Expression of IL-36R in CD4⁺ T-cell subsets and effect of IL-36 on cytokine production by cultured T cells. (A) Quantification of IL-36R mRNA by quantitative RT-PCR in CD4⁺ T cells and in CD4⁺ T-cell subsets. Total mRNA was isolated from total splenic CD4⁺ T cells, Th1 cells, Th2 cells, and Th17 cells for quantitative RT-PCR analysis. Results represent IL-36R mRNA levels normalized for GAPDH expression. Error bars represent the SD of the mean of 3 independent experiments. (B-D) Dose-dependent effect of IL-36 in CD4⁺ T cells isolated from WT and IL36R^−/− mice. Total splenic CD4⁺ T cells (1 × 10⁵ cells/well) were seeded in 96-well plates precoated with anti-CD3/anti-CD28 mAb (0.5 μg/mL) and cultured in the absence (Unstimulated) or presence of the indicated concentrations of IL-36Ra, IL-36α, IL-36β, and IL-36γ for 72 hours. IFN-γ (B), IL-4 (C), and IL-17 (D) levels were measured in culture supernatants by ELISA. Error bars represent the SD of the means of triplicates in the same experiment. *P < .05 (Student t test), IL-36 or IL-1β stimulation significantly differs from unstimulated cells.