Figure 2
Figure 2. Importance of IL-12 for the effect of IL-36 on Th1 differentiation. FACS-sorted naive CD4+T cells from WT (A-B) or IL-36R−/− (A) mice were activated with anti-CD3/anti-CD28 mAbs and cocultured with BMDCs from either WT (A-B), IL-36R−/− (A), or IL-12p35−/− (B) mice in the absence or presence of IL-36R ligands, IL-1β, IL-33, IL-18, and LPS (100 ng/mL). Three days later, IFN-γ was measured in the supernatant by ELISA (A-B). Data are shown from one of 3 independent experiments with similar results. Error bars represent the SD of triplicates in the same experiment. *P < .05 (Student t test), IL-36, IL-1β, IL-33, IL-18, or LPS stimulation significantly differs from unstimulated cells. (C-D) FACS-sorted naive CD4+ T cells were activated (Medium) or not (Naive) with plate-bound anti-CD3/anti-CD28 mAbs and stimulated for 72 hours with IL-12 (10 ng/mL), IL-18 (100 ng/mL), IL-36β (100 ng/mL) either alone or in various combinations as indicated. Total mRNA was isolated for analyses by quantitative RT-PCR. Results represent T-bet (C) and IL-12Rβ2 (D) mRNA expression levels relative to GAPDH. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), IL-36, IL-12, and IL-18 stimulation alone or in combination significantly differs from unstimulated cells. (E) IFN-γ secretion was measured in culture supernatants by ELISA. 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-36, IL-12, and IL-18 stimulation alone or in combination significantly differs from unstimulated cells. (F) Flow cytometric analysis of intracellular staining of IFN-γ in CD4+ T cells obtained from WT mice and cultured for 3 days with priming (anti-CD3/anti-CD28 mAbs) and different stimulations as indicated. FACS profiles shown in panel F represent one of 3 independent experiments with similar results for which quantitative data are shown in panel G. (G) Results represent the percentage of IFN-γ–positive CD4+ T cells for each condition. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), percentage of IFN-γ–positive CD4+ T cells after IL-12/IL-18 stimulation significantly differs from IL-12/IL-36β stimulation.

Importance of IL-12 for the effect of IL-36 on Th1 differentiation. FACS-sorted naive CD4+T cells from WT (A-B) or IL-36R−/− (A) mice were activated with anti-CD3/anti-CD28 mAbs and cocultured with BMDCs from either WT (A-B), IL-36R−/− (A), or IL-12p35−/− (B) mice in the absence or presence of IL-36R ligands, IL-1β, IL-33, IL-18, and LPS (100 ng/mL). Three days later, IFN-γ was measured in the supernatant by ELISA (A-B). Data are shown from one of 3 independent experiments with similar results. Error bars represent the SD of triplicates in the same experiment. *P < .05 (Student t test), IL-36, IL-1β, IL-33, IL-18, or LPS stimulation significantly differs from unstimulated cells. (C-D) FACS-sorted naive CD4+ T cells were activated (Medium) or not (Naive) with plate-bound anti-CD3/anti-CD28 mAbs and stimulated for 72 hours with IL-12 (10 ng/mL), IL-18 (100 ng/mL), IL-36β (100 ng/mL) either alone or in various combinations as indicated. Total mRNA was isolated for analyses by quantitative RT-PCR. Results represent T-bet (C) and IL-12Rβ2 (D) mRNA expression levels relative to GAPDH. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), IL-36, IL-12, and IL-18 stimulation alone or in combination significantly differs from unstimulated cells. (E) IFN-γ secretion was measured in culture supernatants by ELISA. 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-36, IL-12, and IL-18 stimulation alone or in combination significantly differs from unstimulated cells. (F) Flow cytometric analysis of intracellular staining of IFN-γ in CD4+ T cells obtained from WT mice and cultured for 3 days with priming (anti-CD3/anti-CD28 mAbs) and different stimulations as indicated. FACS profiles shown in panel F represent one of 3 independent experiments with similar results for which quantitative data are shown in panel G. (G) Results represent the percentage of IFN-γ–positive CD4+ T cells for each condition. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), percentage of IFN-γ–positive CD4+ T cells after IL-12/IL-18 stimulation significantly differs from IL-12/IL-36β stimulation.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal