RESUMO
Deregulation of signaling pathways is a hallmark of malignant transformation. Signaling-associated phosphoproteins can be degraded to generate cancer-specific phosphopeptides that are presented by major histocompatibility complex (MHC) class I and II molecules and recognized by T cells; however, the contribution of these phosphoprotein-specific T cells to immune surveillance is unclear. 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 human leukocyte antigen-matched primary leukemia cells ex vivo. Notably, healthy individuals showed robust 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. This reduction correlated with clinical outcome; however, immunity was restored after 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.
Assuntos
Imunidade/imunologia , Leucemia/imunologia , Complexo Principal de Histocompatibilidade , Fosfopeptídeos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Humanos , Linfócitos T/imunologiaAssuntos
Proteínas de Ciclo Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Quinases Ativadas por p21/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Fator de Crescimento Epidérmico/farmacologia , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Neoplasias/enzimologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Fatores de Troca de Nucleotídeo Guanina Rho , Quinases Ativadas por p21/químicaRESUMO
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.
Assuntos
Aminopeptidases/metabolismo , Apresentação de Antígeno , Citosol/enzimologia , Retículo Endoplasmático/metabolismo , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Monofenol Mono-Oxigenase/imunologia , Peptídeo Hidrolases/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Retículo Endoplasmático/enzimologia , Glicosilação , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Antígenos de Histocompatibilidade MenorRESUMO
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.