| Background:Immune tolerance is an important reason for the persistence of hepatitis B virus(hepatitis B virus,HBV)infection,it's also an obstacle to the ideal curative effect of chronic hepatitis B(chronic hepatitis B,CHB)patients.In the current clinical practice,most CHB patients have achieved continuous virological suppression underwent long-term nucleos(t)ide analogues(NAs)treatment,but less than 3%of them exhibit loss of hepatitis B surface antigen(HBsAg),which has been considered a“clinical cure”for HBV infection.Long-term administration of NAs has raised several problems that can not be ignored,such as psychological pressure,economic and social burden.In the previous study,we found that NAs lead-in followed by pegylated interferon alpha(Peg IFN-?)in virally suppressed patients can peomote the early repair of immune function by targeting natural kiiler cells(NK)and regulatory T cells(Treg),and significantly improve the clinical efficacy.NK cells are one of the key players of innate immune system and bridge the innate immunity and adaptive immunity.In addition to antiviral effects,more and more attention has been paid to the immunomodulatory function of NK cells.Treg is an important cell subset for the maintenance of peripheral immune balance.Its negative regulation on HBV-specific immune response is one of the important mechanisms of immune tolerance in CHB.Although the regulatory effect of NK cells on Treg cells(hereinafter referred to as"NK-Treg regulatory pathway")has been reported in the infection models for Mycobacterium tuberculosis and Chlamydia pneumoniae,its role in the progression and treatment of HBV infection,as well as the related mechanism were still unclear.Methods:Based on GEO database,the expression profile of immune-related genes in peripheral blood mononuclear cells(PBMC)of NAs-treated CHB patients prior to and at 6 hours after the first Peg IFN?injection was collected and analyzed with bioinformatics tools such as WGCNA,GO,KEGG,GSEA and ss GSEA.Further,a total of 116 patients with CHB were randomLy assigned to either continue with entecavir therapy(NA group)or switch to Peg IFN-?-based treatment(IFN group).The number,phenotype,and functions of NK cells,Treg cells,and HBV-specific CD8~+T cells were evaluated both in CHB patients and in vitro culture systems.NK cell depletion and antibody-mediated blockade of IFN-?were conducted both in vitro and in an HBV-carrier mouse model to investigate the regulatory effects of NK cells on Treg cells and the underlying molecular mechanisms.For Treg differentiation,human naive CD4~+T cells isolated from PBMC were cultured for up to 3 days alone,or with autologous NK(at 1:2 ratio)or/and anti-IFN-?(10?g/mL)in vitro.Results:By combining a series of bioinformatics tools,the results suggest that Peg IFN-?theatment activates the immune response of NAs-treated CHB patients,which is mainly related to CD~+8 T cell,NK cell,Th2 cell,IFN-?,JAK-STAT pathway,adaptive immune response,leukocyte differentiation,innate immune regulation,binding of cytokines and receptors,etc.As for the clinical cohort,an inverse correlation between the peripheral frequencies of NK cells and Treg cells was found in patients receiving Peg IFN-?-based treatment.In a transwell culture system wherein NK cells were separated from PBMC using a membrane,we found a significant increase in the proportion,number,proliferation of Treg cells,as well as the transcription level of Foxp3 in PBMC.Further,the differentiation of Naive T cells into Tregs was suppressed when they were co-cultured with NK cells.Neutralization of IFN-?partially restored the proliferative capacity and differentiation potential of Tregs,resulting in a significant increase in the relative and absolute number of Tregs.A more powerful regulatory effect of NK cells on Tregs was observed in patients of the IFN group,especially in responders(patients acheive HBe Ag seroconversion and/or HBsAg loss in 48 weeks)or those with a marked reduction of HBsAg(>0.5 lg IU/mL).Moreover,patients with a high inhibitory ratio of Tregs exhibited a significantly higher frequency of HBV-specific IFN-?-secreting CD8~+T cells.NK cell depletion or IFN-?blockade resulted in a notable increase in frequencies of hepatic Tregs and elevated serum HBsAg levels in the HBV-carrier mouse model.Conclusion:This study focused on the immunological characteristics and clinical dilemmas of NAs-treated CHB patients.By combining bioinformatics tools,clinical cohorts and animal models,the immunosuppressive effect of NK cells on Treg cells in the progression and treatment of HBV infection has been elucidated.In the peripheral circulation of NAs-treated CHB patients and the liver of HBV-carrier mouse model,we identified a novel mechanism by which NK cells suppressed the proliferation and differentiation of Tregs through the secretion of IFN-?in a cell contact-independent manner.Additionally,the inhibition could be enhanced by Peg IFN-?treatment,which was correlated to more vigorous HBV-specific T cell responses and marked reduction in HBsAg.Our study reveals a novel immunomodulatory mechanism of NK cells and provides a theoretical basis for Peg IFN-?as an immunotherapy agent in treating patients with CHB. |
PDF Full Text Request