Fig. 3
Processing of NY-ESO-1 HLA-Cw3 restricted epitopes by melanoma cells expressing standard or immunoproteasomes. Selected melanoma cell lines were incubated in presence or absence of IFNγ for 72 h to induce expression of the immunoproteasome. Cells were plated in wells of a 96 well flat-bottom plate at 104/well. T-lymphocyte clones specific for NY-ESO-1 HLA-Cw3 restricted epitopes 92–100, 96–104, or 124–133 were added to each cell line at the effector:target (E:T) ratios shown (a–f) or at 5 × 104/well (g, h). Cytotoxicity assays, (a–f): Samples were incubated for 4 (a–d) or 24 h (e, f) at 37 °C. T-lymphocyte mediated cytotoxicity was determined by calcein release (a–d), or by MTS assay (e, f) and normalized to maximum /spontaneous lysis control wells. Asterisks in the IFNγ treated panels (b, d, f) indicate statistical significance of the change in killing between the equivalent cell line under steady state conditions (a,c,e). Intracellular cytokine stain, (g, h, i): Samples were incubated for 4 h in presence of brefeldin A. T-lymphocytes were incubated with fluorescent antibodies for CD3 and CD8 and stained intracellularly with anti-TNFα antibody, followed by FACS analysis. Error bars represent SEM (n = 3). Ns = not significant >0.05; * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001