Single-Cell Immune Phenotyping Protocol

Single-cell suspensions were stained with antibodies against surface molecules (Supplementary Table 2) at RT for 30 min. For intracellular staining, cells were stimulated with cell stimulation cocktail (plus protein transport inhibitors) containing PMA, ionomycin, brefeldin A and monensin (1:500 dilution, eBioscience Cat#00-4975-93) at 37^oC in RPMI media containing 10% heat-inactivated FBS (R&D systems, Cat#S11150H) and 0.05 mM 2-mercaptoethanol (ThermoFisher Scientific, Cat#21985023) for 4 hr prior to staining with Zombie NIR (BioLegend, Cat#423105). Next, cells were stained with antibodies against surface proteins followed by fixation and permeabilization and staining with antibodies against intracellular proteins. Samples were analyzed by a BD LSRFortessa SORP equipped to detect 17 or 15 fluorescent parameters. Compensation and data analysis were carried out using FACSDiva and FlowJo software. Gating strategy is shown in Supplementary Figs. 8 and 9.