Study On The Usage Of T-cell Receptor α And β Chains Of The Hbv-specific Cd8~+ T Lymphocytes In The Patients With Acute Hepatitis B

More than 350million people throughout the world are estimated to be chronically infected with the hepatitis B virus (HBV), which is the primary source of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in endemic areas. Although the pathogenesis of chronic liver disease is not well understood and may associate with factors from virus and host, there is a consensus that liver damage is mediated by immune responses. Among these, specific cytotoxic T lymphocyte (CTL) response plays a crucial role in viral clearance and clinical recovery during acute hepatitis, and also a contributor of pathologic damage. Indeed, patients with acute HBV infection, who clear circulating virus, present a strong and multispecific CTL response that persists in the circulation without further liver damage. By contrast, this response is barely detectable in the circulation of chronic patients despite clinical and histological signs of liver damage, except after resolution when viral replication is controlled. The CTL belong to CD8 T lymphocytes. T lymphocytes use specific T cell receptor (TCR) to recognize complexes of peptide bound to major histocompatibility complex (MHC) glycoprotein on the surface of infected cells. The diversity of T cell receptor is generated by the combinatorial pairing of differentαandβchains. This project aimed at analysis the usage of T-cell receptorαandβchains of the HBV-specific CD8~+ T lymphocytes in patient with acute hepatitis B (AHB), and obtaining the information of specific CD8~+ T lymphocytes receptor variable region sequence. The results will provide helpful information for further clarification of pathogenesis and virus clearance mechanisms in relation to CTL response and support immune therapy against HBV infection.Objective 1. To develop an effective assay for amplifying human T-cell receptor variable region ofαchain (Vα) andβchain (Vβ) genes.2. Analysis the frequency of the HBV-specific CD8~+ T lymphocytes in the peripheral blood of patients with acute hepatitis B.3. Analysis the T-cell receptorαandβchains usage of the HBV-specific CD8~+ T lymphocytes in patients with acute hepatitis B.Methods 1. Forty-two sets of TCR Vαouter and inner-sense primers were designed, basing on the property of 32 subfamilies of human TCR Vαgene sequence. Thirty-four sets of outer and 37 sets of inner sense primers were designed, basing on the property of 25 subfamilies of human TCR Vβgene sequence. In addition, a set of outer and inner antisense primers located in constant region were designed. And a sequencing primer and a sense primer were also designed for sequencing the polymerase chain reaction (PCR) products. RNA of CD8~+ T lymphocytes of a healthy donor was extracted using RNeasy mini kit, and cDNA was synthesized using reverse transcriptase and oligo dT primer, followed by a nested polymerase chain reaction (PCR) for the amplification of 32 subfamilies of human TCR Vαgene and 25 subfamilies of human TCR Vβgene. Jurkat T lymphoma cells were used as control. Target genes were cloned into T-easy vector and sequenced.2. The frequency of HBsAg335-343 and HBsAg183-191 specific CD8~+ T lymphocytes in the peripheral blood mononuclear cells (PBMC) from patients with acute hepatitis B and chronic hepatitis B (CHB) were quantified by flow cytometry using MHC pentamer complex. The relationship between the frequency of specific CD8~+ T lymphocytes with the level of ALT and the virus load were analyzed, respectively. The frequency of HBsAg335-343 specific CD8~+ T lymphocytes in patients infected with acute hepatitis B was dynamic analysis.3. Peripheral blood mononuclear cells were isolated from three patients with acute hepatitis B, whose HLA subtype was all HLA*0201. HBsAg335-343 restricted epitopic peptides and cytokines were used for induction of positive CD8~+ T lymphocytes expansion, followed by sorting of the pentamer-positive CD8~+ T lymphocytes into 96 cells by flow cytometry, after culturing 10-14 days. RNA was extracted from purified T cells using the RNeasy Micro Kit, and cDNA was synthesized by reverse transcription with oligo dT primer. TCR Vαand Vβgenes were then amplified by PCR. Non-specific CD8~+ T lymphocytes from three patients were used as controls. T-easy vector was employed to detect DNA sequencing of the cloned target genes.Results 1. All the subfamilies of the Vαand Vβgenes were obtained from CD8~+ T lymphocytes of a healthy donor, while only the Vα1 and the Vβ8 subfamily was obtained from Jurkat cells as anticipated. The results were confirmed by clonal sequence analysis. High sensitivity of the assay was presented by a lower limit of amplification for TCR encoding genes from only 10 cells.2. The average of the frequency of HBsAg335-343 and HBsAg183-191 specific CD8~+ T lymphocytes in AHB were 0.45%±0.52% and were 0.19%±0.22%, respectively. The average of the frequency of HBsAg335-343 and HBsAg183-191 specific CD8~+ T lymphocytes in CHB were 0.05%±0.04% and 0.08%±0.10%, respectively. The frequency of HBsAg335-343 and HBsAg183-191 specific CD8~+ T lymphocytes had no association with either ALT level or virus load (P >0.05) at acute phase of illness. However, the number of the HBV specific CD8~+ T lymphocytes was significantly reduced at the convalescence phase.3. HBsAg335-343 epitope specific CD8~+ T lymphocytes of three patients with AHB used different T cell receptors. While they commonly used the same Vαsubfamilies of Vα2,Vα3,Vα15,Vα20 and the same Vβsubfamilies of Vβ2,Vβ3,Vβ5,Vβ7,Vβ13,Vβ14,Vβ16. HBsAg 335-343 epitope specific CD8~+ T lymphocytes in the three patients had multiple CDR3 lengths and sequences.Conclusion 1. An effective method for amplifying human T cell receptor variable region ofαandβchains genes was developed, which is helpful for the cloning and spectrum usage studies of the TCR on antigen-specific CD8~+ cytotoxic T lymphocytes (CTL).2. The property of predominant TCR Vαand Vβusage of HBV-specific CTL was found in patients with acute hepatitis B. It was suggested that the biased usage of TCR was due to preferential proliferation of CTL clone targeting the same epitope. The founding offered novel evidence for the hypothesis that antiviral CTL response had preferential TCR usage that favored virus clearance.3. TCR Vαand Vβencoding genes from preferentially-proliferated HBV-specific CTL were firstly cloned from Chinese patients, which offered materials for further CTL functional and gene theray studies.