Stat5 inhibitor

For in vitro ILC differentiation, sorted CLPs or αLPs (1 × 10⁶ cells/well) were co-cultured with OP9-DL1 cells (pre-treated with 50 ng/ml mitomycin C for 25 min) in completed DMEM in the presence of cytokines in 6-wells 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 6-well plates. Half of the culture medium was replaced every 3 days, and cultured cells were harvested on day 10–12. When indicated, Stat3 inhibitor VI (100 μM, Santa Cruz), Stat5 inhibitor (200 μM, Cayman Chemical), and Stat6 inhibitor (AS1517499, 250 nM, Cayman Chemical) were added to culture.

All recombinant cytokines were obtained from Peprotech except for hIL-12, mIL-12, TSLP, GM-CSF, TL1-A, and hTGF-β (R&D). Details of antibody clones and validation are provided in Source Data. Tofacitinib, FK506, BAY11, CAY10571, STAT5-INHIBITOR, and STAT6 inhibitor AS1517499 were purchased from Cayman and reconstituted in DMSO to 100 mM, 10 mM, 50 μM, 100 mM, 50 μM, and 50 nM stock concentrations, respectively.