Flow cytometry

For staining, cells were stained with Zombie Aqua Viability Dye (#423102, BioLegend, San Diego, CA) in PBS for 10 min on ice, then Fc receptors were blocked with Human BD Fc Block™ from BD Biosciences (#564219, BD Biosciences, San Jose, CA) for an additional 10 min. After centrifugation, the supernatant was removed and cells were stained with a surface antibody cocktail containing in FACS buffer (PBS, 2 mM EDTA, 2% FBS) and, if needed, Brilliant Stain Buffer Plus from BD Biosciences (#566385) for 20 min on ice. After surface staining, cells were washed in FACS buffer and fixed for 20 min on ice with Fixation/Permeabilization Buffer from BD Biosciences (#554722). For intracellular cytokine analysis, single cell suspensions were incubated in RPMI + 5% HS and 10 ug/mL GolgiPlug from BD Biosciences (#555029) at 37 °C for 4 h. Cells were then stained as described above and afterwards cells were incubated with intracellular antibodies for 30 min on ice. All samples were resuspended in FACS buffer and acquired on a BD Fortessa or a BD LSRII flow cytometer. Data was analyzed using FlowJo software from Tree Star, v10.5.

The following antibodies were purchased from BioLegend (San Diego, CA); CD25-BV711 (M-A251), CD107a-PE (H4A3) and TNF-α BV605 (Mab11). CD11c-PE/Cy7 (B-ly6), IFN-γ BV650 (B27), CD25-APC (M-A251), CD69 APC Cy7 (FN50), Granzyme-B BV421 (GB11), CD14-AF700 (M5E2), Perforin PE-CF594 (δG9), Vγ9-PE (B3), CD38-PE (HIT2), CD40-PE (5C3), CD86-APC (2331), CD40L-PE/Dazzle™ 594 (24–31) and HLA-DR-V450 (G46-6) were obtained from BD Biosciences (San Jose, CA). TCR Vδ2-FITC (123R3) was obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) and CD3-AF700 (UCHT1) was obtained from Thermo Fisher Scientific, Waltham, MA.