Deregulation of signaling pathways involving phosphorylation is a hallmark of malignant transformation. Degradation of phosphoproteins generates cancer-specific phosphopeptides that are associated with MHC-I and II molecules and recognized by T-cells. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8 T-cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and HLA-matched primary leukemia cells ex vivo. Healthy individuals showed surprisingly high levels of CD8 T-cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients, which correlated with clinical outcome, and was restored following allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T-cell adoptive transfer immunotherapies..
Alterations in phosphorylation of cellular proteins are a hallmark of malignant transformation. Degradation of these phosphoproteins could generate cancer-specific class I MHC-associated phosphopeptides recognizable by CD8 ؉ T lymphocytes. In a comparative analysis of phosphopeptides presented on the surface of melanoma, ovarian carcinoma, and B lymphoblastoid cells, we find 5 of 36 that are restricted to the solid tumors and common to both cancers. Differential presentation of these peptides can result from differential phosphorylation of the source proteins. Recognition of the peptides on cancer cells by phosphopeptide-specific CD8 ؉ T lymphocytes validates the potential of these phosphopeptides as immunotherapeutic targets.tandem mass spectrometry ͉ immobilized metal-affinity chromatography
Although multiple components of the class I MHC processing pathway have been elucidated, the participation of nonproteasomal cytosolic enzymes has been largely unexplored. In this study, we provide evidence for multiple cytosolic mechanisms in the generation of an HLA-A*0201-associated epitope from tyrosinase. This epitope is presented in two isoforms containing either Asn or Asp, depending on the structure of the tyrosinase precursor. We show that deamidation of Asn to Asp is dependent on glycosylation in the endoplasmic reticulum (ER), and subsequent deglycosylation by peptide-N-glycanase in the cytosol. Epitope precursors with N-terminal extensions undergo a similar process. This is linked to an inability of ER aminopeptidase 1 to efficiently remove N-terminal residues, necessitating processing by nonproteasomal peptidases in the cytosol. Our work demonstrates that processing of this tyrosinase epitope involves recycling between the ER and cytosol, and an obligatory interplay between enzymes involved in proteolysis and glycosylation/deglycosylation located in both compartments.
CD8+ T cells have been identified as potent effectors of the adaptive anti-tumor immune response. So far, only a small number of tumor antigens have been objectively linked to the oncogenic processes. Alteration in phosphorylation status of cellular proteins is a hallmark of malignant transformation and a proven important therapeutic target. Phosphorylated antigens thus represent attractive targets for immunotherapeutic development based on the hypothesis that immune escape through mutation may be more difficult without compromising the malignant phenotype. Using a mass spectrometry approach phosphopeptide display was analyzed in CLL, AML, ALL, hairy cell leukemia and mantle cell lymphoma samples and compared against normal tissue. 109 or more primary tumor cells from HLA-A*0201+ or HLA-B*0702+ patients were lysed and peptides extracted from the surface HLA molecules and phosphopeptides isolated using in-line immobilized metal ion affinity chromatography and analyzed using tandem mass spectrometry using previously established methodology (Zarling A.L. et al PNAS 2006). 55 HLA-B*0702 phosphopeptides and 12 HLA-A*0201 phosphopeptides have been characterized. Many of these phosphopeptides are derived from phosphoproteins known to function in signaling cascades implicated in neoplastic transformation including MAP kinase pathway signaling proteins, c-Myc, NFAT and Bcl-11 with many representing novel phosphorylation sites. Peptide binding assays reveal IC50 binding affinities ranging between 4 and 300nM within the immunogenic range for the subset of phosphopeptides investigated. To investigated the mechanism of binding X-ray crystallography was performed in combination of peptide binding assays. Six HLA-phosphopeptide structures were resolved and demonstrate upward facing phosphate moiety directly interacting with HLA alpha helix residues yet with potential for direct recognition by the TCR. Peptide binding data support contributions from phosphate group in binding to the HLA in some but not all phosphopeptides tested and these data support a novel non-canonical binding mechanism for phosphopeptides complexed with HLA-A*0201. We have used mice transgenic for HLA-A*0201 and HLA-B*0702 to demonstrate in vivo immunogenicity of a subset of these peptides in a dendritic cell-based vaccination model. T cells generated differentially recognized phosphopeptides over non-phosphopeptide counterparts. Phosphopeptide-specific primary T cell lines were also generated ex-vivo from healthy lab donors which bound HLA-phosphopeptide tetramers and recognized HLA-matched primary tumor samples. In summary this work characterizes phosphopeptides that are differentially presented on primary leukemia and lymphoma samples by class I MHC molecules. These post-translationally modified peptide antigens represent distinct antigenic determinants which may overcome barriers of immune tolerance and autoimmunity inherent with other tumor antigens. These phosphopeptides therefore represent attractive novel candidates for future cancer immunotherapy.
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