Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy.
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.
Depontieu
Florence R
FR
Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
Qian
Jie
J
Zarling
Angela L
AL
McMiller
Tracee L
TL
Salay
Theresa M
TM
Norris
Andrew
A
English
A Michelle
AM
Shabanowitz
Jeffrey
J
Engelhard
Victor H
VH
Hunt
Donald F
DF
Topalian
Suzanne L
SL
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
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