Effect And Antigenic Specificity Of CD4~+Foxp3~+Tregs In Chronic HBV Infection

Hepatitis B virus (HBV) is a hepatotropic non–cytopathic DNA virus that infects about350million people worldwide, and approximately1million people die annually fromHBV-related liver disease. It has been demonstrated that multiclonal HBV-specific CD4~+andCD8~+T-cell responses are required for the control of an acute self-limiting infection. Incontrast, patients with chronic HBV infection are characterized by weaker or undetectableHBV-specific T cell responses. The precise mechanism responsible for this T-cellhyporesponsiveness is not completely understood. High levels of viral antigens, liverimmunological features, dendritic cell defects and CD4~+CD25~+regulatory T cells (Tregs)have been reported to contribute to immuno-hyporesponsiveness in chronic HBV infection.CD4~+CD25~+Tregs, which characteristically express the forkhead/winged helix familytranscription factor (Foxp3), play a critical role in inducing peripheral tolerance andpreventing autoimmunity. Many studies suggest that CD4~+CD25~+Tregs also contribute toregulating specific anti-virus immune response. CD4~+Foxp3~+Tregs have been linked tochronicity of HBV infection and were inducible from human PBMCs exposed to HBVantigen. However those findings on Tregs mainly based on CD25and/or Foxp3expressiondon’t seem to be quite reliable, as accumulating evidence indicates that CD25as well asFoxp3may not be a good marker for Tregs, which can be induced in conventional human Tcells upon mere TCR stimulation. So either CD4~+CD25~+T cell subset or CD4~+CD25~+Foxp3~+T cell population is inevitably a mixture of Tregs and recently activated effector T cells,especially in pateints with persistent viral antigen exposure.Recently, Miyara et al. showed that human CD4~+Foxp3~+T cells were composed of threephenotypically and functionally distinct subpopulations: CD45RA~+Foxp3loresting Treg cells(rTregs) and CD45RA-Foxp3hiactivated Treg cells (aTregs), and cytokine-secretingCD45RA-Foxp3lononsuppressive T cells (non-Tregs). They suggested that the dissection ofTreg cell subsets based on the combination of CD25, CD45RA and Foxp3expression ishighly informative in assessing the dynamics of Tregs differentiation under physiological and disease conditions. Thus it is necessary to reexamine the role of Tregs by detection theproportion of rTregs, aTregs and non-Tregs subpopulations in chronic HBV infection.In this study, we found that CD4~+Foxp3~+populations could be distinctly separated bythe combination of Foxp3and CD45RA staining in PBMC of patients with chronic HBVinfection; i. e., rTregs, aTregs, and non-Tregs. Among them, aTregs can be considered as keyeffector of immune suppression in chronic HBV pathogenesis. Taken together, thephenotypes and cytokine profiles of three distinct subsets of CD4~+Foxp3~+T cells in PBMC ofpatients with chronic HBV infection are in agreement with the study of Miyara et al.Compared with healthy controls, all patients showed significantly increasedCD4~+CD25~+Foxp3~+T cell frequencies. We also found circulating and intrahepatic aTregswere selectively increased in patients with chronic active hepatitis B (CAH) andacute-on-chronic liver failure (ACLF), but not in asymptomatic carriers (AsCs); the aTregsfrequency was strongly correlated with HBV DNA load, but not liver damage or TGF-β1,suggesting that robust levels of aTregs could likely inhibit the anti-HBV immune response,thus facilitating HBV replication, but could play a minor role in the protection of liver injuryin chronic HBV infection. Both in PBMCs and livers, ACLF patients showed a dramaticallyelevated frequency of IL-17A-secreting non-Tregs which were shown to be associated withsevere liver damage.Since the activation, proliferation, retention and even suppressive activity of Tregs at theflamed local site were considered as specific antigen-dependent, antigen-specificity of theseactivated Tregs was further studied in chronic HBV infection. Interestingly, we found HBVcore antigen (HBcAg)-derived peptides that stimulated Tregs varied between individuals, forexample, one peptide HBcAg81-105was able to preferentially expand the CD4~+CD25~+Foxp3~+Tcells, as well as the aTregs, in CAH patients carrying an HLA-DR9allele. HBcAg-derivedpeptide induced Tregs required the peptide-specific re-activation for suppressor function,suggesting that some of the HBcAg-derived peptide ligands may play a critical role inactivating, proliferation and retention of CD4~+Tregs in HBV-infected liver tissue and thatsuch HBV specifically activated Tregs may be much more efficient at blocking effector CD4~+or CD8~+T cells that recognize the same antigen. This study highlighted the importance ofdiscriminating different Foxp3-expressing subsets, and the findings demonstrate that thedelineation of CD4~+Foxp3~+T cells among the three activated Treg, resting Treg and non-Treg cell subsets can better distinguish different chronic HBV infection states. We also proposedthat HBcAg-derived peptide ligands may play a critical role in activating CD4~+Tregs, andsuch HBV antigen activated Tregs may be much more efficient at control anti-HBV immuneresponse, but not liver damage.