Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy

Florence R. Depontieu, Jie Qian, Angela L. Zarling, Tracee L. McMiller, Theresa M. Salay, Andrew Norris, A. Michelle English, Jeffrey Shabanowitz, Victor H. Engelhard, Donald F. Hunt, Suzanne L. Topalian

Research output: Contribution to journalArticlepeer-review

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-DRβ1*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.

Original languageEnglish (US)
Pages (from-to)12073-12078
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number29
DOIs
StatePublished - Jul 21 2009

Keywords

  • Tumor antigen
  • Tumor immunology

ASJC Scopus subject areas

  • General

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