Expression Hierarchy of T Cell Epitopes from Melanoma Differentiation Antigens: Unexpected High Level Presentation of Tyrosinase-HLA-A2 Complexes Revealed by Peptide-Specific, MHC-Restricted, TCR-Like Antibodies

Abstract

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8؉ T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes

“…The physiological significance of this study may be correlated with the findings of Michaeli et al (59), who demonstrated that a tyrosinase-derived class I HLA-A2 peptide is presented at several thousand complexes/cell. In light of our results, such a dose induces anergy and tolerance, which might explain the low frequency of tyrosinase-specific CTLs in tumor-infiltrating lymphocytes isolated from melanoma patients (60).…”

Melanoma cells present high levels of HLA‐A2‐tyrosinase in association with instability and aberrant intracellular processing of tyrosinase

“…This observation was confirmed using immunological and biochemical tools such as mass spectrometry, flow cytometry, immunohistochemistry, and microscopy and using a unique molecular tool that is a specific anti-T cell receptor-like (TCR-like) antibody. This antibody binds the HLA-A2 and the Tyr (369)(370)(371)(372)(373)(374)(375)(376)(377) peptide in a way that mimics T-cell specificity [12].…”

“…Other studies have measured the degradation rate of total protein in the cell and did not investigate the exact link between the processing degradation rate of a single protein and the presentation level of peptides derived from that protein. Here, for the first time, we show the relationship between native processing of an endogenous, wild-type, protein in tumor cells and the presentation level of peptides derived from the same protein.We have recently shown that HLA-A2-Tyr (369)(370)(371)(372)(373)(374)(375)(376)(377) complexes are present on the surface of cells with thousands of copies [12]. In the current study, we examined the mechanisms that contribute to this high presentation by comparing the intracellular processing of tyrosinase in cells that present different levels of the HLA-A2-Tyr (369-377) complex, and monitoring HLA-A2-Tyr (369-377) presentation levels with anti-HLA-A2-Tyr (369-377) TCR-like antibody.…”

“…This observation was confirmed using immunological and biochemical tools such as mass spectrometry, flow cytometry, immunohistochemistry, and microscopy and using a unique molecular tool that is a specific anti-T cell receptor-like (TCR-like) antibody. This antibody binds the HLA-A2 and the Tyr (369)(370)(371)(372)(373)(374)(375)(376)(377) peptide in a way that mimics T-cell specificity [12].Notably, most studies evaluating the source of MHC-restricted peptides were performed by transfection [13,14], thereby not addressing the native processing of the endogenous protein. In the current study, we used a TCR-like antibody specific to HLA-A2-Tyr (369)(370)(371)(372)(373)(374)(375)(376)(377) as a direct molecular tool to evaluate the level of Tyr (369)(370)(371)(372)(373)(374)(375)(376)(377) peptide presentation.…”

“…a shorter half-life) than in low HLA-A2-Tyr (369)(370)(371)(372)(373)(374)(375)(376)(377) presenting cells. Furthermore, increasing the folding efficiency and stability of tyrosinase with L-DOPA, a cofactor of tyrosinase that stabilizes the protein [10], has previously been shown to decrease the number of HLA-A2-Tyr (369)(370)(371)(372)(373)(374)(375)(376)(377) complexes presented on melanoma cells [12] and our findings, in accordance with other studies [10], revealed that adding L-DOPA led to a correct intracellular transport of tyrosinase suggesting that protein processing and stability plays a major role in the presentation of MHC class I-restricted peptides.Our results suggest that proteins with distinct intracellular localization and stability produce MHC-restricted peptides with different efficiencies. Multiple steps in the MHC class I processing machinery may be influenced by protein characteristics such as glycosylation patterns, folding states, etc.…”