Figure 4.
KLRG1
+
ILCs and NKp46
+
ILCs show multipotent cytokine production and TF profile. (A) Representative histogram of expression of CD25 or RANKL on KLRG1+ ILCs, ILC2s, and NKp46+ ILCs after culture on OP9-DL1 cells in the presence of IL-2 and IL-7 with or without TSLP and IL-33 or IL-1β and IL-23 for 7 d. (B) Quantification of CD25 or RANKL expression on after culture as in A (n = 3–8). (C) Representative flow cytometric analysis of intracellular IL-5, IL-13, IFN-γ, and IL-17A in KLRG1+ ILCs, ILC2s, and NKp46+ ILCs, after 7 d culture as in A and subsequently stimulated by PMA/ionomycin for 3 h. (D) Quantification of cytokine production by ELISA in culture supernatants from cells stimulated as in A. The concentration is adjusted to 5,000 cells. (E) Quantification of IFN-γ production by ELISA of ILC subsets cultured on OP9-DL1 cells in the presence of IL-2 and IL-7 with IL-1β and IL-23 or IL-1β and IL-12 for 7 d. (F) Representative flow cytometry of the expression of CD117, NKp44, and CD94 on KLRG1+ ILCs, ILC2, and NKp46+ ILCs after culture as in E. Filled histograms represent isotype control. (G) Representative flow cytometric analysis of intracellular expression of EOMES and T-bet in KLRG1+ ILCs, ILC2s, and NKp46+ ILCs after culture as in E. Data in A, C, F, and G are representative of at least three donors from more than three independent experiments. Cytokines used in all experiments are IL-2 (20 U/ml), IL-7, TSLP, IL-33, IL-1β, IL-23, and IL-12 (all 20 ng/ml). **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001 (one-way ANOVA).