CD8^low T-cell Subpopulation Is Increased In Patients With Chronic Hepatitis B Virus Infection And Establishment Of An Approach To Amplify Monoclonal Epitope-specific TCR From Human T-cell Repertoire

Hepatitis B virus (HBV) is known as a hepatotropic, noncytopathic DNA virus, infecting more than300million people worldwide and readily establishing chronicity. In patients chronically infected with HBV, specific antiviral T-cell response is weak or undetected, resulting in persistence of infection and development of severe liver diseases. Although the mechanisms responsible for T cell hyporesponsiveness or tolerance to HBV antigens are not fully elucidated, substantial studies suggest both CD4+and CD8+T cells of regulatory property would play an important role in inhibiting HBV-specific T-cell responses.CD3+CD8low T cells are recognized as a subset of CD8+T cells with down-regulated CD8expression, whose increase is observed in chronic and persistent antigen exposure, e.g. patients infected with HIV, patients underwent transplantation and mouse chronically infected with parasite. The CD8low T cells are reported to be type-2polarized and poorly cytolytic, and even exhibit suppressive function via IL-10 or membrane-bound TGF-β1(mTGF-β1).Given that chronic HBV infection is also a persistent antigen exposure, in this study, we explored the role of CD8low T cells in the impaired CD8+T cell response in patients with chronic HBV infection.1. Frequency of CD8low T cells is increased in peripheral blood of chronic HBV patients19healthy individuals and47chronic HBV patients were selected. PBMC were isolated and flow cytometry was performed to determine the circulating CD8low T-cell frequency. There was a significantly higher frequency of circulating CD8low T cells in chronic HBV patients than that in healthy controls (the CD8low cells in CD8+T cell:15.20%±1.04%vs.5.66%±0.57%[mean±SEM];p<0.001). we also found the patients with the disease course longer than5years (n=21) showed a markedly higher frequency of CD8low T cells compared to those shorter than5years (n=10)(the CD8low cells in CD8+T cell:18.64%±1.70%vs.10.37%±1.84%[mean±SEM]; p<0.01).2. Soluble HLA class I plasma levels show a positive relationship with frequency of CD8low T cells in chronic HBV patientsThe concentrations of plasma HLA class I molecules from47chronic HBV patients and19healthy donors were measured. In comparison to healthy donors, soluble HLA class I plasma levels were significantly higher in chronic HBV patients (ng/mL,850.1±79.77vs.263.1±29.67[mean±SEM];/p<0.001). There was a positive correlation between soluble HLA class I plasma levels and the percentage of CD8low cells in CD8+T cells (R2=0.2155;p=0.001). In contrast, no correlation was found in healthy donors.3. Phenotype of CD8low T cells in chronic HBV patientsThe CD8low T cells in chronic HBV patients and healthy donors were typed for the commonly used effector and suppressive markers. We found CD8low T cells in the patients, compared to that in healthy donors, seemed phenotypically to be type-2polarized (more IL-4and less IFN-y expression) and of regulatory/suppressive properties (elevated expression of suppressive markers, especially mTGF-β1).4. CD8low T-cell frequency is negatively correlated with HBc18-27-specific CD8+T-cell responses in chronic HBV patientsCD8+T-cell responses to the HBc18-27peptide were evaluated with HLA-A2+ve samples by IFN-y Elispot assay. The CD8+T cells of chronic HBV patients showed significantly lower IFN-y production in response to the HBc peptide (15.39±1.59vs.36.78±10.01[mean±SEM];p<0.01). Notably, a significantly negative correlation was observed between frequency of CD8low T cells and HBc-specific CD8+T-cell responses in chronic HBV patients (r2=0.376,p=0.003). In contrast, no correlation was found in healthy donors (r2=0.195,p=0.234).5. ConclusionCD8low T-cell subpopulation is significantly increased in chronic HBV patients, which is type-2polarized and expresses elevated levels of suppressive markers. The prevalence of CD8low T cells in patients with chronic HBV infection may be one of the factors that resulting in impaired CD8+T-cell response. T cells execute cellular immune response and immuno-regulatory functionthrough interaction of their membrane TCRs with the antigenic peptides presented bythe major histocompatibility complex molecule on APCs. Investigation of TCRmolecules could not only dissect the molecular mechanism of antigen recognition, butalso develop immunotherapeutic approaches based on TCRs. Recent studies haveindicated T cells transduced with specific TCR gene display specificimmuno-regulatory or cytotoxic characteristics, suggesting their great potential intreatment of malignant tumors, autoimmune diseases, chronic viral infections, etc.Obtaining epitope-specific monoclonal TCR is cruial for investigation of TCR genetherapy.The diversity of TCR repertoire is amazingly enormous (reaches1018) because ofvarious and complex gene