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. 2021 Feb 22;10:e57417. doi: 10.7554/eLife.57417

Figure 3. Combined IL-2 and IL-4 increase the suppressive ability of Tregs.

(A–C) Proliferation (flow cytometry) and cytokine output (ELISA) of freshly isolated and Celltrace-stained Tconv co-cultured with αCD3ε and the indicated ratios of WT Tregs separately and previously stimulated with αCD3ε and all cytokine conditions for 3 days, washed, and normalized for cell number at the time of co-culture. The flow histograms were gated on Tconv cells. N = 3 for all graphs and histograms are representative. (D–F) Proliferation (flow cytometry) and cytokine production (ELISA) of freshly isolated Tconv cells co-cultured with αCD3ε and the indicated ratios of IL-10-/- Tregs separately and previously stimulated with αCD3ε and all cytokine conditions for 3 days, washed, and normalized for cell number at the time of co-culture. The flow histograms were gated on Tconv cells. N = 3 for all graphs and histograms are representative. (G–I) Proliferation (flow cytometry) and cytokine production (ELISA) of freshly isolated Tconv cells from WT mice co-cultured with αCD3ε and the indicated ratios of IL-10 cKO Tregs separately and previously stimulated with αCD3ε and all cytokine conditions for 3 days, then washed. The flow histograms were gated on Tconv cells. N = 3 for all graphs and histograms are representative. (J–L) Proliferation (flow cytometry) and cytokine production (ELISA) of freshly isolated Tconv cells co-cultured with αCD3ε and the indicated ratios of WT Tregs separately and previously stimulated with αCD3ε and all cytokine conditions for 7 days. Co-culture ratio was based on the number of Tregs prior to cytokine stimulation to incorporate their proliferation. The flow histograms were gated on Tconv cells. N = 3 for all graphs and histograms are representative. (M) Percentage of the overall culture comprising of WT or IL-10 cKO Tregs and Tconv over the course of time as determined by flow cytometry. The Tregs were purified and stimulated with αCD3ε and all cytokine conditions for 3 days, washed, then placed in co-culture with freshly isolated Tconv cells at a 1:8 Treg:Tconv starting ratio (line). N = 3. For all panels, mean ± SEM are indicated. *p<0.05, ****p<0.0001.

Figure 3—source data 1. Numeric values of the heatmaps in Figure 3B–C (A), Figure 3E–F (B), Figure 3H–I (C), and Figure 3K–L (D).
Values were obtained by analyzing the suppression assay supernatants by ELISA.

Figure 3.

Figure 3—figure supplement 1. Colon lengths of conditional and global Il10 knockout mice.

Figure 3—figure supplement 1.

(A and B) Mouse colon photographs and their lengths as measured by the distance between anus (at 0) and cecum.