Studying On The Virus-specific Cytotoxic T Lymphocyte Response In Hepatitis B Infection

Objects1.To develop a method to detect specific CTL-HLA classⅠ/peptide tetrameficcomplexes, and provide the experiment grounding of investigating to epitope-specific CTLeffect in vivo and understanding the role of HBV-specific CTL in the immunopathogenesisof HBV infection.2.To investigate the frequencies and the effects of epitope-specific CTLs inself-limited and chronic HBV infections by tetrameric complexes.3.To analyse the mechanism of HBV-specific T-cell exhaustion in chronic HBVpatients, and develop the immune-based therapies.Methods1.Proteins HLA-A~*0201-BSP, HLA-A~*2402-BSP, HLA-A~*1101-BSP andβ2m wereobtained by effective prokanyotical expression.After being purified, the proteins wererefolded into complexes in the presence of common antigenic peptides by dilution method.The complexes then were biotinylated by BirA enzyme and purified via the gel-filtrationchromatography.Tetramers were generated by mixing the complex with PE-Streptavidin ata molar ration of 5:1. 2.The biotinylated HLA-A~*0201-, HLA-A~*2402- and HLA-A~*1101-β2m-peptidecomplexes were identified by Western blot and were shown to have the naturalconformation by Dot-ELISA.The specificity of the tetramers was tested by flow cytometricanalysis.3.PBMCs were isolated from fresh heparinized venous blood by Ficoll-Hypaquedensity gradient centrifugation and HLA allele was performed by PCR-SSP.4.Hepatitis B surface antigen (HBsAg), anti-HBs, total and IgM anti-HBc, hepatitis Be antigen (HBeAg), anti-HBe, anti-HCV, and anti-HIV were determined by commercialenzyme immunoassay kits.HBV DNA was quantified by the Roche Amplicor Monitorsassay.5.The analysis of stained PBMCs using the tetramers and other antibodies wasperformed by flow cytometry.6.Data are analysed by Statistical Package for Social Sciences (SPSS) version 13.0.Results1.Constructing the HLA-A~*0201 core18-27, po1575-583, env335-343; HLA-A~*2402core117-125, po1756-764; HLA-A~*1101 core88-96, preS1 10-17 tetramers.Using thesetetramers can detect the specific CTLs.2.The frequencies and characterization of HBV-specific cytotoxic T lymphocytes inself-limited and chronic HBV infection in Chinese population:(1) The virus-specific CD8~+T cell responses during chronic HBV infection are weakor absent whereas in resolved infection these responses are generally vigorous andmultispecific.In self-limited HBV infected individuals the frequencies and expansionability of core-specific CD8~+T cells is higher than that of pol- and env-specific CD8~+Tcells.(2) In patients with chronic infection the frequencies of HBV-specific CD8~+cells afterin vitro expansion is inversely proportional to the level of HBV DNA.A lower level of expansion and IFN-γ-production is present in patients with HBV DNA levels of <10~7copies/mL, and no expansion and IFN-γ-production is present in patients with HBV DNAlevels of>10~7 copies/mL.(3) The frequencies of HBV-specific CD8~+T cells are higher in heterozygote patientswith self-limited infection than in patients with chronic infection, and the frequencies ofHBV-specific CD8~+T cells in patients with chronic infection are low, furthermore, nocorrelation is detected between the frequencies and the HLA allele.3.The expression of CD127 on CD8~+T cells in self-limited and chronic HBV patientsis no difference, but the expression of CD127 on HBV-specific CD8~+T cells in chronicpatients is higher than that in self-limited patients.4.In self-limited, chronic, uninfected HBV patients, no significant correlation is foundamong the frequencies of CD3~+CD4~+T cells, CD3~+CD8~+T cells and CD4~+/CD8~+ in thesethree groups.5.There is no significant correlation in the frequencies of IL-10-, TGF-β-, IFN-γ-producingCD4~+, CD8~+T cells in self-limited or chronic HBV patients.6.Either the IL-1 OR blockade or the TGF-βblockade can increase the frequencies andcharacterization of HBV-specific CTLs in self-limited and chronic HBV patients, especially,the function of the TGF-βblockade is stronger.Conclusions1.Using these constructed tetramers can detect the specific CTLs.2.The frequencies, the ablity of expansion and IFN-γ-production of HBV-specificCD8~+T cells were higher in self-limited HBV patients than in chronic patients; thefrequencies of HBV-specific CD8~+T cells in chronic HBV patients were low, only a lowerlevel of expansion and IFN-γ-production is present in patients with low HBV DNA levels,and no expansion and IFN-γ-production is present in patients with high HBV DNA levels. 3.IL-10 and TGF-βsuppress cellular immune responses in HBV infection, so theblockade of the receptor for IL-10 or TGF-βcan restore the antiviral immune response andresult in viral clearance.