Screening and identification of HLA-A0201 restricted CTL epitope from hepatitis B virus X protein in vitro / 第二军医大学学报

Objective: To identify the HLA-A0201 restricted CTL epitopes derived from hepatitis B virus X protein predicted by computer program and general principles in vitro. Methods: HBx gene sequences of Hepatitis B virus genotypes B/C and serotypes adw/adr, with the highest frequencies in Chinese, were computed and analyzed by screening service offered by Internet combined with peptide supermotif, extended motif and quantitative motif prediction. Four most ideal nine-peptides (HBx1, HBx2, HB3x3, and HBx4) were selected as candidate peptides. Using flow cytometry, the fluorescence index of both control and experimental groups were detected and the 4 nine-peptides were evaluated with T2 binding assay and DC50 assay. Results: The nine-peptides VLCLRPVGA (HBx1), CLFKDWEEL (HBx2), VLHKRTLGL (HBx3) and HLSLRGLPV (HBx4) were selected as candidate targets. Among the 4 candidate peptides, HBx2 showed higher HLA-A0201 affinity and HBx2, HBx4 showed better stability. Conclusion: Our study indicates that CLFKDWEEL might be a potential HLA-A0201 restricted CTL epitope from hepatitis B virus X protein; further study is needed for verification of its immunity in vivo.

Frequencies and Characterization of HBV-specific Cytotoxic T Lymphocytes in Self-limited and Chronic Hepatitis B Viral Infection in China / 华中科技大学学报（医学）（英德文版）

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differ-ences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononu-clear cells (PBMCs) from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8+ T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8+ T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8+ T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immuno-therapeutic approaches should be aimed at not only boosting a HBV-specific CD8+T response but also improving its function.

HLA-restricted and Antigen-specific CD8+ T Cell Responses by K562 Cells Expressing HLA-A*0201

BACKGROUND: Identification of antigen-specific T cells has yielded valuable information on pathologic process and the disease state. Assays for quantification of inflammatory cytokines or lytic-granule molecules have been generally used to evaluate antigen specific T cell response, however their applicability have been hampered due to the limited source of autologous antigen-presenting target cells (APC). METHODS: K562, a leukemic cell line deficient of human leukocyte antigen (HLA), was transfected with a gene encoding HLA-A*02 (K562/A*02) and its function as stimulator cells in inducing activation of HLA-matched T cells was evaluated by IFN-gamma enzyme linked immunospot (ELISPOT) assay. RESULTS: The stable transfectant K562/A*02 pulsed with HLA- A*02 restricted peptide could specifically induce IFN-gamma secretion by CD8+ T cells compared to no detectable secretion by CD4+ T cells. However, CD56+ NK cells secreted IFN-gamma in both K562/A*02 with peptide and without peptide. The number of IFN-gamma secreted CD8+ T cells was increased according to the ratio of T cells to K562 and peptide concentration. Formalin-fixed K562/A*02 showed similar antigen presenting function to live K562/A*02. Moreover, K562/A*02 could present antigenic- peptide to not only A*0201 restricted CD8+ T cells but also CD8+ T cells from A*0206 donor. CONCLUSION: These results suggest that K562/A*02 could be generally used as target having specificity and negligible background for measuring CD8+ T cell responses and selective use of K562 with responsder matched HLA molecules on its surface as APC may circumvent the limitation of providing HLA-matched autologous target cells.

The Study of MHC class I Restricted CD8+ T Cell Mediated Immune Responses against Mycobacterium tuberculosis Infection: Evidence of M. tuberculosis Specific CD8+ T Cells in TB Patients and PPD+ Healthy Individuals

BACKGROUND: The protective immunity against tuberculosis (TB) involves both CD4+ T cells and CD8+ T cells. In our previous study, we defined four Mycobacterium tuberculosis derived peptide epitopes specific for HLA-A*0201 restricted CD8+ T cells (ThyA30-38, RpoB127-135, 85B15-23, PstA175-83). In this study, we investigated the immune responses induced by these peptide specific CD8+ T cells in latently and chronically infected people with TB. METHODS: We characterized these peptide specific CD8+ T cell population present in PBMC of both TB patients and PPD healthy people using IFN-gammaelispot assay, intracellular staining and HLA-A2 dimer staining. RESULTS: The frequency of peptide specific CD8+ T cell was in the range of 1 to 25 in 1.7x10(5) PBMC based on ex vivo IFN-gamma elispot assay, demonstrating that these peptide specific CD8+ T cell responses are induced in both TB patients and PPD people. Short term cell lines (STCL) specific for these peptides proliferated in vitro and secreted IFN-gamma upon antigenic stimulation in PPD+ donors. Lastly, HLA-A*0201 dimer assays indicated that PstA175-83 specific CD8+ T cell population in PPD+ healthy donors is heterogeneous since approximately 25~33% of PstA175-83 specific CD8+ T cell population in PPD+ healthy donors produced IFN-gamma upon peptide stimulation. CONCLUSION: Our results suggest that MHC class I restricted CD8+ T cell mediated immune responses to M. tuberculosis infection are induced in both TB patients and PPD+ people; however, the CD8+ T cell population is functionally heterogeneous.

Screening and identification of HLA-A0201 restricted CTL epitope from hepatitis B virus X protein in vitro / 第二军医大学学报

Objective:To identify the HLA-A0201 restricted CTL epitopes derived from hepatitis B virus X protein predicted by computer program and general principles in vitro.Methods:HBx gene sequences of Hepatitis B virus genotypes B/C and serotypes adw/adr,with the highest frequencies in Chinese,were computed and analyzed by screening service offered by Internet combined with peptide supermotif,extended motif and quantitative motif prediction.Four most ideal nine-peptides(HBx1,HBx2,HBx3,and HBx4)were selected as candidate peptides.Using flow cytometry,the fluorescence index of both control and experimental groups were detected and the 4 nine-peptides were evaluated with T2 binding assay and DC50 assay.Results:The nine-peptides VLCLRPVGA(HBx1),CLFKDWEEL(HBx2),VLHKRTLGL(HBx3)and HLSLRGLPV(HBx4)were selected as candidate targets.Among the 4 candidate peptides,HBx2 showed higher HLA-A0201 affinity and HBx2,HBx4 showed better stability.Conclusion:Our study indicates that CLFKDWEEL might be a potential HLA-A0201 restricted CTL epitope from hepatitis B virus X protein;further study is needed for verification of its immunity in vivo.

Cloning of HLA-A~*0201 and its transient expression on COS-7 cells / 中国免疫学杂志

Objective:To clone HLA A *0201 cDNA and express it transiently on COS 7 cells.Methods:HLA A cDNA isolated from the HLA A *0201 positive lymphocytes by using RT PCR was cloned into pBluescript II SK vector directionally.After the sequence was confirmed,the cDNA was inserted into mammalian expression vector pcDNA3.The recombinant vector was transfected into COS 7 cells by cation liposome.The transient expression on the cells was measured by flow cyteometer.Results:The cDNA was identical with HLA A *0201 cDNA published on Genebank.Determined by flow cytemeter,the expressing rate was recorded for 57%.Conclusion:We have cloned HLA A *0201 successfully and expressed it transiently on COS 7 cell,which would be potentially useful in research on killing tumor restricted by HLA A2.