Rmil 7

FACS-isolated hepatic ILC2 (1 × 10⁴) were cultured in the presence of rmIL-33 (10 ng/ml), rmIL-25 (10 ng/ml), rmIL-1β (10–200 ng/ml; all BioLegend), rmIFNγ (10 ng/ml) and rmIL-12 (10–200 ng/ml; both R&D Systems, Wiesbaden, Germany) for 4 days. For ILC2 maintenance, all cultures were done in the presence of rmIL-2 (10 U/ml) and rmIL-7 (10 ng/ml; both R&D Systems). For CD4⁺ T-cell activation, hepatic ILC2 (2 × 10⁴) were co-cultured with FACS-isolated, OVA-specific CD4⁺ T cells (1 × 10⁵) or OVA-specific Th1 cells (1 × 10⁵) in the presence of OVA_323-339 peptide (5 µg/ml) for 4 days. For blocking IL-2 or IFNγ, co-cultures were done in the presence of an anti-IL-2 (JES-1A12; 10 µg/ml; BD Pharmingen) and anti-IFNγ (R4-6A2; 10 µg/ml; BioXCell, West Lebanon, NH) antibody, respectively.

For short-term culture of sorted γδ thymocytes, OP9-DL1 feeder cells were used. OP9-DL1 cells (gift from J.C. Zúñiga-Pflücker^{55 (link)}) were maintained at 30–80% confluency in OP9 medium (HEPES-buffered Glutamax MEM-α (Life Technologies) containing 20% FBS, penicillin/streptomycin, Na-pyruvate, gentamycin and β-mercaptoethanol) in 10 cc culture dishes (Nunc, Roskilde, Denmark). On the day before the experiments, OP9-DL1 cultures were set up by adding 3 × 10³ OP9-DL1 cells per well of a flat-bottom 96-well plate (Nunc, Roskilde, Denmark) and allowed to form monolayers overnight. For anti-CD3-treated cultures, the wells were coated with anti-CD3 prior to seeding OP9-DL1 cells by adding 5 µg per mL anti-CD3 antibody (145-2C11 clone; BioLegend) in PBS to each well and incubated for at least 4 h at 37 °C. The medium was removed, and 2–10 × 10³ sorted γδ thymocytes were suspended in 200 µL OP9 medium containing 5 ng per mL rmIL-7, and 5 ng per mL rhFlt-3L (R&D Systems Inc., Minneapolis, USA) and added to each well. OP9-DL1 co-cultures are known to induce artificial phenomena such as fate switching^{27 (link), 31 (link), 56 (link)}. To avoid these phenomena and limit the risk of selective outgrowth of minor populations, the cells were only cultured for 2 days before analysis by flow cytometry.