Clonal analysis of CD117⁺ PB ILC subsets shows broad functional differentiation capacities. (A–E) Single KLRG1⁺ ILCs, ILC2s, or NKp46⁺ ILCs from PB were index sorted by FACS into 96-well round-bottom plates preseeded with OP9-DL1 and stimulated with IL-2 (20 U/ml), IL-7, IL-1β, and IL-23 (20 ng/ml each). (A) After 14–21 d, KLRG1⁺ ILC cultures were analyzed for intracellular cytokine production (IL-5, IL-13, IFN-γ, IL-17A, and IL-22) after PMA/ionomycin stimulation. Representative flow cytometric analysis of clones producing various combinations of cytokines as indicated. (B) Summary of numbers and type of cytokines produced by KLRG1⁺ ILC clones and ILC2 clones. Three KLRG1⁺ ILC clones did not produce any of the measured cytokines. (C) Correlation of IL-13 production with GATA3 expression and IL-22 production with EOMES expression in KLRG1⁺ ILCs. (D) Representative flow cytometric analysis of NKp46⁺ ILC clones producing various combinations of cytokines as indicated. (E) Representative flow cytometric analysis of intracellular RORγt, T-bet, and EOMES expression of selected clones obtained from NKp46⁺ ILCs upon culture as in A. (F) Summary of numbers and type of cytokines produced by NKp46⁺ ILC clones. 28 NKp46⁺ ILC clones did not produce any of the measured cytokines. Data in A, C, and D are representative of at least four donors. MFI, mean fluorescence intensity.