Bdca2 pe

Purity of pDCs and mDCs after isolation and the phenotype of the pDC populations were determined by flow cytometry. The following primary monoclonal antibodies (mAbs) and the appropriate isotype controls were used: anti-BDCA1-FITC, BDCA2-PE, BDCA4-PE and CD123-APC (all Miltenyi Biotec); mIgG1-PE, mIgG1-APC, anti-CD11c-FITC or -APC, anti-HLA-ABC-PE (W6/32), anti-CD80-PE, or -APC, or -PeCy7, anti-CD86-PE, or -APC (all BD Bioscience Pharmingen, San Diego, CA, USA) anti-PD-L1-APC, anti-PD-L2-PE; anti-CD40-PE, anti-CD83-PE (Beckman Coulter, Mijdrecht, the Netherlands); anti-MHC-II-APC (eBioscience).
The phenotype of the DC populations after treatment with vemurafenib, dabrafenib, trametinib or a combination was determined by staining with the following antibodies and appropriate isotype controls: anti-CD80-PE-Cy7, anti-CD86 APC, anti-PD-L1-PE, anti-HLA-ABC-V450, anti HLA-DR-BV510, mIgG1-PE-Cy7, mIgG1-PE (all BD biosciences) and mIgG1-APC (eBioscience).

Venous blood was drawn into an ethylenediaminetetraacetic acid (EDTA) S-monovette (Sarstedt, Nümbrecht, Germany) for immunophenotyping. Whole blood (100 µl per flow cytometry panel) was directly stained in FACS tubes with fluorescence-labeled antibodies (BD-Biosciences, Franklin Lakes, United States: CD8-PerCP-Cy5.5, CD5-PE, CD45RA-PE-CF594, CD19-PE-Cy5, CD4-PE-Cy7, CD45RO-APC, CD38-A700, CD3-APC-H7, CD138-BV421, CD10-BV510, CD27-BV605, CD14-PE-CF594, CD19-PE-Cy5, CD25-PE-Cy7, CD16-A700, CD3-APC-H7, CD11b-BV421, HLADR-BV510; Biolegend, San Diego, United States: CD11c-BV605; Miltenyi Biotec, Bergisch-Gladbach, Germany: BDCA1-APC, BDCA2-PE) for 15 min in the dark at room temperature (RT). After washing, red blood cell (RBC) lysis was performed with 2 ml ACK lysing buffer (Thermo Scientific, Waltham, United States) for 7 min in the dark at RT. 2 ml of FACS buffer (phosphate buffered saline, 3% fetal calf serum, 1% sodium azide) was added to wash the samples twice. Subsequently, cells were resuspended in 400 µl FACS-buffer. After the staining procedure, cells were measured by flow cytometry (LSR Fortessa cytometer, BD Biosciences, Franklin Lakes, United States) and analyzed with FACS-Diva Software (BD Biosciences, Franklin Lakes, United States). The gating strategy is available in the supplementary data section of this paper (Supplementary Figure S7).

PBMCs were stimulated with IL‐29 (500 ng/ml) or IFN‐α (500 ng/ml, both from Peprotech) or left unstimulated for 20 min at 37°C. Cells were harvested, washed with ice‐cold staining buffer, blocked with Fc blocking reagent, and surface stained with BDCA‐2 PE (both from Miltenyi Biotec) and CD123 FITC (Thermo Fisher Scientific) for 20 min at 4°C. Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with PERM Buffer III (BD Biosciences) according to the manufacturer's protocol and stained for 30 min with anti‐pSTAT1(pY701) Alexa Fluor 647 (4a, BD Biosciences). Cells were washed twice with staining buffer and immediately analyzed by flow cytometry. For total STAT1 protein expression, unstimulated PBMCs were analyzed using the total STAT1 ELISA kit (Abcam, Cambridge, UK) according to the manufacturer's instructions.