OP9-DL1 culture for ILCs

For in vitro ILC differentiation, sorted CLPs or αLPs (1 × 10⁶ cells per well) were cocultured with OP9-DL1 cells [pretreated with mitomycin C (50 ng/ml) for 25 min] in complete Dulbecco’s minimum essential medium (DMEM) in the presence of cytokines in six-well plates. Recombinant murine IL-7, stem cell factor (SCF), and IL-15 (all 20 ng/ml) were used for ILC1 differentiation. IL-7, SCF, and IL-33 were used for ILC2 differentiation. IL-7, SCF, and IL-23 were used for ILC3 differentiation in six-well plates. Half of the culture medium was replaced every 3 days, and cultured cells were harvested on days 10 to 12. When indicated, Stat3 inhibitor VI (100 μM; Santa Cruz Biotechnology), Stat5 inhibitor (200 μM; Cayman Chemical), and Stat6 inhibitor (250 nM; Cayman Chemical, AS1517499) were added to the culture.