Experimental Studies On CD4+CD25+ Regulatory T Cells And Chronic Hepatitis B

Hepatitis B virus (HBV) infection is a global public health problem. Of the approximately 2 billion people who have been infected worldwide. 350 million people suffer from chronic HBV infection, and approximately 1 million people die annually from HBV-related liver disease. Considerable numbers of chronic HBV carriers suffer from progressive liver diseases.Chronic hepatitis B virus (HBV) infection is characterized by a weak immune response to HBV. Over the past decade,there have been tremendous advances in our understanding of the basic processes that control immune tolerance. CD4+CD25+regulatory T cells (Treg) have been shown to maintain peripheral tolerance against self and foreign antigens. It can suppress the function of effector T cells and may be play a key role in this impaired immune response.In most vitro models,the suppression function of Treg is caused by a cell-cell contact dependent mechanism and is a dose-dependent manner. So the number of Treg maye explain the decreased antiviral response in chronic HBV patients,and might have implications for offer immunotherapeutic approaches to HBV treatment in future. To determine whether CD4+CD25+Treg play a key role in the persistence of a chronic HBV infection, the personnel first compared the percentage of Treg present in the peripheral blood of chronic HBV patients with healthy controls ,but,the result come from different study groups is not same. For instance, Stoop studies suggest that patients with chronic HBV infection contained a higher percentage of Treg in their peripheral blood compared the health control,whereas others suggest that the number of these cells is normal in CHB patients.A major reason for this problem is that the study and application of human setting Treg has been lack of specific cell surface biomarkers to define and separate Treg cells from other regulatory or effector T cell subsets.Although many studies indicate that CD25 is a crucial cell surface marker for the regulatory subset, unlike the mouse, several studies have suggested that only the CD4+T cell subset expressing the highest levels of CD25 have in vitro suppressive activity. In addition, other markers such as CTLA-4 and GITR, which have been reported to be expressed on CD4~+CD25~+Treg cells, are also expressed on effector T cells, and These markers have proven problematic for uniquely. That is the reason why different study groups has several disparate reports of CD4~+CD25~+Treg cell quantification in CHB patients.The transcription factor, Foxp3 is the most sensitive marker for defining this specialized T cell subset in humans,but because it is a kind of protein in internal of cell, so we need to perforate on cell surface ,this make the results not so stableTo solve this problem, Weihong,L.and his partner found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4~+CD25~+T cells in peripheral blood. They demonstrate that the majority of these cells are FoxP3+,including those that express low levels or no CD25. A combination of CD4, CD25, and CD127 resulted in a highly purified population of Treg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact, CD127 can be used to identify CD4~+CD25~+Treg as a biomarker.In the fist part of our study, to investigate the proportion of CD4~+CD25~+Treg in CHB patients and determine whether the proportion of CD4~+CD25~+Treg was related to the clinical parameter of these patients. Fresh isolated peripheral blood mononuclear cells (PBMCs) of 48 patients with CHB, 29 cases of HBV carriers and 23 healthy donors were analyzed for the proportion of CD4~+CD25~+Treg using flow cytometry by surface staining for CD4-PC5, CD25-FITC, CD127-PE. Serum HBV markers were evaluated each subject.HBV DNA levels were measured using real-time RT-PCR. In this part we found that the proportion of CD4~+CD25~+CD127low/-Treg (9.39±2.56%) and CD4+CD25high Treg (8.00±4.75%) in CHB patients were showed a statistically significant increase compared to health controls (4.29±1.77%, P<0.01), (1.20±0.72%, P<0.01), within their population of CD4+T cells in peripheral blood. There was no significantly difference between the HBV carriers and that of health controls; and there was no significantly difference between the proportion of CD4~+CD25~+CD127low/-Treg and CD4+CD25highTreg of HBeAg negative CHB and HBeAg positive CHB. As the same result between HBeAg negative HBV carrier and HBeAg positive carrier.We get the conclusion that the level of CD4~+CD25~+Treg in CHB patients is higher than health control and HBV carriers. The weak immune condition in CHB patiens was related to the raising level of CD4~+CD25~+Treg.So far, Foxp3 is still the most sensitive marker, for this reason, We investigated the level of Foxp3 mRNA in the PBMC of patients with CHB. Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine Foxp3 mRNA expression in the PBMC from 15 cases with CHB patients, 16 cases of HBV carrier, and 16 normal controls. The result of our study show that Foxp3 mRNA expression was higher in patients (0.42±0.08) with CHB than that in the controls (0.32±0.07, P<0.01), while Foxp3 mRNA expression in HBV carrier stage (0.30±0.08) was not significantly different from that in the controls (0.32±0.07, P>0.05). So we can get a conclusion that Foxp3 mRNA expression in the PBMC may be involved in the pathogenesis and activity of CHB.In the last part of our study, we want to identify impacts of nucleos (t) ide analogue therapy on the proportion of circulating CD4~+CD25~+ regulatory T cells as well as lymphocyte subsets in patients with chronic hepatitis B virus (HBV) infection. For the base line of this group, we found, the 28 cases of CHB patients with high level of alanine amino transferase [ALT] (ALT>100 U/L) had a higher fraction (4.26%±3.10%) of CD4+CD25high Treg in peripheral blood than those patients with low ALT levels (ALT<100U/L ) (2.52%±2.72%, P<0.05). The percentage of CD4+CD25high Treg was correlated with ALT levels, (R=0.44, P<0.05). 28 CHB patients treated with nucleos (t) ide analogue and 24 healthy individuals as controls were enrolled in our study. Fresh isolated peripheral blood mononuclear cells (PBMCs) of these patients,who were treated with nucleos (t) ide analogue were analyzed for the proportion of CD4~+CD25~+Treg as well as lymphocyte subsets before and after 12 weeks treatment using flow cytometry. The result show that after 12 weeks nucleos (t) ide analogue therapy, both of the proportion of CD4+CD25high Treg (1.94%±0.56%) andCD4~+CD25~+CD127low/- Treg (2.56%±1.05%) in CHB patients within their population of CD4+T cells in peripheral blood were showed a statistically decrease compared baseline. (3.78%±2.82%, P<0.05), (4.16%±1.83%, p<0.01). The propotion of CD4+CD25highTreg (1.93%±0.56%) and CD4~+CD25~+ CD127low/-Treg (2.67%±1.24%) in 12 HBeAg positive patients showed a statistically decrease compared baseline. (4.33%±3.05%, P<0.05), (4.60%±1.98%, P<0.01). The conclusion for this part is the proportion of CD4~+CD25~+regulatory T cells in the patients with chronic HBV infection decreased.In summary, the level of CD4~+CD25~+Treg and Foxp3 mRNA in CHB patients is abnormal. The proportion of CD4~+CD25~+Treg decreased under the treatment of nucleoside analogue. The expression of CD25 could be influenced by the level of ALT. As a specific cell surface marker, CD127 can helpful to obtain pure CD4~+CD25~+Treg. CD4+CD2 5+Treg could contribute to an inadequate immune response against the virus, leading to chronic infection.