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. 2022 Oct 31;10(10):e005326. doi: 10.1136/jitc-2022-005326

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© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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Characterization of sort-purified Tregs. (A) MFI of FOXP3-PE in sorted Tregs and T_rest from the three donors following intracellular staining. (B–F) RT-qPCR analysis of the expression of IL2RA (B), TNFRSF18 (C), IKZF2 (D), CTLA4 (E), or IL10 (F) in the Tregs and T_rest. The expression of each Treg signature gene was normalized to the housekeeping gene POL2RA and presented as fold change versus T_rest with bars showing the mean±SD of technical triplicates. (G) RT-qPCR analysis of PDCD1 expression in Tregs and T_rest. Data is represented as relative expression to the housekeeping gene POL2RA (arbitrary units) with bars showing the mean±SD of technical triplicates. (H) In vitro Treg suppression assay. (Left) Percentage of undivided CD8⁺ T cells after 5 days of co-culture with sort-purified Tregs (+Treg) or T_rest (+T_rest). Bars indicate the mean of three technical triplicates (depicted as dots). **P=0.0069 (unpaired t-test). (Middle) Proliferation index of CD8 T cells after 5 days of coculture with Tregs or T_rest. Bars indicate the mean of three technical triplicates (depicted as dots). **P=0.0022 (unpaired t-test). (Right) CFSE staining of CD8⁺ T cells cocultured with Tregs or T_rest for 5 days. The gray bar depicts the undivided population. CFSE, carboxyfluorescein succinimidyl ester; IL, interleukin; MFI, mean fluorescence intensity; Treg, regulatory T cell; T_rest, resting T cell.