For intracellular staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA; final concentration 25 ng/mL; Merck, Darmstadt, Germany) and ionomycin (final concentration 1 µg/mL; Merck) for 18 hours. After 2 hours, Brefeldin A (5µg/mL; Merck) and Monensin (1µg/mL; Merck) were added. First, surface antigens were stained as described above. Cells were then permeabilized with fixation/permeabilization solution (Cytofix/Cytoperm; BD Biosciences) and stained with fluorochrome-conjugated antibodies for 30 minutes at 4°C. The following anti-human monoclonal antibodies were used: anti-IL-2, anti-CD4, anti-IL-10, anti-TCR-γ/δ (BD Biosciences), anti-TCR-Vδ2 (Beckman Coulter Life Sciences), anti–IFN-γ, anti-TNF-α, anti-CD8, anti-TGF-β, anti-CD39, anti-Granzyme-B, anti-CD19, anti-CD3, anti-CD73, and anti-CD14 (all BioLegend), see also Supplementary Table 3
For kinetic studies of CD39 surface expression, cells were stimulated as previously described with small adaptations (109 (link)). Briefly, cryopreserved PBMC were plated into 48-well plates and stimulated with rhIL-2 (20 U/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), PMA (5 ng/mL), ionomycin (0,5 µg/mL), anti-CD3/CD28-Dynabeads (ratio 1:1; ThermoFisher Scientific, Waltham, USA) or combinations thereof. Cells were cultured for up to 6 days before FACS analysis.
For kinetic studies of CD39 surface expression, cells were stimulated as previously described with small adaptations (109 (link)). Briefly, cryopreserved PBMC were plated into 48-well plates and stimulated with rhIL-2 (20 U/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), PMA (5 ng/mL), ionomycin (0,5 µg/mL), anti-CD3/CD28-Dynabeads (ratio 1:1; ThermoFisher Scientific, Waltham, USA) or combinations thereof. Cells were cultured for up to 6 days before FACS analysis.