS and CD40 L molecules.According to the secretion of IFN-?,IL-4,or IL-17,Tfh cells are divided into three subsets: Tfh1(IFN-?~+CD4~+CXCR5~+ T cells),Tfh2(IL-4~+CD4~+CXCR5~+ T cells),and Tfh17(IL-17A~+CD4~+CXCR5~+ T cells).It is reported that these cell subgroups play different roles in the differentiation,maturation and immunoglobulin class switching of B cells.Studies have shown that Tfh cells are involved in the humoral immune process after the HBV invasion.To date,no studies have characterized the status,biological significance,or regulatory mechanisms of Tfh cells on B-cell responses during the treatment of CHB patients with PEG-IFN-?.Objective:Research the functional changes of Tfh cells and B cells in the antiviral immune response of patients with CHB to pegylated interferon alpha(PEG-IFN-?).To explore the pathway of Tfh cells regulating B cells to produce protective neutralizing antibodies,so as to improve HBs Ag seroconversion and provide theoretical basis and guidance for the clinical treatment of hepatitis B.Methods:A total of 59 patients with CHB and 15 age-and gender-matched healthy controls(HC)were enrolled into this study.CHB patients were treated with PEG-IFN-? for 48 weeks and followed up to 96 weeks.According to the treatment response,CHB patients were classified into the following three groups: Complete Response Group(CRG),Incomplete Response Group(ICRG)and Non-Response Group(NRG).Flow cytometry were used to explain the surface markers expression of circulating Tfh cells(CD3/CD4/CXCR5/CD40L/ICOS/PD-1)and B cells(CD19/CD20/CD21/CD38/CD138)from CHB with interferon therapy for 0,12,48,and 96 weeks and control group.To validate the function of Tfh cells on the differentiation of B cells and the autoantibodies production,and the effects of IFN-? or HBV on Tfh cells function,the Tfh cells and B cells in the peripheral blood or tonsil of the patients or healthy person were obtained by flow cytometry sorting technology,and to build a Tfh-B co-culture system in vitro.Microarray technology was used to detect the gene expression of Tfh cells in peripheral blood of CHB patients,and immunofluorescence technology was used to detect the expression level of Tfh cell surface marker CD40L/ICOS in liver tissues.Cytometric Bead Array(CBA)was used to detect the expression levels of cytokines(IFN-?,TNF-?,IL-2,IL-4,IL-6,IL-10,IL-17A),and immunoglobulin(Ig G,Ig A,Ig M).Results:1.Of the 59 patients with CHB enrolled into this study,only 42 finished the 48-week PEG-IFN-? treatment and followed up to 96 weeks.According to the treatment response,14 patients were classified into CRG,8 into ICRG,and 20 into NRG.2.Using flow cytometry,it was found that in CHB patients before treatment with PEG-IFN-?,the frequencies of CD38hiCD138~+ plasma cells,Ig G~+CD21~+ B cells,CD4~+CXCR5~+ Tfh cells,ICOS~+CD4~+CXCR5~+ Tfh cells,CD40L~+CD4~+CXCR5~+ Tfh cells,PD-1~+CD4~+CXCR5~+ Tfh cells,as well as serum Ig G and Ig M levels were all higher than in HC group,and the frequency of Tfh1 cells was lower than in HC group,while the frequencies of Tfh2 and Tfh17 cells have no significant difference.Dynamic analysis of the frequencies of circulating Tfh cells and B cells subsets,as well as the changes of serum immunoglobulin in patients before and after treatment with PEG-IFN-? at 12,48 and 96 weeks,found that the frequencies of ICOS~+CD4~+CXCR5~+ Tfh cells,CD40L~+CD4~+CXCR5~+ Tfh cells,Tfh1 cells,CD38hiCD138~+ plasma cells,Ig G~+CD21~+ B cells,and serum Ig G,Ig M levels in CRG showed a time-dependent increase during the treatment and peaked at 48 weeks.Moreover,after 48 weeks of PEG-IFN-? treatment,the frequencies of ICOS~+CD4~+CXCR5~+ Tfh cells,CD40L~+CD4~+CXCR5~+ Tfh cells,Tfh1 cells,CD38hiCD138~+ plasma cells,Ig G~+CD21~+ B cells,and serum Ig G and Ig M levels were all significantly higher in CRG than those in NRG and ICRG.3.Correlation analysis found that the frequency of CD40L~+CD4~+CXCR5~+ Tfh cells was significantly and positively correlated with serum HBs Ab concentration,CD38hiCD138~+ plasma cells,Ig G~+CD21~+ B cells,and serum Ig G level,while ICOS~+CD4~+CXCR5~+ Tfh cells were not correlated with any of these parameters.4.Using immunofluorescence confocal technique,we found that the frequencies of CD40L~+CD4~+CXCR5~+ and ICOS~+CD4~+CXCR5~+ Tfh cells increased in liver tissue of CHB patients,and CXCR5~+CD40L~+ and CXCR5~+ICOS~+ Tfh-like cells were adjacent to B cells in position.5.In the in vitro experiment,Tfh cells from different response groups and healthy controls were co-cultured with B cells from the same healthy donor.Compared with NRG and HC group,Tfh cells from CRG group had stronger ability to promote B cell differentiation to memory B cells and to produce antibodies.The co-culture of Hep G2.215 cells and peripheral blood mononuclear cells using the Transwell system showed that HBV inhibited the expression of CD40 L on Tfh cells;the high expression of CD40 L and ICOS on Tfh cells was stimulated by the addition of recombinant human protein IFN-? in co-culture system;Blocking the CD40L-CD40 pathway with anti-CD40 antibody is more effective in reducing the production of Ig G~+ B cells than blocking the ICOS-ICOSL pathway with anti-ICOS antibody.6.Gene chip technology was used to analyze the gene expression profile of peripheral blood Tfh cells in different response groups.We found that the expression of CD40 L and interferon stimulating factor gene in CRG were all higher than that in NRG and ICRG,while there was no significant change in ICOS.Conclusion:1.The frequencies of CD40L~+CD4~+CXCR5~+ Tfh and ICOS~+CD4~+CXCR5~+ Tfh cells increased in peripheral blood and liver of CHB patients,and the location of Tfh-like cells in liver tissues was close to that of B cells,suggesting that Tfh cells may directly regulate the activity of B cells,which may be closely related to liver damage,verifying that Tfh cells directly participate in the pathogenesis of CHB.2.The frequency of circulating Tfh1 cell significantly decreased in patients with CHB,and significantly increased in CRG after PEG-IFN-? treatment,suggesting that Tfh1 cells may be involved in the pathogenesis of CHB and play an important role in the process of HBV clearance.3.After PEG-IFN-? treatment,the frequencies of CD40L~+CD4~+CXCR5~+ Tfh and ICOS~+CD4~+CXCR5~+ Tfh cells increased in CRG patients,and Tfh cells had a stronger ability to promote the differentiation of B cells and the production of antibodies,indicating that PEG-IFN-? treatment enhanced the function of Tfh cells assisting B cells to produce antibodies in CRG patients,thereby promoting HBs Ag seroconversion.4.In vitro experiment found that the presence of HBV inhibited the expression of CD40 L on Tfh cells,which may be one of the reasons for the formation of immune tolerance state,while IFN-? can indirectly induce the expression of CD40 L and ICOS on Tfh cells.5.It was found and proved that CD40L~+CD4~+CXCR5~+ Tfh cells were more effective in activating B cells than ICOS~+CD4~+CXCR5~+ Tfh cells,promoting the production of HBs Ab,and achieving HBs Ag seroconversion.Looking for effective ways to stimulate the expression of CD40 L,and then to effectively control the activation of Tfh-B cells axis in immune tolerance state,may be as new therapeutic means or an adjunctive therapy for the treatment of CHB patients. |
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