Ril 7

Foxp3^eGFP reporter mice were treated with 100 ng of rmIL-33 (R&D Systems) i.n. every 3 days for 2–3 weeks. Lin⁻ CD45⁺CD90⁺CD25⁺IL-33R⁺ ILC2s and/or CD45⁺CD3⁺CD5⁺CD4⁺Foxp3⁻IL-33R⁺ T_H2 cells were sort-purified from the lung and rested for 18 h in DMEM Complete Media (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Denville Scientific), 1% l-glutamine (GIBCO), 1% penicillin/streptomycin (GIBCO), 25 mm HEPES buffer, and 55 μm 2-β-mercaptoethanol (Sigma-Aldrich)) on ice. For Arg1 inhibitor studies only, cells were then cultured for additional 24 hin DMEM Complete Media with 20 ng/ml rIL-2, 20 ng/ml rIL-7 and 50 ng/ml rIL-33 (all cytokines from R&D Systems) at 37°C. Cells were plated at 200,000 cells per well and OCR and ECAR measured in XF media (non-buffered RPMI 1640 containing 25 mM glucose, 2 mM l-glutamine, and 1 mM sodium pyruvate) under basal conditions, in response to 1 μM oligomycin, 1.5 μM fluoro-carbonyl cyanicide phenylhydrazone (FCCP) and 100 nM rotenone + 1 μM antimycin A (Sigma-Aldrich) and as indicated after DMSO or 500 μM nor-NOHA injection using a 96 well Extracellular Flux Analyzer (Seahorse Bioscience).

Lin⁻CD45⁺Thy1⁺Red5⁺ ILC2s from lung and skin were FACS sorted and 2500 (lung) or 1000 (skin) cells per well were cultured in RPMI-1640 (Sigma) containing 10% FBS, 10 mM HEPES (Sigma), 100 μM non-essential amino acids (Sigma), 1 mM sodium pyruvate (Gibco), 100 μU/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 50 μM 2-mercaptoethanol (Gibco), 10 ng/ml rIL-7 (R&D Systems), 10 ng/ml rIL-2 (R&D Systems), and 10 ng/ml rIL-33 (BioLegend) in 96-well round bottom plates at 37 °C under 5% CO₂. After 3 days, supernatants were collected and the concentration of CXCL2 was measured by ELISA (ThermoFisher, EMCXCL2).