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Native gp100, a glycoprotein highly expressed in the majority of melanomas, contains several immunogenic peptides that are recognized by cytotoxic lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I molecules. The objective of this study was to evaluate the ability of dendritic cells (DCs) from melanoma patients to take up gp100 protein and stimulate specific autologous CTL. The gp100 used in this study was a recombinant molecule with diminished hydrophobicity, HR-gp100, produced in Escherichia coli bacteria and in Pichia pastoris yeast. Stimulation of CD8+ T cells from melanoma patients with HR-gp100-loaded DC was visualized by confocal microscopy using stained target cells, and was quantitatively measured by the production of IFN-gamma using an ELISPOT assay. The results showed that HR-gp100 protein, produced either in bacteria or in yeast, when loaded on DC from melanoma patients, stimulated autologous CD8+ lymphocytes. By direct visualization, these lymphocytes were found in close contact with dead melanoma cells, and to contain membrane material transferred from stained melanoma cells; in cultures containing control lymphocytes stimulated with unloaded DC, no melanoma cell killing was observed. In ELISPOT assays, increased number of IFN-gamma-producing CD8+ T lymphocytes from patients, but not from healthy controls, were measured upon stimulation with HR-gp100-loaded DC. HR-gp100 could represent a useful tool to load DC with multiple immunogenic epitopes/antigen-derived epitopes for the immunotherapy of melanoma.