Expression And Clinical Significance Of Circulating CD4~+Foxp3~+T Cell Subsets In Patients With Hepatitis B

Chronic hepatitis B is a significant medical problem worldwide, it is estimated that 350 million people suffer from a chronic HBV infection in the global. As the most affected area by HBV infection, China alone has 130 million carriers of HBV and 23 million patients with chronic hepatitis B(CHB). Infection with HBV in adults usually results in a self-limiting, acute hepatitis, which confers protective immunity and causes no further disease. Ten percent of adults and 90% of children become persistent HBV carriers after the infection. Chronic HBV infection is an important risk factor for the development of liver cirrhosis and hepatocellular carcinoma. Worldwide approximately 1~2 million people die annually from HBV-related disease. Patients with a chronic HBV infection lack such a vigorous multi-specific T-cell response, but instead exhibit a weak or undetectable virus-specific T-cell response. The mechanisms responsible for the T cell tolerance in chronic HBV infection are not completely understood.Regulatory T cells (Treg) were recently identified as a specialized subset of T cells that suppress auto-reactive cells to maintain immunological tolerance and inhibit autoimmunity . Treg have also been shown to control excessive or chronic immune activation after infections with various pathogens . Growing evidence suggests that Treg may play an important role in the suppression of antiviral T-cell responses in the acute and chronic phases of infection. Indeed, the virus-specific induction of Treg may have two very different consequences: suppressing T-cell responses directed against hepatitis viruses or down-regulating the immune responses causing the liver damage. Questions also remain unresolved on which effect of the Treg are more important and how to establish a beneficial balance, mostly due to the difficulties in studying the heterogeneous Treg populations but also due to the problem accessing liver, the principal target of hepatitis viruses. In accordance with such reasons, there are even much controversy in whether circulating CD4+CD25+ Treg frequency is increased in chronic HBV patients and whether the frequency is correlated with HBV replication in several reports on Treg research in chronic HBV infection.Treg cells were generally analysed on the basis of CD4, CD25, CTLA4 and Foxp3 phenotype in recent studies. Identification of Treg cells remains problematic, because accumulating evidence suggests that all the presently-used Treg markers represent general T-cell activation markers, rather than being truly Treg-specific. FoxP3+CD4+ cells may not be a functionally homogenous population. Thus description of Treg in related HBV infection research may not be very precise. So more specific makers were very important in the study of Treg in HBV infection .Sakaguchi study group used various exquisite methods to showed that human CD4+FoxP3+ T cells comprised three distinct subpopulations with a precise phenotype and fate: CD45RA+ FOXP3lo (CD25++) resting Treg cells (rTreg cells), CD45RA-FOXP3hi (CD25+++) activated Treg cells (aTreg cells) and CD45RA- FOXP3lo (CD25++) cytokine secreting non-Treg cells. Resting Treg and activated Treg represented different stages of Treg cell differentiation and are both suppressive in vitro, and cytokine secreting non-Treg cells lacked such suppressive activity. Thus it appeared that these makers are more specific than ever before. We could expect that it would gain some more specific description of Treg status in HBV infection study.We attempted to investigate the expression three CD4+Foxp3+T cell subsets in the peripheral blood of chronic hepatitis B patients and their potential clinical significance, in order to obtain more precise knowledge of Treg in chronic HBV infection and some clues for further study .Asymptomatic HBV carriers (AsC), chronic hepatitis B (CHB) patients, and healthy controls (health) were enrolled in the study. We analyzed the frequencies of rTreg, non-Treg and aTreg of CD4+ T cells in peripheral blood mononuclear cell (PBMC) isolated from anticoagulant peripheral blood of experimental subjects using flow cytometry , and their correlation with HBV DNA load, HBeAg status and ALT level in CHB and AsC, CD4+CD25+Foxp3+Treg was analyzed simultaneously for comparison. Our results shown that the frequency of CD4+CD25+Foxp3+Treg was higher in patients with CAH and AsC than in healthy controls. The frequency of rTreg didn't show significant difference among subjects. Patients with CHB and AsC exhibited a significantly higher frequency of non-Treg compared with healthy controls. The frequency of aTreg in CHB patients was higher than in AsC patients and healthy controls. The aTreg expressed the high CTLA-4 and secret rarely low level of IL-2 and IFN-γin comparison with non-Treg. Our findings suggested that activated Tregs could be more precisely representative of the Treg than CD4+CD25+Foxp3+Treg on HBV research because of its advantage of excluding the unspecific contaminated non-Treg cells. The activated Treg might play a very important role in chronic HBV infection. As the CHB patients had higher ALT level than AsC patients , we suggested that hepatic injury may have a close relation with the increment of the frequency of aTreg, aTreg then down-regulate the immune response causing the liver damage through CTLA-4 related mechanism and other, meanwhile promote the hepatic B virus persistence through hamper T cell response against virus. We also found that the frequency of activated Treg had significant correlation with serum HBV load in patients with CHB and AsC. The possible explanation for these results was that the aTreg generation was caused by a variety of mechanisms. Besides some released self-antigens and/or certain cytokine microenvironment caused by hepatic injury could make rTreg convert to aTreg, the persistently existing HBV antigen may also induce aTreg from CD4+Foxp3- T cells. The antigenic specificity and functional characteristics of peripheral activated Treg, and the correlation between their frequencies and function with liver Treg and liver disease status need to be further investigated..