Anti cd39

Cryopreserved PBMC were isolated and used for immunophenotypic staining as previously described (108 (link)). Cells were stained with Zombie NIR fixable viability stain (BioLegend, San Diego, USA) and the following anti-human monoclonal fluorochrome-conjugated antibodies: anti-CD45RA, anti-CD4, anti-TCR-γ/δ (BD Biosciences, Heidelberg, Germany), anti-TCR-Vδ2 (Beckman Coulter Life Sciences, Indianapolis, USA), anti–HLA-DR, anti-CD27, anti-CD279 (PD-1), anti-TIGIT, anti-CD8, anti-CD28, anti-CD39, anti-CD38, anti-CD19, anti-CD3, anti-CD73 and anti-CD14 (all BioLegend) (Supplementary Table 2). Cells were incubated for 30 minutes at room temperature with the respective antibodies. After washing, cells were fixated with 4% paraformaldehyde. All samples were run on a Becton Dickinson LSR Fortessa flow cytometer with FACS Diva version 8 (BD Biosciences).

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.