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. 2021 Nov 8;12:6446. doi: 10.1038/s41467-021-26844-1

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© The Author(s) 2021

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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Fig. 7 — Panels a and b show results over three independent experiments and from three different donors, except for the groups MOCK-Tregs (two experiments) and 4-1BB CAR-Tregs (one experiment). a Frequencies of CAR-expressing (mCherry+) cells among the total circulating cells assessed by flow cytometry. Statistical significance is observed in the following comparisons: CD28 (n = 14) vs. 4-1BB (n = 10), p < 0,0001 (day 10), p = 0,0010 (day 17), p = 0,0044 (day 24); and 4-1BB vs. 4-1BB + rapamycin/vitamin C (n = 13), p < 0,0001 (day 10), p < 0,0001 (day 17), p < 0,0001 (day 24). PBMCs only n = 16, MOCK-Tregs n = 8. b 3D bioluminescence imaging tomography (DLIT) of recipient mice was performed as described in the Methods. A region of interest (ROI) corresponding to the spleen was determined by coregistration with the Automatic Mouse Atlas (upper left panel), and the absolute number of luciferase-positive (Luc+) cells was calculated. Individual kinetics of the absolute number of Luc+ cells for each mouse corresponding to each group and the number of living animals per timepoint are shown (upper panels). Grouped kinetics are shown in lower left panel. The number of mCherry-positive cells was measured in each group at day 12 (CD28 CAR-Tregs, n = 14; 4-1BB CAR-Tregs, n = 10; 4-1BB CAR-Tregs + rapamycin/vitamin C, n = 13; and MOCK-Tregs, n = 8 living animals) and day 58 (CD28 CAR-Tregs, n = 10; 4-1BB CAR-Tregs + rapamycin/vitamin C, n = 8 living animals) (lower right panel). Statistical significance is observed in the following comparisons: CD28 vs. 4-1BB, p < 0.0001 (day 12); MOCK vs. 4-1BB, p = 0.0238 (day 12); 4-1BB vs. 4-1BB + Rapa/vitC, p < 0.0001 (day 12); and CD28 vs. 4-1BB + Rapamycin/vitamin C, p = 0.0085 (day 58). Data are depicted as mean +/− SEM and compared using two-tailed Mann–Whitney test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).