| 1. Background/AimsHepatitis B virus (HBV) has high prevalence in our country. Around 50% Chinese people once infected by HBV, among whom 93 million people are with chronic infection. HBV infection has wild clinical presentation, including insidious infection, acute hepatitis B, chronic hepatitis B, liver failure, liver cirrhosis and hepatocelluar carcinoma, etc. Until now, the pathologic mechanisms have not been well understood. Plenty of studies suggest that the elimination of HBV and the liver damage caused by the virus infection mainly depend on the host immune status. The virus may indirectly caused hepatocyte impairment through stimulation of immune responses.In the immune responses against HBV infection, HBV-specific cytotoxic lymphocytes (CTL) play a key role in clearing virus and influencing clinical outcome. Patients with acute hepatitis B present a strong and polyclonal CTL response, leading to a quick elimination of infected virus. By contrast, the HBV-specific CTL response is weak and lack of polyclonal CTL responses, leading to incomplete elimination of infected virus. On the other hand, CTL responses are associated with the degree and pattern of immune pathogenesis. Mechanisms of the difference remain classification. Possible influence factors may come from host (e.g. genetic polymorphism of inflammatory factors, HLA subtype and CTL per se) and virus (e.g., virus variation and virus load). The specificity of CTL is decided by T cell receptor (TCR), and the specificity of TCR mainly depends on V regions of a andβchains. Therefore, it is important to clarify molecular consistent feature of a andβchain V region for understanding mechanisms of HBV-specific immune responses. This project aimed at analyses of CTL frequency in different illness categories and the TCRαandβchain usage of the HBV-specific CTL in patient with hepatitis B, acquirement of the information of V region sequence of HBV-specific CTL TCR, identification and construction of matchedαandβchains for artificial expression of the CTL TCR by retroviral vector-mediated gene transfer. The study is helpful to characterize the clinical and functional feateres of HBV-specific CTL for better understanding anti-HBV immune response mechanisms and for assistance ofanti-HBV immune therapy.2. Materials and methods(1) The study enrolled 40 patients with acute hepatitis B and 40 patients with chronic hepatitis B. The patients were hospitalized in our unit (The 302 Hospital of PLA) from January 2008 to December 2010. In addition,15 healthy volunteers were involved as normal control. The diagnosis was made according to the criteria for diagnosis, prevention and cure of virus hepatitis constituted by the Chinese liver disease association in September 2000. Concurrence of hepatitis A, C, E and autoimmune or drugs inducing liver disease was excluded for all enrolled individuals.(2) Blood were sampled from 40 patients with acute hepatitis B (AHB). Among them,18 patients were HLA-A2-positive and their peripheral blood mononuclear cells (PBMC) were isolated at acute phase and convalescent phase if available. In addition,8 HLA-A2-positive patients with chronic hepatitis B (CHB) and 6 HLA-A2-positive normal persons were sampled, too. HBV-specific CD8 T cells were stained by fluorescence-labeled anti-CD3, anti-CD8 and HBV epitope-specific pentamers (HBsAg 183-191, HBsAg335-343) and analyzed by flow cytometry.(3) Genomic DNA was extracted from blood of the patients. HLA-A2 subtype was determined by amplifying and sequencing the related gene fragment. The serum HBV DNA level, HBV antigens/antibodies and biochemical parameters were detected in the Central Clinical Laboratory of the 302 Hospital.(4) PBMC from 6 HLA-A2 positive patients were clutured for 3 to 4 weeks in the presence of stimulator HBsAg335-343 peptide. HBV-specific CD8 T cells were isolated by magnetic activated cell sorting followed by flow florescence activated cell sorting. The sorted cells were expanded with IL-2, anti-CD3 and anti-CD8. Cell mRNA was extracted and used for rapid amplification of cDNA 5'-ends of TCR a andβchains which were analyzed by cloning sequencing (≥50 clones for single chain of each sample).(5) Constituting whole gene of a andβchain from product of rapid amplify cDNA 5'ends (5'RACE) and rapid amplify cDNA 3'ends (3'RACE) by OVER-LAP PCR, then a andβchain gene were ligated to vector SAMEN CMV/SRa.After packaing in incasing cell,retroviral supernatants obtained from incasing cell infected Jakurt,then screening positive cell that expressing special TCR for function test.3. Result(1) Average frequencies were 0.23% for HBsAg183-191-specific CD8 T cells and 0.49% for HBsAg335-343-specific CD8 T cells at acute phase of the 18 HLA-A2-positive AHB patients, significantly higher than those in the 18 HLA-A2-positive patients with chronic hepatitis B whose CTL frequencies were 0.06% for HBsAg183-191-specific CD8 T cells and 0.08% for HBsAg335-343-specific CD8 T cells.(2) The frequency of HBsAg335-343-specific CD8 T cells rather than HBsAg183-191-specific CD8 T cells was significantly positively correlated with ALT level, but not correlated with HBVDNA level.(3) Dynaimical analysis of 8 AHB patient's showed that the CTL frequency was significantly higher at acute phase than that at convalescent phase (0.75±0.42vs 0.42±0.30, P=0.011) (4) The subtype of HLA-A2 was associated with dominance of the CTL epitopic peptide. In HLA-A0201 and A0203 positive AHB patients, HBsAg335-343-specific CTL frequency was higher than HBsAg (0.56±0.63% vs. 0.21±0.27%, P=0.0413). In contrast, in HLA-A0207 and A0206 positive AHB patients, HBsAg183-191-specific CTL frequency was relatively higher(0.17±0.31 vs.0.33±0.47%, P=0.0749)(5) Analysis of 600 more cloned TCR gene sequences showed that the proliferated HBV-specific CD8 T cells from all 6 AHB patients presented predominance in TCR usage ofαandβchains, with 2-4αchain families and 1-4βchain families in each case. Theα2,α14,α15,β3,β13 andβ23 families were detected in one more cases. One case hadβ13 chain for all tested clones.(6) For the sameαchain orβ-chain family, CDR3 sequences tended to be identical in one case but to be different between cases.(7) The construction of TCRαandβchains was successful,but retralviral vector construction and TCR expression were failure.4. Conclusion(1) HBV-specific CD8 T cells play an important role in both virus clearance and liver impairment, while different epitope-specific CD8 T cells may contribute in different extent. HLA-A2 subtypes may have impact on the generation of CD8 T effector cells specific to different epitopes.(2) Subtype of HLA may associated with the outcomes of acute hepatitis B.(3) HBV-specific CD8 T cells with antigenic peptide-induced proliferation had predominance in the usage of TCRαandβchains. This property might be one of the important molecular factors influencing anti-HBV immunity.
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