title: Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy.
abstract: The activation and recruitment of CD4(+) T cells are critical for the development of efficient antitumor immunity and may allow for the optimization of current cancer immunotherapy strategies. Searching for more optimal and selective targets for CD4(+) T cells, we have investigated phosphopeptides, a new category of tumor-derived epitopes linked to proteins with vital cellular functions. Although MHC I-restricted phosphopeptides have been identified, it was previously unknown whether human MHC II molecules present phosphopeptides for specific CD4(+) T cell recognition. We first demonstrated the fine specificity of human CD4(+) T cells to discriminate a phosphoresidue by using cells raised against the candidate melanoma antigen mutant B-Raf or its phosphorylated counterpart. Then, we assessed the presence and complexity of human MHC II-associated phosphopeptides by analyzing 2 autologous pairs of melanoma and EBV-transformed B lymphoblastoid lines. By using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing, and comparative analysis, a total of 175 HLA-DR-associated phosphopeptides were characterized. Many were derived from source proteins that may have roles in cancer development, growth, and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome "fingerprints." We then generated HLA-DRbeta1*0101-restricted CD4(+) T cells specific for a phospho-MART-1 peptide identified in both melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen-presenting cells as well as for intact melanoma cells. This previously undescribed demonstration of MHC II-restricted phosphopeptides recognizable by human CD4(+) T cells provides potential new targets for cancer immunotherapy.
authors: Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
authors: Florence R
authors: FR
authors: Depontieu
authors: Jie
authors: J
authors: Qian
authors: Angela L
authors: AL
authors: Zarling
authors: Tracee L
authors: TL
authors: McMiller
authors: Theresa M
authors: TM
authors: Salay
authors: Andrew
authors: A
authors: Norris
authors: A Michelle
authors: AM
authors: English
authors: Jeffrey
authors: J
authors: Shabanowitz
authors: Victor H
authors: VH
authors: Engelhard
authors: Donald F
authors: DF
authors: Hunt
authors: Suzanne L
authors: SL
authors: Topalian
mesh_terms: D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D006684:HLA-DR Antigens / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D007167:Immunotherapy; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000276:immunology; D010748:Phosphopeptides / Q000737:chemistry / Q000276:immunology*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics
chemical_list: D000951:Antigens, Neoplasm; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D010748:Phosphopeptides; D048493:Proto-Oncogene Proteins B-raf