Supplemental Figures 1 - 8 from BRAFV600E Co-opts a Conserved MHC Class I Internalization Pathway to Diminish Antigen Presentation and CD8+ T-cell Recognition of Melanoma
Supplemental Figure 1. Three melanoma cell lines (CHL1, Mel888, and WM793) were treated with DMSO (0), 10uM, or 50uM of dabrafenib (BRAFi) for 3 hours. Whole cell lysates were prepared, and levels of phospho-ERK and total ERK were analyzed by immunoblotting. Supplemental Figure 2. Mel888 cells were treated with vehicle (DMSO) or 50 uM of BRAFi for 3 hours. Following treatment, cells were stained with fluorescently-labeled antibodies specific for MHC-I (HLA-A,B,C), MHC-II (HLA-DR), PD-L1, or melanoma-associated chondroitin sulfate proteoglycan (MCSP), and analyzed by flow cytometry. Data shown are representative of at least three replicate experiments with similar results. * indicates p < 0.05; ns, not significant. Supplemental Figure 3. HLA-A2-transduced Mel888 melanoma cell lines were treated with vehicle DMSO (0), 10uM, or 50uM of BRAFi for 3 hours. Whole cell lysates were prepared and levels of phospho-ERK and total ERK were analyzed by immunoblotting. Supplemental Figure 4. HLA-A2-transduced Mel888 or WM793 cells were treated with DMSO or MEKi for 3 hours. Following treatment, cells were stained with a fluorescently-labeled HLA-A2-specific antibody and analyzed by flow cytometry. These experiments were repeated at least four times with similar results. *** indicates p < 0.005; ns, not significant. Supplemental Figure 5. T2 cells were pulsed with titrated amounts of MART-1(27-35) peptide, washed, and then used as stimulator cells for MART1-specific CD8+ TILs. Supernatants were collected after 8 hours of co-culture and analyzed by ELISA to measure interferon-gamma (IFNgamma) release. Supplemental Figure 6. Percentage of intracellular IFNgamma-positive TILs following 3 hours of co-culture with DMSO- or BRAFi-treated HLA-A*0201-transduced Mel888 cells or MART-1(27-35) peptide-pulsed WM793 cells, as measured by flow cytometry. All data are representative of experiments performed at least 3 times with similar results. *, p<0.05; ns, not significant. Supplemental Figure 7. Mel888 cells were treated with 50 uM BRAFi for the indicated times, and cell lysates prepared. Cellular expression of MART-1 protein at each time point was assessed by immunoblotting and quantified using densitometry, with beta-actin levels measured as a loading control. Supplemental Figure 8. Mel888 cells were treated with DMSO, BRAFi, or MEKi for 2 hours and then labeled with 35S-Methionine for 20 min. Cells were placed back into culture at 37 C with continued exposure to drug or vehicle. Cells lysates were prepared at each of the indicated time points and total HLA-A2 was immunoprecipitated and analyzed using polyacrylamide gel electrophoresis. Immunoprecipitated HLA-A2 was then analyzed for 35S content by Phosphor-imaging.
