Article Figures & Data
Figures
- Figure 1.
MAPK pathway inhibition induces rapid upregulation of MHC class I surface expression in BRAF-mutant melanoma cell lines. A, flow-cytometric analysis showing HLA-A, B, and C surface expression in human dermal melanocytes transduced with lentiviral vectors expressing BRAFV600E and GFP or GFP alone. B, summary of MHC-I expression in three melanocyte lines, each transduced in triplicate; MFI, mean fluorescence intensity. C, melanoma cell lines harboring V600E-mutated BRAF (Mel888 and WM793) or WT BRAF (CHL1) were treated for 3 hours with either DMSO, dabrafenib, 50 nmol/L (BRAFi), or trametinib, 50 nmol/L (MEKi), then stained for total HLA-A, B, and C and analyzed by flow cytometry. D, confocal microscopy images of DMSO- or BRAFi-treated Mel888 and CHL1 cells following staining with fluorescently labeled HLA-A, B, and C–specific antibody. E, quantification of confocal imaging, with MHC-I surface expression expressed as mean pixel counts for at least 40 imaged cells within each treatment group. F, time-course experiment showing total MHC-I upregulation in Mel888, WM793, and CHL1 melanoma cells treated with dabrafenib for 30 minutes, 60 minutes, or 3 hours, as measured by flow cytometry. All experiments were performed at least four times with similar results; **, P < 0.01; ns, not statistically significant.
- Figure 2.
Modulation of MHC-I expression by the MAPK pathway is mediated by conserved MHC-I tail phosphorylation site Serine-335. A, Western blot analysis showing HLA-A*0201 immunoprecipitated from Mel888 and WM793 melanoma cells lentivirally transduced to express WT HLA-A*0201 or one of three cytoplasmic tail mutants of HLA-A*0201: ΔTail (ΔT), Y320A, or S335A. B, transduced Mel888 and WM793 melanoma cells were treated for 3 hours with either DMSO or dabrafenib, 50 nmol/L (BRAFi), then stained for HLA-A*0201 and analyzed by flow cytometry. C, quantification of BRAFi-induced changes in HLA-A*0201 surface expression, as measured by flow cytometry; MFI, mean fluorescence intensity. D, 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-A1–specific antibody and analyzed by flow cytometry. E, IFNγ release by MART-1–specific CD8+ TILs during 8 hours of coculture with HLA-A*0201–transduced Mel888 cells pretreated for 3 hours with DMSO or dabrafenib, 50 nmol/L, as measured by ELISA. F, the percentage of intracellular IFNγ-positive TILs following 3 hours of coculture 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 from representative experiments performed at least three times with comparable results; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, not statistically significant.
- Figure 3.
BRAFV600E promotes rapid MHC-I internalization and intracellular sequestration that is reversible with MAPK pathway inhibition. A, WM793 and CHL1 melanoma cells were surface-labeled with Alexa488-conjugated HLA-A, B, and C–specific mAb (green, 1° label) and then cultured at 37°C. At the indicated time points, cells were stained with an Alexa568-conjugated secondary antibody (red, 2° label) to double label the remaining cell surface MHC-I (yellow), and analyzed by confocal microscopy. White arrows indicate MHC-I (green) that was internalized during time in culture. B, same experiment as in A, except WM793 cells were pretreated with either DMSO or dabrafenib, 50 nmol/L for 2 hours before HLA-A, B, and C surface labeling, and then cultured at 37°C for 90 minutes with continued exposure to DMSO or BRAFi. C, time course of MHC-I surface expression in Mel888 (BRAFV600E) or MeWo (BRAF WT) melanoma cells following removal of dabrafenib from cell culture medium, as measured by flow cytometry. D, confocal images showing cellular distribution of total HLA-A, B, and C in permeabilized WM793 cells following a 2-hour treatment with DMSO or dabrafenib. White arrows indicate increased cell surface localization following BRAFi treatment. E, double staining of total MHC-I (green) and the endolysosomal marker LAMP-1 (red), with white color indicating overlap of the two markers. F, the percentage of pixels showing green/red (MHC-I and LAMP-1) colocalization, quantified from at least 40 imaged cells within each treatment group. All data are from representative experiments performed at least three times with comparable results; *, P < 0.05.
- Figure 4.
The proposed model of melanoma cell MHC class I trafficking in the context of MAPK pathway activation or inhibition. Oncogenic activating BRAF mutations (e.g., V600E) in melanoma cells drive the rapid and constitutive internalization of MHC-I molecules from the cell surface and their subsequent sequestration within LAMP1-positive endolysosomal compartments (left). This redistribution process requires the conserved Serine-335 phosphorylation site (S) found within the cytoplasmic tail of MHC-I. Pharmaceutical inhibition of MAPK signaling can inhibit MHC-I internalization, thus altering the equilibrium and resulting in reduced endolysosomal sequestration and enhanced MHC-I surface expression within hours (right). This augmented MHC-I surface expression in turn leads to increased tumor cell recognition and function of melanoma antigen-specific CD8+ T cells.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplemental Figures 1 - 8 - 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.
- Supplemental Methods and Figure Legends - Supplemental Methods and Figure Legends
Volume 3, Issue 6
