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. 2020 May 20;10:8350. doi: 10.1038/s41598-020-65212-9

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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 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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Infection-experienced T_regs show trend for higher in vitro suppressive capacity. CD4+GFP+ T_regs were sorted from naive or LCMV-experienced Foxp3-GFP reporter mice and co-cultured with CD4+GFP- effector cells isolated from acutely LCMV infected donor mice in the presence of soluble anti-CD3 (1 µg/ml) and irradiated splenic APCs isolated from naive mice for 2 days, then [³H]thymidine was added for 18–22 h and [³H]thymidine incorporation was quantified to determine proliferation. Target cell proliferation (A) and calculated T_reg-mediated suppression (B) are shown. (C) The concentration of IFN-γ in the suppression assay supernatants harvested after 48 h of co-culture was determined by cytometric bead assay. (Mean ± SD; biological replicates: no T_regs, naive T_regs, memory T_regs n = 6; 2 independent experiments) (one way ANOVA and multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001). (D) Splenic T cells from naive or LCMV-experienced mice (>30 days post infection) were stained using gp66 tetramers to detect LCMV-specific CD4+Foxp3+ T_regs or CD4+Foxp3- effector T cells. Frequencies and absolute numbers are shown. (Mean ± SD; biological replicates: naive n = 8, LCMV-experienced n = 9; 3 independent experiments) (t Test, ****p < 0.0001).