Human ril 2

Cells were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 Medium (Invitrogen, Carlsbad, CA) supplemented with Golgi Stop (BD sciences, Le Pont de Claix, France). For ILC1 stimulation, 50 ng/ml of rat rIL-12 (Cat# 1760-RL) from R&D systems Europe (Lille, France) was used. For the ILC2 stimulation cocktail, human rTSLP, human rIL-33 and rat rIL-25 were purchased from Peprotech (Neuilly-sur-seine, France) and used at 50 ng/ml concentration. For ILC3 stimulation, 50 ng/ml of rat rIL-23 (Cat# 3136-RL) from R&D systems Europe (Lille, France), rat rIL-1β and human rTGFβ1 from Peprotech, human rIL-7 and 50 U/ml of human rIL-2 from Miltenyi Biotech (Paris, France), were used. PMA (Phorbol 12-myristate 13-acetate), Ionomycin, DNaseI, EDTA, SVF, Hank's balanced salt solution (HBSS), Collagenase D and Collagenase II were purchased from Sigma-Aldrich (Saint-Louis, MI); all reagents indicated are at the final concentration of use.

Human ex vivo CD127^loCD25⁺CD4⁺ Tregs were flow sorted using a BD FACSAria cell sorter (BD Biosciences) after staining with: CD39-BB515 (clone Tu66, BD), CD25-PE (clone MA251, BD) or CD25-PE (BD), CD4 APCFire/750 (Clone A161A1, Biolegend) or CD4-ECD (Clone SFCI12T4D11, Beckman Coulter), CD73-BV421 (clone AD2, Biolegend), CD8 V500 (RPA-T8, BD), CD3-BV605 (SK7, Biolegend), CD127-AF647 (clone HIL-7R-M21, BD) or CD127-PEcy7 (eBioRDR5, ebioscience/Thermofisher) and CD45RA-BV786 (clone Hi100, BD), then expanded with 500–1000 U/mL human rIL2 (Miltenyi Biotec/Chiron) ±100 nM rapamycin (Miltenyi Biotec) and human Treg expansion kit anti-CD3/CD28 beads (Miltenyi Biotec/Invitrogen), using x-vivo 15 cell medium (Lonza) or RMPI-1640 medium supplemented with L-glutamine, penicillin-streptomycin (both PAA Laboratories, UK), sodium pyruvate (Gibco, UK) and 2–10% human AB pooled serum and media exchanged with fresh IL-2 every 3–5 days. For additional experiments, either memory Tregs (mTregs) were sorted as CD4⁺CD25⁺CD127^loCD45RA⁻, naïve Tregs were sorted as CD4⁺CD25⁺CD127^loCD45RA⁺ or CD39⁻CD73⁻ double negative Tregs were sorted as CD4⁺CD25⁺CD127^loCD39⁻CD73⁻. Beads were removed using MACSibead magnet (Milteny Biotec) prior to phenotyping or use in functional assays. Example sorts shown in Supplementary Figs. 1a and 2b).