combination rearrangement process. To select randomepitope-specific TCR from this huge T cell repertoire is like looking for a needle in ahaystack. At present, single-cloning following in vitro expansion of specific T cells isthe main method to obtain monoclonal TCRs. However, the difficulty of long-termsingle-cloning expansion in vitro and time-consuming culture limited the widespreaduse of this method. In this study, we chose HLA-A2restricted melanoma-relatedantigenic peptide gp100as an example. A combination of in vitro epitope-specificalloreactive T cell expansion, pMHC tetramer sorting and single cell RT-PCRtechnique was employed to clone single epitope-specific TCR with high-affinity. TheTCR gene was then expressed in a vector and identified. The method proposed hereprovides an efficient and practical way to employ the TCR-based immune intervention.1. Design of primers for TCR α and β chain amplification and validation of thedesigned primersSequences corresponding to the functional variable and constant region genes forTCR alpha, beta chains were obtained from IMGT/Gene-DB (http://imgt.cines.fr/).After analysis and alignment of all the sequences,59forward primers and4reverseprimers were designed with the aid of the Primer Primer5.0software, which wereannealed to the variable region and the constant region, respectively. To furthervalidate all the primers we designed, RT-PCRwere performed. For each reaction,cDNA derived from PBLs of healthy donors was used as template. Nearly each pair ofprimer of TCR α chain produced bands of expected size. Similarly, each pair ofprimer of TCRβ chain obtained the positive results. It suggested that the primers wedesigned were suitable for cloning TCR genes.2. Establishment of single cell RRTT--PPCCRRfor cloning TCR2.11Amplification of TCR α and β chain from single jurkat cellsTo explore the optimum condition of single cell RT-PCR, we chose a humanlymphoblastic T jurkat cell line which expresses TCR and single cloned it. Singlejurkat cells were isolated using serial dilution method and lyzed to obtain RNA. ThenRNA was reverse transcribed to cDNA using constant-specific primers (2in total) forα and β chain. TCR α and β chain of single jurkat cells were amplified using35pairsof mix primers for α chain and24pairs of mix primers for β chain. The amplifiedPCR products were cloned to T-easyvector for sequencing and aligned to NCBI. Asexpected, sequence we amplified was right. Variableregion of TCR α and β chain ofjurkat cells were TRAV8-6and TRBV12-3, respectively.2.2Amplification of TCR α and β chain from single CD8+T cells frompheripheral blood of healthy donorsPheripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. And then CD8+T cells were enriched by negative selection using a naive CD8+T CellIsolation Kit and diluted at one cell per PCR tube. TCR α and β chain of each cell wasamplified using modified single cell RT-PCR for jurkat cells. We successfullyamplified5TCR α chains and8TCR β chains from12single T cells. Sequences ofamplified TCR α and β chain were obtained after sequencing.3. Expansion of single epitope-specific T cells through mixed lymphocytecoculture in vitroT2cells (TAP deficient, only express HLA-A2molecule) pulsed with gp100peptide were used as stimulators and named as T2/gp100. Cytoxic T cells specific toT2/gp100epitope (CTL-T2/gp100) were induced by coculturing HLA-A2-ve PBLswith T2/gp100for14days. The bulk cells were tested for their binding ability to thegp100/HLA-A2tetramer or irrelevant HBc/HLA-A2tetramer. The frequency ofT2/gp100tetramer-stained CD8+T cells was significantly higher compared with thatof irrelevant HBc/HLA-A2tetramer-stained CD8+T cells. It suggested CTL specificto T2/gp100were successfully induced.4. Cloning TCR from sorted single gp100/HLA-A2specific T cells using singlecell RT-PCRGp100/HLA-A2tetramer-stained CTL-T2/gp100were sorted by flow cytometryand diluted to single cell per tube. Then α and β chain of epitope-specific TCR wereamplified by single cell RT-PCR decribed above. We determined5α and5β chainsequences from10single cells after sequencing and alignment (4pairs).5. Expression and identification of soluble gp100/HLA-A2specific TCROne pair of α and β chain gene of gp100/HLA-A2specific TCR was selected andtransduced to pfast-dual expression vector after codon optimization for expression ofsoluble gp100/HLA-A2specific TCR molecule. Western-blot detection indicated theconformation and molecular size of the protein were right. Unfortuately, specificitydetection of the protein using ELISA with gp100/HLA-A2tetramer was not desirable. It is supposed that the TCR we chose may have an intrincially affinity not highenough for detection, or maybe the concentration of produced TCR molecule was toolow for detection.6. ConclusionWe establish an approach to amplify epitope-specific monoclonal TCR fromhuman T cell repertoire which is not restricted to epitope, providing basis for furtheremployment of TCR-based immunotherapy.