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. 2018 May 17;8:7799. doi: 10.1038/s41598-018-26088-y

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© The Author(s) 2018

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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NTG-A-009 reduces Th1, Th17 cells differentiation in vitro. Naïve CD4⁺ T cells isolated from spleen and lymph nodes were stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) under Th1, Th17 and T_reg inducing conditions with NTG-A-009 (1 μM), Tofacitinib (1 μM) or Triamcinolone (1 µM). (a) Chemical structure of NTG-A-009. (b,c) The percentage of IFN-γ⁺ Th1, IL-17A⁺ Th17 and FoxP3⁺ T_reg cells was determined by flow cytometry. (d) Different concentration of NTG-A-009 was used to find its effect in Th1 and Th17 cells in anti-CD3 antibody coating system. (e,f) The antigen specific differentiation of Th1, Th17 and T reg cells was done by the isolation of CD4⁺ T cells and irradiated antigen presenting cells from 6–10 weeks OTII mice and stimulated under Th1, Th17 and T_reg polarizing conditions in the presence of OVA _323–339 (0.1 μM) and incubated for 72 hour for Th1 and Th17 and 96 hours for T_reg cells differentiation. Cells were then restimulated with PMA, ionomycin and golgistop and analyzed by FACS through intracellular staining. (g) Different doses of NTG-A-009 were cultured under Th1, Th17 condition with the cells from OT-II transgenic mice. Data were quantified in bar diagram. Data represent three independent experiments. Mean ± SEM of the triplicates are shown. ***P < 0.001.