Analysis Of The CD4～+CD25～(high) Regulatory T Lymphocytes In Chronic Persistent Hepatitis B Virus Infection

Despite the existence of effective vaccines against hepatitis B virus (HBV) for many years and massive prophylactic vaccination campaigns, HBV infection remains to be an important health problem worldwide. Approximately 370-million world population is persistently infected with HBV, carrying a greatly increased risk of developing chronic liver inflammation leads to cirrhosis and hepatocellular carcinoma. Mechanism of virus chronic infection remains discussed and can produce new clue and theory basis for clinical therapy.About the mechanism of HBV persistence, there are many hypothesizes such as anergy and exhaustion of T cells, decreased quality and function of dendrtic cells etc. Which one is more important in the immune tolerance of HBV infectionis is not very clear yet. In the past, much more attention focus on the clone clearance, immune tolerance and immune ignorance. The notion of immune regulation has changed after CD4⁺CD25^high regulatory T cells (Treg) was introduced by Sakaguchi in 1995. More study showed Treg can maintenance immune tolerance by"initiative"manner which supply clue for our study in HBV persistent infection.Recent study show Treg can have a role on T lymphocytes to implement immune regulation. Since the virus is preferentially hepatotropic and not cytopathic, HBV-specific cytotoxic T-lymphocytes (CTLs) are thought to play key roles in viral control and liver damage. It was reported that complete recovery from acute HBV infection required cellular immune responses, especially multispecific and polyclonal CTL response whereas these responses were weak or absent in chronic HBV (CHB) carriers and inadequate CTL responses might contribute to liver pathology. What is the real reason of epitope-specific CTL immune hyporesponse has been unclear yet. At the basis of these, we produce our hypothesis that whether Treg can downregulate epitope-specific CTL in CHB infection carriers.According to the hypothesis, we detected the quality and function of Treg and CD4⁺CD25^high Foxp3⁺ T cells. The results showed the frequency of Treg and CD4⁺CD25^high Foxp3⁺ T cells were higher in CHB infection carriers and expressed functional phenotypes which imply Tregs have an important role in HBV persistent infection. We found CD4⁺CD25^highFoxp3⁺ T cells and CD4⁺CD25^highCD127low T cells have a positive correlation, so CD127 can act as a new specific phenotype marker of Treg. Since CD127 expressed on the surface of lymphocytes which can be detected easily but without the damage to cells, it can be an excellent specific marker especially used in identification and sorting of Tregs.It was further to study the function status of the epitope-specific CTLs by using pentamers and intracellular cytokine staining (ICCS) both directly ex vivo and after in vitro expansion in parallel. Our result found that the frequency of CTLs can be detected but defected. Large mount of perforin or granzyme B positive cells displayed in FACScan dot plots. However, most parts of the perforin or granzyme B positive cells were existed in the non-pentamer positive cells. The epitope-specific CTLs were displayed with low levels of perforin, granzyme B and IFN-γ. These results suggest that the balance between the quantity of virus and the quality efficiency is likely to be an important issue determining viral control or persistence. It is possible that when virus-specific CTLs are faced with quantity of virus exceeding a certain threshold, a cascade of events are trigger that lead to liver damage, mainly through the action of non-antigen specific cells. Despite readily detectable virus-specific CD8⁺ T cells in most HBV chronic infected patients, immune surveillance is eventually lost, leading to persist infection. Our study demonstrated that the epitope-specific CTLs function was defected. Interestingly, in contrast to previous study, our result found that direct ICCS assys using peptide core 18-27 were able to visulize a population of IFN-γin the absence of specific pentamer binding in patients. The study imply the function of immune-surveillance in CTLs has lost which result in HBV persistent existence.An interesting phenomenon occurred during our study. There was a negative correlation between the frequency of Treg and epitope-specific CTLs which supply us a new clue. Then we study the mechanism of the Treg effect on the CTLs in vitro. The result showed the Treg can downregulate the proliferation and function of epitope-specific CTLs by the manner of cell-cell contact and cytokines secretions which may result in the persistent existence of virus.Although many scientists contributed themselves to the therapy of HBV infection during the last 30 years after HBV was firstly found, the patients suffered with HBV infection can not be cured to this day. Mimogen is a new therapeutic vaccine strategies based on epitopes whose aim is to excite active immunity. We followed and observed several patients treated with mimogen and found that there was a correlation between Treg and epitope-specific CTLs. A phenomenon was observed when the frequency of CTLs increased, the frequency of CTLs decreased which imply Treg can downregulate CTLs in vivo. In conclusion, our study supply new theory for the mechanism of HBV persistence and can produce new therapeutic strategies for CHB patients by controlling the effect of Treg.