Importance of endogenous IL-36 in the Th1 response. (A) FACS-sorted naive CD4⁺ T cells were activated (Medium) or not (Naive) with plate-bound anti-CD3 and anti-CD28 mAb and stimulated with IL-12 (10 ng/mL), IL-18 (100 ng/mL), IL-36β (100 ng/mL) either alone or in various combinations as indicated for 72 hours. Total mRNA was isolated from naive T cells for analyses by quantitative RT-PCR. Results represent IL-36β mRNA expression levels relative to GAPDH. Error bars represent the SD of the mean of 3 independent experiments. (B-C) IL-2 (B) and IFN-γ (C) production profiles in WT and IL-36R^−/− naive CD4⁺T cells after different stimulations as indicated. Data are shown from one of 3 independent experiments with similar results. Error bars represent SD of triplicates in the same experiment. *P < .05 (Student t test), IL-12 + IL-18 stimulation in WT Th0 cells significantly differs from the same stimulation in IL-36R^−/− Th0 cells. (D-E) Activated WT and IL-36R^−/− Th0 cells were stimulated or not with IL-12/IL-18 and IL-12/IL-36β combinations and then treated with PMA plus ionomycin for 5 hours. IFN-γ–expressing CD4⁺ T cells were detected by intracellular staining and quantified by flow cytometry. FACS profiles shown in panel D represent one of 3 independent experiments with similar results for which quantitative data are shown in panel E. (E) Results represent the percentage of IFN-γ⁺CD4⁺ T cells for each condition. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), IL-12 + IL-18 stimulation in WT cells significantly differs from the same stimulation in IL-36R^−/− cells.