Adult lung IL-18Rα⁺ ILCs have similar differentiation properties as BM ILCPs. BM ALPs (Lin⁻Flt3⁺CD127⁺Ly6D⁻CD25⁻Thy1⁻), ILCPs (Lin⁻Thy1⁺CD127⁺PD-1⁺α4β7⁺CD25⁻), or lung IL-18Rα⁺ ILCs (Lin⁻YFP⁺Thy1⁺CD127⁺IL-18Rα⁺ST2⁻) were purified by from papain-treated RORα-YFP mice (CD45.2⁺) and injected (2,000 cells per mouse) intravenously into lethally irradiated Pep3b mice (CD45.1⁺). The tissues of the recipient mice were analyzed 6 wk after transplantation. (A) Donor-derived lung ILC2s were sequentially gated by CD45.1⁻CD45.2⁺Lin⁻T-bet⁻Thy1⁺ST2⁺CD127⁺GATA-3⁺ (pink gates). The Lin cocktail in this analysis included anti-CD3e, TCRαβ, TCRγδ, CD19, NKp46, CD11b, CD11c, Gr-1, and Ter119. Donor-derived Lin⁺T-bet⁺ cells were also gated and analyzed for Thy1 and CD127 expression. (B) Donor-derived liver cells were gated by CD45.1⁻CD45.2⁺Lin⁻NKp46⁺ and further divided into T-bet⁺Eomes⁻ ILC1s and T-bet⁺Eomes⁺ NK cells. The Lin cocktail in this analysis included anti-CD3e, TCRαβ, TCRγδ, CD19, Gr-1, and Ter119. (C) Absolute numbers of donor-derived lung ILC2s, liver ILC1s, and NK cells. (D) BM ILCPs or lung IL-18Rα⁺ ILCs were purified as in A, and 1,000 cells each were cultured on OP9-DL1 with IL-7 and SCF for 1 wk. The progenies were analyzed for Thy1 and NKp46 expression. Thy1⁺NKp46⁻ cells were further analyzed for ICOS expression. Frequencies of the indicated subsets are shown in pie charts. (E) BM ILCPs or lung Lin⁻Rorc(γt)-EGFP⁻Thy1⁺CD127⁺IL-18Rα⁺ST2⁻ cells were purified from papain-treated Rorc(γt)-EGFP^+/− mice and cultured on OP9-DL1 with IL-7 and SCF for 1 wk. The progenies were analyzed for Thy1 and NKp46 expression and Thy1⁺NKp46⁺ cells were further analyzed for Rorc(γt)-EGFP expression. Data in A–C are representative of four independent experiments with three or more mice per group in each experiment, and data in D and E are representative of ≥10 replicates per experimental group in two independent experiments (mean ± SEM). *, P ≤ 0.05 by two-tailed Student’s t test.