Research On Regulatory T Cells In Immuosuppression Of Hepatocellular Carcinoma With HBV Infection

Previous studies have shown that unresolved immune responses, such as chronic inflammation could promote the carcinogenesis and progression of cancer, and traditional wisdom holds that immune escape from host immunosurveillance may contribute to tumor growth. Chronic hepatitis B (CHB) that leads to the multistep development of hepatocellular carcinoma (HCC) is well known and it is estimated that CHB contributes to more than80% of HCC cases throughout the world. Regulatory T cells (Tregs) are the main T cell subgroups that possess the characteristic of immunosuppression and anergy, which are considerd as an important member of immunosuppressive network in tumor. Recent studies have indicated that increased Tregs in the peripheral blood from patients with HCC correlates with poor prognosis and is involved in the persistence of hepatitis B virus infection. However, all of these studies conducted CHB and HCC independently of each other and the roles of Tregs in the development of HCC by CHB have remained largely unknown.Objective:The aim of the present study was to evaluate the distribution of Tregs in peripheral blood and liver tissue from CHB and HCC patients, and to investigate the correlation between Tregs and the context of chronic hepatitis B in HCC patients. Moreover, we also explored the mechanisms by which Tregs were involved in the immunosupression of HCC with hepatitis B virus infection and for silibinin efficacy against HCC as the immune response modifier.Methods:1. The liver resident Tregs and CD8+T cells on core biopsy were examined using immunohistochemistry staining in clinical samples of human liver tissues, including47samples of CHB,16samples of atypical hyperplasia,137samples of HCC, and25samples of healthy controls. Circulating Tregs were detected in the above patients by flow cytometry.2. Immunohistochemistry was performed to analyze of tumor-infiltrating Tregs presence as well as CD34, a vascular endothelial marker, and hepatitis B virus core antigen expression.3. MTT and ELISpot assays were performed to determine that the inhibitory effect of Treg on CD8+cytotoxic T cells(CTL) in HepG2and HepG2.2.1.5cells.4. Flow cytometry and immunofluorescence staining were performed to detect the apoptosis of HepG2and HepG2.2.1.5cells induced by silibinin whereas MTT and ELISpot assays were performed to investigate whether silibinin functions as the immune response modifier. Results:1.The numbers of tumor-infiltrating and circulating FoxP3+Tregs in patients with HCC were significantly higher than those in patients with CHB or atypical hyperplasia (P<.001and P<0.001, respectively). However, there were fewer intratumoral Tregs in patients with advanced HCC than in patients with early stage HCC (P=0.043). In contrast, the circulating Tregs frequency increased during the progression of HCC (P=0.024). Increased tumor-infiltrating and circulating FOXP3+Tregs were associated with poor overall survival (P=0.041and p=0.002, respectively) and a shorter time to recurrence (P=0.049and P=0.002, respectively) in patients with early stage HCC.2. Tumor-infiltrating Foxp3+Tregs were related to the natural history of CHB and CD34expression in HCC (P=0.012).3. The circulating Treg suppressed the proliferation of CDS CTL.4. Silibinin induced the apoptosis of hepatoma carcinoma cell and promoted the secretion of IFN-y by CD8+CTLConclusions:1. Increased FOXP3+Tregs may represent a prognostic predictor in patients with early stage HCC.2. The natural history of CHB may affect the density of tumor-infiltrating Tregs in HCC patients with CHB infection.3. Tregs play a pivotal role in the suppression of CTL.4. Silibinin is able to regulate tumor immunity by increasing the production of IFN-y by CD8+CTL and induce the apoptosis of hepatocellular carcinoma cell.