Ifn α2b

Thawed cells were labeled with viability dye in PBS for 20 min at 37°C, followed by surface staining for 15 min at room temperature in PBS 2% FBS. After stimulation with IFN-α2b 40.000 IU/ml (Miltenyi Biotec) for 15 min at 37°C, cells were fixed and permeabilized using Cytofix Fixation Buffer (BD Biosciences) pre-warmed to 37°C and Perm Buffer III (BD Biosciences) pre-cooled to −20°C, according to the instructions of the manufacturer. Fc Block (Human TruStain FcX, Biolegend) in PBS was used to prevent the unwanted binding of Fc receptor CD16 antibody. Finally, intracellular staining was performed for 30 min at room temperature in PBS with 0.5% BSA. The following panel was used: Viability Dye Aqua Brilliant Violet 510 (L34957 A+B, Invitrogen), CD3 APC-Cy7 (317341, Biolegend), CD19 APC-eFluor780 (47-0199-42, Invitrogen), CD56 APC-eFluor780 (47-0567-42, eBiosciences), CD14 BB700 (566466, BD Biosciences), CD16 Alexa Fluor 647 (557710, BD Biosciences), HLA-DR PE-CF594 (562304, BD Biosciences), and phospho-STAT1 Brilliant Violet 421 (566238, BD Biosciences).

For the analysis of the protein levels of selected ISGs (ISG15, PKR, and MX1), PBMCs were thawed at 37°C, counted, and seeded in a 96-well plate, and then analyzed directly or stimulated overnight at 37°C with or without IFN-α2b 40.000 IU/ml (Miltenyi Biotec). After stimulation, cells were labeled with a viability dye for 30 min at room temperature in PBS, followed by surface staining in PBS 2% FBS for 20 min at 4°C. Cells were then fixed and permeabilized using the FOXP3/Transcription Factor Fixation/Permeabilization Buffers (eBioscience) according to the instructions of the manufacturer. Fc Block (Human TruStain FcX, Biolegend) in PBS was used to prevent the unwanted binding of Fc receptor CD16 antibody. Finally, intracellular staining was performed for 30 min at room temperature in Permeabilization Buffer (eBioscience). The flow cytometry panel was the following: Viability Dye eFluor780 (65-0865-14, Invitrogen), CD14 Brilliant Violet 421 (563743, BD Biosciences), MXA Alexa Fluor 488 (AMab237298, Abcam), PKR Alexa Fluor 647 (AMab224921, Abcam), and ISG15 PE (IC8044P, R&D Systems).