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. 2022 Nov 9;12:19112. doi: 10.1038/s41598-022-23601-2

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© The Author(s) 2022

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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FH535 effects on Treg cells are replicated by the N-arylbenzene-sulfonamide derivative Y3. (A) Short-time effect of Y3 on Treg cell expansion. In vitro expansion of Treg cells exposed to vehicle control (no drug), Rapa, FH535 or Y3 for 3 days. Results are pooled data collected from four independent experiments and depicted as mean ± SD of percentage of experimental values with respect to control (no drug) cells (dotted line, 100% reference value). (B) Representative time-course experiment of in vitro expansion of Treg cells placed in culture in Treg cell media supplemented with the corresponding drug treatment. Results show the relative fold increase of cell numbers with respect to the original sample (day 0). Dotted line shows the response of the cells to the standard culture conditions with Rapa. (C) Comparative fold increase of Treg cell numbers generated from pooled results of four different donors are shown as mean ± SD. (D) The suppressor activity of Treg cells after 7 day-exposure to drug treatments was evaluated in 4-day MLR assays. Representative panels show the corresponding histograms of 1:3 ratio of Treg: CFSE-labeled T cells overlaid to standard treatment with Rapa (R, dark profile) or to the absence of Tregs (empty profile). Results show the percentages of proliferating responder cells for each condition. Individual experiments were confirmed at least three times. Data was analyzed by ANOVA with Dunnett’s test or Student’s t test for pairwise comparisons. Significance with respect to control is shown by *(ρ < 0.05) and **(ρ < 0.005); NS no significant differences.