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. 2023 Aug 23;13:13762. doi: 10.1038/s41598-023-40925-9

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© The Author(s) 2023

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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Ex vivo suppressive activity of Tregs expanded by scR2agoTNF-Fc. (A) WT-Tregs and (B) TNFR2^−/−Treg suppression assays were performed using pre-expanded Tregs. WT-CD4⁺CD25⁺ Tregs and TNFR2^−/−CD4⁺CD25⁺ Tregs were expanded by scR2agoTNF-Fc (100 ng/ml) for 72 h. Then, each Treg population was cultured with CFSE-labeled CD4⁺CD25⁻ Tconvs for another 72 h. CFSE division was measured by FCM. Data are the mean ± SD (n = 4). (C) A Treg suppression assay was performed under the conditions where CD4⁺CD25⁺ Tregs and CFSE-labeled CD4⁺CD25⁻ Tconvs were co-incubated. A mixture of Tregs, CFSE-Tconvs, and APC-containing 90.2⁻ cells were cultured and stimulated by scR2agoTNF-Fc (0, 1, 10, 100 ng/ml) for 72 h. Then, CFSE division was measured by FCM. Data are the mean ± SD (n = 4). (D) LN cells were stimulated with immobilized anti-mouse CD3 mAb and soluble recombinant IL-2 (10 U/ml). Then, TNFR2 expression levels of the activated T cell subsets, CD4⁺CD25⁺ Tregs, CD4⁺CD25⁻ Tconvs, and CD4⁻CD25⁻ cells, were measured. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Turkey multiple comparison test).