Identification of melanoma-specific peptide epitopes by HLA-A2.1-restricted cytotoxic T lymphocytes.
HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and allogeneic HLA-A2.1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.
Department of Immunology, Shanghai Jiaotong University School of Medicine, Shanghai Institute of Immunology, Shanghai 200025, China. gehailiang@yahoo.com
Hai-Liang
HL
Ge
Ying
Y
Wang
Shu-Jun
SJ
Wang
Yong
Y
Zhang
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000627:therapeutic use; D017252:CD3 Complex / Q000276:immunology / Q000494:pharmacology; D019496:Cancer Vaccines / Q000276:immunology / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008545:Melanoma / Q000276:immunology*; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D019496:Cancer Vaccines; D000939:Epitopes; D015789:HLA-A2 Antigen; D010455:Peptides; D007770:L-Lactate Dehydrogenase