Figure 3
Figure 3. Different contributions of IL-2 signaling to the effect of IL-36β on Th0 cell survival, proliferation, and Th1 polarization. FACS-sorted naive WT and IL-2−/− CD4+T cells were labeled with CFSE, activated with plate-bound anti-CD3/anti-CD28 mAb, and stimulated or not for 72 hours with IL-36β, IL-12 + IL-36β, IL-12 + IL-18 or IL-2. When specified, WT CD4+T cells where cultured in presence of blocking anti-CD25 and anti-IL-2 mAbs or isotype-matched control mAbs. (A-B) FACS analyses of viable 7AAD− CD4+ T cells. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), cytokine stimulation in IL-2−/− or in presence of neutralizing treatment significantly differs from WT or isotype control treatment, respectively. (C-D) Analysis of cell divisions in WT and IL-2−/− Th0 cells. (C) Histogram profiles of CFSE-labeled cells. Data shown are representative of one of 3 independent experiments with similar results. (D) Percentage of divided cells as assessed by CSFE labeling obtained in panel C. Error bars represent the SD of the mean of 3 independent experiments.*P < .05 (Student t test), cytokine stimulation in WT Th0 cells significantly differs from the same stimulation in IL2−/− Th0 cells. (E) IFN-γ secretion in WT and IL-2−/− naive CD4+ T-cell supernatants after 72 hours of stimulation with IL-12 + IL-18 and IL-12 + IL-36β. 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), cytokine stimulation in WT naive CD4+ T cells significantly differs from the same stimulation in IL-2−/− naive CD4+ T cells. (F) Quantitative RT-PCR analysis of IL-12Rβ2 mRNA expression in WT naive CD4+ T cells stimulated or not for 72 hours with IL-12 + IL-18 and IL-12 + IL-36β in presence of blocking anti-CD25 and anti-IL-2 mAbs or isotype-matched control mAbs. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), cytokine stimulation in presence of neutralizing treatment significantly differs from isotype control treatment

Different contributions of IL-2 signaling to the effect of IL-36β on Th0 cell survival, proliferation, and Th1 polarization. FACS-sorted naive WT and IL-2−/− CD4+T cells were labeled with CFSE, activated with plate-bound anti-CD3/anti-CD28 mAb, and stimulated or not for 72 hours with IL-36β, IL-12 + IL-36β, IL-12 + IL-18 or IL-2. When specified, WT CD4+T cells where cultured in presence of blocking anti-CD25 and anti-IL-2 mAbs or isotype-matched control mAbs. (A-B) FACS analyses of viable 7AAD− CD4+ T cells. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), cytokine stimulation in IL-2−/− or in presence of neutralizing treatment significantly differs from WT or isotype control treatment, respectively. (C-D) Analysis of cell divisions in WT and IL-2−/− Th0 cells. (C) Histogram profiles of CFSE-labeled cells. Data shown are representative of one of 3 independent experiments with similar results. (D) Percentage of divided cells as assessed by CSFE labeling obtained in panel C. Error bars represent the SD of the mean of 3 independent experiments.*P < .05 (Student t test), cytokine stimulation in WT Th0 cells significantly differs from the same stimulation in IL2−/− Th0 cells. (E) IFN-γ secretion in WT and IL-2−/− naive CD4+ T-cell supernatants after 72 hours of stimulation with IL-12 + IL-18 and IL-12 + IL-36β. 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), cytokine stimulation in WT naive CD4+ T cells significantly differs from the same stimulation in IL-2−/− naive CD4+ T cells. (F) Quantitative RT-PCR analysis of IL-12Rβ2 mRNA expression in WT naive CD4+ T cells stimulated or not for 72 hours with IL-12 + IL-18 and IL-12 + IL-36β in presence of blocking anti-CD25 and anti-IL-2 mAbs or isotype-matched control mAbs. Error bars represent the SD of the mean of 3 independent experiments. *P < .05 (Student t test), cytokine stimulation in presence of neutralizing treatment significantly differs from isotype control treatment

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