Fig. 5

Surface MHC class I expression and immunogenicity of melanoma cells is augmented by RSK inhibition. A, B MHC class I protein levels and their localization at the cell surface of melanoma cells following RSK inhibitor treatment for 72 h. Immunoblot analysis was performed on whole cell protein lysates using Vinculin as loading control (A). Cell surface staining was conducted with FITC-coupled HLA-ABC- or APC-coupled HLA-A*02-specific antibodies, quantified by flow cytometry (mean fluorescence intensity, MFI) and normalized to the respective solvent control (mean ± SD, N ≥ 2) (B). Significance was determined by unpaired t-tests and subsequent Holm-Šídák’s multiple comparisons test. C Schematic workflow for non-adherent co-culture experiments with HLA-A*02-restricted gp100-TCR or Melan-A-TCR T cells and signaling pathway inhibitor pre-treated melanoma cells. D, E T-cell activation assessed by IFNγ secretion (D) and CD25 surface staining of gp100- or Melan-A-specific T cells (E) after 24 h co-culture of T cells with RSK inhibitor pre-treated melanoma cells (PMD-026, 72 h). Basal T-cell activation was assessed using the respective T cells without melanoma cell co-culture. Significance was assessed by one-way ANOVA with Dunnett’s correction for multiple comparison (mean ± SD; N ≥ 2). F AnnexinV-/ 7-AAD-based cell death staining of the RSK inhibitor pre-treated melanoma cells with and without 24 h co-culture with gp100- or Melan-A-specific T cells. The reduction of viable cell fraction (AnnexinV-negative / 7-AAD-negative population) compared to the control-treated melanoma cells without T-cell co-culture is indicated and significance was evaluated by two-way ANOVA with subsequent Bonferroni’s multiple comparisons test (mean ± SD; N ≥ 2)