Fig. 5

Processing of NY-ESO-1 HLA-Cw3 restricted epitopes following LMP7 inhibition in melanoma cell lines. The enzymatic activity of the immunoproteasome subunit LMP7 was inhibited by incubation of melanoma cell lines with the small molecule inhibitor PR-924, in presence or absence of IFNγ. Following 72 h, T-lymphocytes were added and activation (a–c) or cytoxicity (d–f) was assessed for each of the treatment conditions. a–c T-lymphocytes were incubated with melanoma cells for 4 h in presence of Brefeldin A and cytokine secretion was determined by intracellular staining, followed by FACS analysis. d–f T-lymphocytes were incubated with melanoma cells at a 1:1 effector:target ratio for 24 h at 37 °C, then washed off. T-lymphocyte mediated cytotoxicity was determined by MTS assay and normalized to control wells with no T-lymphocytes. Error bars represent SEM (n = 3). ns = not significant >0.05; * < 0.05; *** < 0.001