Flow cytometry and cell sorting

Gating information for ILC progenitors and mature ILCs (NK, ILC1, ILC2, and ILC3) is described in table S1. Cells were stained with antibodies to surface antigens, such as CD25/3C7, CD45.2/104, CD45.1/A20, CD90.2/53-2.1, CD127/A7R34, Sca-1/D7, KLRG-1/2F1, α4β7/DATK32, Flt3/A2F10, c-kit/2B8, NKp46/29A1.4, IL-23R/12B2B64, and lineage antigens. The lineage-specific antibody cocktail (Lin) included antibodies to CD3ε, CD4, CD8, T cell receptor γδ, CD11b, CD11c, CD19, B220, Gr-1, NK1.1, and Ter119. For ILC1P and NKP cells, anti-NK1.1 was omitted. Cells were fixed and permeabilized with Transcription Factor Staining Buffer Kit (Tonbo Biosciences) for further staining for intracellular antigens (T-bet/eBio-4B10, GATA-3/TWAJ, RORγt/AFKJS-9, PLZF/9E12, Eomes/Dan11mag, Batf/D7C5, phospho-Stat3/D3A7, and/or phospho-Stat5/C11C5). Most of the antibodies were from BioLegend or eBioscience unless indicated otherwise. Antibodies to detect protein phosphorylation were from Cell Signaling Technology. Stained cells were acquired on a NovoCyte Flow Cytometer (ACEA Biosciences Inc.), and data were analyzed with FlowJo version 10.0.7 (FlowJo). For intracellular staining of cytokines, such as IL-22, IL-13, IL-5, and IFN-γ, cells, isolated from various tissues and stained for surface antigens, were activated with phorbol myristate acetate and ionomycin in the presence of monensin for 4 hours, and the cells were fixed in 1% paraformaldehyde and permeabilized with saponin buffer, followed by staining with antibodies to cytokines (BioLegend). For cell sorting, Lin⁻CD127⁺SCA-1⁺Flt3⁺ CLP and Lin⁻CD127⁺SCA-1⁺Flt3⁻α4β7⁺ αLP were sorted from BM cells and Lin⁻CD127⁺CD90⁺KLRG-1⁻ ILC3 was sorted (~95% pure) from intestinal lamina propria cells by a flow cytometry sorter (FACSMelody, BD Biosciences).