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 tumorderived 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-DR␤1 * 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.tumor antigen ͉ tumor immunology

Phosphorylated alpha-enolase induces autoantibodies in HLA-DR8 pancreatic cancer patients and triggers HLA-DR8 restricted T-cell activation

“…Synthetic peptides were chosen as Ser 419 -ENOA phosphorylation seems to be pancreatic specific [3,18], suggesting that ENOA would not be properly phosphorylated when expressed into EBV-transformed cells. As reported in previous studies in which T cells could discriminate phosphopeptides from their unphosphorylated counterparts [9,10,11,12], the HLA-DR8-restricted CD4 + T cell clones were specific for the phosphate moiety of the ENOA-derived peptide presented in the context of HLA-DR8. The crystal structure of phosphopeptide-MHC complexes shows, in fact, that the phosphomoiety is exposed to the TCR, suggesting a direct interaction between the phosphorylated residue and the TCR [8,17,26].…”

“…Among the post-translationally modified peptides identified to date, phosphopeptides are of particular interest, as deregulated phosphorylation is one of the major hallmarks of malignant transformation. T cell discrimination of phosphopeptides versus their unphosphorylated counterparts has also been observed, indicating that phosphorylation can influence peptide immunogenicity [9,10,11,12]. Depending on the position within the peptide, phosphorylation tends to stabilize the complex, without greatly altering the peptide-binding mode.…”

Heat Shock Protein 90-Mediated Peptide-Selective Presentation of Cytosolic Tumor Antigen for Direct Recognition of Tumors by CD4+ T Cells

“…In previous studies of endogenous MHC-II presentation, intracellular viral Ags or ectopically expressed model Ags were used, for which specific CD4 + T cells have strong TCR affinity and where Ag expression is relatively high. In contrast, TCR affinity for self or tumor Ags, except for mutated Ags, breakpoint of fusion proteins, or Ags with tumor-specific modification such as phosphorylation (32), is considered to be lower because of the expression of these Ags in the thymus (33). Therefore, analyses of tumor-recognizing CD4 + T cells are required for the evaluation of the role of previously characterized pathways in the tumor immunosurveillance by tumor-recognizing CD4 + T cells.…”

Interleukin-2 Receptor β Thr-450 Phosphorylation Is a Positive Regulator for Receptor Complex Stability and Activation of Signaling Molecules

“…Importantly, IL-2 also induces threonine phosphorylation of the receptor. Previously, we have shown induction of IL-2R␤ threonine phosphorylation in response to IL-2 stimulation and CA treatment via phosphoamino acid analysis (18), whereas other groups identified Thr-79, Thr-256, Thr-507, and Thr-522 as phosphosites through proteomic discovery mode mass spectrometry (20,57,58), although no site-specific characterization was performed. This work indicates that phosphorylation of IL-2R␤ Thr-450 provides an additional positive regulatory mechanism that modulates IL-2 signaling.…”