Study On The Peptide-specific CTL In The Patients With HBV Infection

Hepatitis B virus (HBV) may cause both acute and chronic infection. Chronic infection is the major causes of chronic liver inflammation, leading to cirrhosis, hepatic decompensation, and hepatocellular carcinoma. The pathogenesis of HBV infections is not well understood, most studies indicate that HBV is not cytopathic for the infected hepatocyte, the cellular immune response is thought to be critical in determining the outcome of infection in terms of both viral clearance and liver cell injury. Therefore, understanding the antigen-specific immune response against HBV is important for the development of successful therapy and vaccine for this serious public health problem.The antigen-specific T cells in patients with HBV infection, especially the chronic infection, is so little that it can not be indetected by conventional methods, such as chromium-release assays, limiting dilution assays (LDA), and enzyme-linked immunosorbent spot assay (ELISPOT). Study on the specific CTL had long been hampered by lack of suitable technques. Since Altman published the first paper in 1996 describing the use of major histocompatibility complex (MHC) class peptide tetrameric technology for direct visualization and quantification of antigen-specific cytotoxic T cells, there have been many tetramer-based studies in both basic and clinical immunology. The tetrameric technology is a powerful tool in researching on antigen-specific T cells, regardless of their function after in vivo or in vitro antigen stimulation, and without the need of in vtro expansion, it have greatly enhanced the opportunities for directly studying antigen-specific T cells.The tetrameric technology involves the engineering of a biotinylation sigal sequence onto the C terminus of a recombinant MHC class I molecule which, after complexing with a specific peptide is bound to avidin at a 4:1 ratio. This results in a tetrameric peptide-MHC complex that recognizes T cell receptors (TCRs) on lymphocytes specific for the particular epitope, the particular peptide determines the specificity of tetramer. So, ifyou can synthesis one tetramer, it is easy for other one.Although the tetrameric technology was used in the research of HBV infection for several years, but a few epitopes and a few cases of patients with HBV infection were involved, these results were not enough confidenty. In order to study extensively on the HBV-specific CTL using tetramers, we tried to construct the HBV-peptide tetramer, and we used four HLA-A2 tetramers, specific for HBV core (18-27), HBV envelope (183-191), (335-343) and polymerase (575-583), to investigate the frequency of epitope-specific CTL in PBMCs of chronical hepatitis B, to evaluate the correlation between the specific T cells and the outcome of infectionThe main research results are as follows:1. The construction of HBV-peptide tetramer.Upon addition of IPTG, the recombinant HLA-A2-BSP and P -2 microglobulin was produced in E.coli BL21(DE3)pLysS transformed by the expression plasmid. The 6x His-tagged fusion protein was purified using Nickel Chelated Agarose, HLA-A2-BSP was folded in the presence of 0 -2M and a HBV peptide to form a peptide-MHC complex, and the MHC complex were bitotinylated using BirA enzyme. Tetramer were generated by mixing bitotinylated protein complex with streptavidin-PE at a molar ratio of 4:1.2. Establish the method of using MHC- peptide tetramer in detecting antigen- specific CTLs.HLA-A2+ HBV-negative blood donors and HLA-A2- negative patients with chronic hepatitis B were tested as negative controls, we used the tetramers to test peripheral blood from the HLA-A2+ patients with chronic HBV infection and controls. The frequency of tetramer- positive T cells exceeded 0.02% circulating CD8+ cells which represent the maximum staining observed in the controls, so the cutoff for tetramer-positive was set at 0.02%. The frequency of tetramer- positive T cells in the A2+ chronic HBV infection was 0-2.41%.3. Research on the frequency of antigen- specific T cells in the peripheral blood from patients with hepatitis B.The frequency of HBV-specific CTL in the circulation was determined by the tetramer in 2 individuals with self-limited HBV infection, 36 chronically infection patients and 5 serious hepatitis. The frequencyof of tetramer-positive T cells was 0-2.41% in patients withHBV infection, numbers of tetramer- positive cells was above the control level, and more than one epitope specific CTL in 34 out of 36 chronic patients was demonstrated. But the positive rate of whole 4 tetramer cells was only higher in two patients with self-limitited HBV infection. Although tetramer-positive cells specific for one peptide (especially envelope 183-191) were found at a high frequency in a few chronically infection patients, the frequency of tetramer- positive T cells was low in most of these patients. The results were coincidence with that by intracellular staining and ELISPOT for Y -IFN. The number of tetramer binding cells was not different in chronic infection and serious hepatitis.4. Research on the antigen- specific T cells in the peripheral blood from patients with hepatic flares36 chronically infection patients were divided into four groups according to the levels of serum alanine aminotransferase (ALT). 1st group: ALT< 1 x ULN(upper limit of normal), 2nd group: ALT^2xULN, <5xULN, 3rd group: ALT2?5xULN, <10xULN, 4th group: ALT^lOxULN. We studied the number and function of the specific cytotoxic T lymphocytes in each group. The results indicated that frequency of three epitope specific CTL (C18-27, El83-191 and P575-583) was higher in 3rd group.6 patients who had elevated serum alanine aminotransferase (ALT) were monitored for 9 monthes, the frequency of tetramer- positive T cells was reduced as the ALT descended in 3 patients. In one patients, the number of tetramer- positive T cells was fluctuated following the changes of ALT.Conclusion:1. Purified the recombinant protein of HLA-A2-BSP and 0-2 microglobulin, and completed the construction of one HBV peptide tetramer.2. The tetrameric technology is a specific tool in enumerating antigen-specific T cells, the HBV- HLA-A2 tetramers was only used in HLA-A2-positive HBV infections.3. The frequency of tetramer-positive cells was different between the self-limited HBV infection and chronically infection. Self-limited HBV infection mount a multispecific polyclonal CTL response to several HBV-antigens. In contrast, the HBV-specific immune response is weak in the blood of chronically infected patients.4. Virus and antigen specific CTLs coexist in the chronic HBV infection. The presence of HBV-specific T cells are not determinant of immune-mediated protection inHBV infection.5. The frequency of HBV-specific T cells was highly when hepatitic flares occur. It indicated that the specific cellular immune was boost up, and the HBV-specific T cells may contribute to liver injury in chronic infection.