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. 2020 Oct 21;10:17872. doi: 10.1038/s41598-020-74808-0

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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In vitro differentiation of CD4⁺ T regulatory cells under varying concentrations of Mito-ATOs and controls. After six days of culture, cells were stained for flow cytometry analysis. (left) Live/dead staining to assess the percentage of live CD4 + T cells within the lymphocyte gate. (middle) T_eff cell function is shown as the frequency of IFNγ-YFP positive cells within the live CD4⁺ T cells. (right) The percentage of T_reg cells is shown as the frequency of FoxP3⁺CD25⁺ cells within the live CD4⁺ T cells. (A, bottom) Representative plot using FlowJo Software, effects of Mito₁₀-ATO on T_reg (FoxP3⁺CD25⁺) cells within the live CD4⁺ T cell gate (contour plots depict gating corresponding to panel A, upper right)³⁹. Mito₄-ATO (B) effectively suppressed T_reg while Mito₁₂-ATO (C), Butyl-ATO (D), Decyl-ATO (D), and ATO (E) did not suppress T_reg cells.