Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely b...Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely because of their costly and time-consuming production. In this report we utilized an economic strategy to construct HLA/peptide tetramers with recombinant peptide-linked β2 microglobulin (β2m). The HLA-A2-restricted, melanoma antigen MARTI-derived pep- tide MART127-35( AAGIGILTV) was fused to the N terminus of human β2m through a 15-amino acid (aa)-long linker before being refolded with the recombinant biotinylated HLA-A2 heavy chain ectodomain. The resulted 2-component (2C) monomer was then tetramerized with phycoerythin-labeled streptavidin. The experimental result showed that the 2C HLA-A2/ MART127-35 monomer was shown to bind to the HLA class complex-specific monoclonal antibody W6/32 and the HLA-A2/ MART127-35 complex-specific single chain antibody fragment (scFv) 8.3, suggesting the correctness of its specificity. Fur- thermore, the 2C HLA-A2/MART127-35 tetramer detected a specific CD8+ T cell population in HLA-A2-restricted melanoma infiltrating lymphocytes as the conventional 3C HLA-A2/MART127-35 tetramer. The yield of 2C HLA-A2/MART127-35 monomer was 2. 5 times more than that of the conventional 3C monomer. Taken together, these data indicate that the HLA-A2/ MART127-35 tetramer can be generated conveniently through the use of MART127-35 peptide-β2 m fusion proteins, which can fa- cilitate the monitoring of HLA-A2-restricted, MART1-specific CTL responses in patients with melanoma.展开更多
In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from...In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the target cells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%, 25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-terminal of the viral nucleocapsid protein.展开更多
文摘Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely because of their costly and time-consuming production. In this report we utilized an economic strategy to construct HLA/peptide tetramers with recombinant peptide-linked β2 microglobulin (β2m). The HLA-A2-restricted, melanoma antigen MARTI-derived pep- tide MART127-35( AAGIGILTV) was fused to the N terminus of human β2m through a 15-amino acid (aa)-long linker before being refolded with the recombinant biotinylated HLA-A2 heavy chain ectodomain. The resulted 2-component (2C) monomer was then tetramerized with phycoerythin-labeled streptavidin. The experimental result showed that the 2C HLA-A2/ MART127-35 monomer was shown to bind to the HLA class complex-specific monoclonal antibody W6/32 and the HLA-A2/ MART127-35 complex-specific single chain antibody fragment (scFv) 8.3, suggesting the correctness of its specificity. Fur- thermore, the 2C HLA-A2/MART127-35 tetramer detected a specific CD8+ T cell population in HLA-A2-restricted melanoma infiltrating lymphocytes as the conventional 3C HLA-A2/MART127-35 tetramer. The yield of 2C HLA-A2/MART127-35 monomer was 2. 5 times more than that of the conventional 3C monomer. Taken together, these data indicate that the HLA-A2/ MART127-35 tetramer can be generated conveniently through the use of MART127-35 peptide-β2 m fusion proteins, which can fa- cilitate the monitoring of HLA-A2-restricted, MART1-specific CTL responses in patients with melanoma.
文摘In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the target cells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%, 25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-terminal of the viral nucleocapsid protein.
