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. 2017 Sep 12;7:11315. doi: 10.1038/s41598-017-11803-y

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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IL-4 induces IL-10 production in polarised Th1 cells. (a–c) Polarised Tg4 Th1 cells (Primary stimulation) were stimulated for a further cycle (Secondary stimulation) with irradiated B10.PL splenocytes APCs with MBP Ac1-9[4Y] in the presence of IL-12 (Control) or IL-12 and exogenous IL-4 (+IL-4). Intracellular cytokine staining was carried out on day 7 of each stimulation following PMA and ionomycin stimulation. (a) Representative FACS plots of three independent experiments are gated on live CD4⁺ cells and show IFN-γ, IL-4 and IL-10 expression. (b) Pooled quantitation of IL-10⁺, IFN-γ⁺ and IL-4⁺ live CD4⁺ cells from three independent experiments as shown in (a). Error bars are mean ± SEM. n.s (not significant) p > 0.05, *p ≤ 0.05 (Student’s paired t-test). (c) Tissue culture supernatants were taken on day 7 of each stimulation and analysed for IL-10, IFN-γ and IL-4 by ELISA. Data are mean + SD from triplicate wells from one experiment, representative of two independent experiments. n.s. (non-significant) p > 0.05, ***p < 0.001, ****p < 0.0001 (Student unpaired t-test). (d) Polarised CD4⁺ selected Th1 cells were stimulated with anti-CD3 for 20 minutes in the presence of IL-4. Histograms of IL-4 receptor and phospho-STAT6 staining gated on live CD4⁺ cells and graphs of geometric mean fluorescence are shown and are representative of two independent experiments. (e) Polarised CD4⁺ selected Th1 cells (Primary stimulation) were stimulated for a further cycle of 7 days (Secondary stimulation) with anti-CD3 and anti-CD28 in the presence of IL-12 (Control) or IL-12 and exogenous IL-4 (+IL-4). Intracellular cytokine staining for IFN-γ, IL-4 and IL-10 was carried out on day 7 of each cycle of stimulation following PMA and ionomycin stimulation. Graphs show pooled quantitation of IL-10⁺, IFN-γ⁺ and IL-4⁺ live CD4⁺ cells from four independent experiments. Error bars are mean ± SEM. n.s (not significant) p > 0.05, *p ≤ 0.05 (Student paired t-test).