Human skin ILC2s stimulated with IL-33 produce high levels of type-2 cytokines. (A) Cultured skin ILC2s were stimulated with PMA/ionomycin or IL-25, IL-33, and TSLP alone or in the indicated combinations and concentrations. Supernatant was collected after 24 h. Levels of IL-4, IL-5, IL-6, and IL-13 were measured by multiplex cytokine analysis. Statistical comparisons are compared with the negative (no cytokine) control. (B) Skin-derived ILC2s were stimulated with PMA/ionomycin or IL-33, and levels of IL-13, IL-17A, and IL-22 expression were measured using intracellular cytokine staining. Data are representative of four independent experiments on cultured cells. (C) ILC2s were stimulated for 16 h with IL-25, IL-33, or TSLP in isolation or combination at the indicated combinations. Expression of IL1RL1 (IL-33R) was measured by RT-PCR. Data are representative of three independent experiments. Statistical comparisons are compared with the negative (no cytokine) control. (D) Chemotaxis of skin-derived ILC2s toward indicated concentrations of IL-33 and TSLP, relative to baseline, was measured after 1 h using transmigration across a 5 µM pore membrane. Data are representative of three independent experiments (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test (mean and SEM in A, C, and D).