Novel approach to the characterization of melanoma associated-peptide-specific CTL lines from Japanese metastatic melanoma patients.
Melanoma-associated antigens, MART-1, tyrosinase, gp100 and MAGEs, are typical melanoma-specific tumor antigens which can potently induce immune responses in metastatic melanoma patients treated with peptide vaccines. In the present study, we established a dendritic cell (DC)-based HLA-A2 melanoma-associated peptide (MART-1 or gp100)-specific CTL induction method and characterized the CTLs using HLA-A2 tetramer staining in 6 cases of HLA-A2+ melanoma treated with DC vaccines. Peripheral blood mononuclear cells (PBMC) from patients were stimulated twice with MART-1 A2 peptide-pulsed DCs in the presence of a low dose of IL-2. To boost CTL populations, CTL lines were further stimulated twice with MART-1 A2 peptide-pulsed T2 cells. The frequency of MART-1 A2 tetramer-positive CTLs increased from 0.16% (prior to stimulation) to 2.15% (after DC stimulation), and reached 46.5% on average (after additional T2 stimulation) in 4 cases which showed a successful expansion. The absolute numbers of MART-1 A2 tetramer-positive CTLs increased from 187- to 619-fold (average, 415-fold) compared to prior to DC stimulation. CTL assays using MART-1-specific CTL lines demonstrated potent killing activity against MART-1 peptide-pulsed T2 cells or HLA-A2+ melanoma cell lines in accordance with the frequency of tetramer-positive CTLs. Finally, we were successful in identifying melanoma peptide-specific T-cell receptor (TCR) cDNAs in 2 cases for MART-1 and 1 case for gp100 using the anti-TCR MoAb-based sorting as a novel approach instead of a conventional cell cloning, and confirmed peptide-specific IFN-gamma production in TCR cDNA-transduced naïve T cells. The results showed that cloned TCR cDNAs were efficient in reconstituting tumor-specific cytotoxicity and good candidates for novel immunotherapy.
Akiyama
Yasuto
Y
Immunotherapy Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan. y.akiyama@scchr.jp
Maruyama
Kouji
K
Tai
Sachiko
S
Takikawa
Masako
M
Ohshita
Chie
C
Yamamoto
Akifumi
A
Yamazaki
Naoya
N
Kiyohara
Yoshio
Y
Yamaguchi
Ken
K
D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology*; D019496:Cancer Vaccines / Q000276:immunology* / Q000627:therapeutic use; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D007564:Japan; D008213:Lymphocyte Activation / Q000276:immunology; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology; D009363:Neoplasm Proteins / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014161:Transduction, Genetic / Q000379:methods; D014407:Tumor Cells, Cultured; D058951:gp100 Melanoma Antigen
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D015789:HLA-A2 Antigen; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D011948:Receptors, Antigen, T-Cell; D058951:gp100 Melanoma Antigen; D007371:Interferon-gamma