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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. 1 — a CD28 and 4-1BB CAR structures. b Gating strategies for CD4+ CD45RA+ CD45RO- CD25hi CD127low naive Tregs and CD4+ CD45RA+ CD45RO- CD25low CD127hi Tconvs. c Process timeline for CD28 or 4-1BB CAR-T-cell production. T cells were sorted, stimulated with anti-CD3/CD28 beads, transduced with CAR-encoding lentiviruses, expanded, and sorted based on reporter gene expression, followed by activation with anti-CD3/CD28 beads and expansion for up to 21 days. d On day 11, CD28 and 4-1BB CAR-Tregs were incubated with or without anti-CD3/CD28 beads (TCR stim.) or HLA-A2-positive or HLA-A2-negative irradiated splenocytes, and CD69 and EGFR expression was measured by flow cytometry. Contour plots are representative of two experiments. e Fold expansion of untransduced Tregs and CAR-Tregs (left panel) or their respective Tconv populations (right panel) from TCR restimulation with anti-CD3/CD28 beads on day 11 (arrow) to day 21 of culture. The median +/− interquartile range (IQR) is shown. A total of 16 independent experiments are shown including the following cell lines: CD28 CAR-Tregs (blue full circle, n = 10), 4-1BB CAR-Tregs (red full triangle, n = 11), untransduced (UT) Tregs (black full diamond, n = 8), CD28 CAR-Tconvs (blue open circle, n = 8), 4-1BB CAR-Tconvs (red open triangle, n = 9), UT Tconvs (black open diamond, n = 8). Two-tailed Mann–Whitney test comparing 4-1BB and CD28 CAR-Tregs indicates significant differences at day 18 (p = 0.0079) and day 21 (p = 0.0012). f FSC-A ratios of 4-1BB/CD28 CAR-Tregs and CAR-Tconvs are calculated at the time of CAR-positive cell sorting (D11). Data are depicted as mean +/− SEM and compared using two-tailed Mann–Whitney test. Statistical significance is found between Tconv and Treg populations: ***p = 0.0004. N = 16 donors for Tregs and n = 12 for Tconvs, from 16 independent experiments. Color code: gray full square = ratio 4-1BB/28 CAR-Tregs, gray square = ratio 4-1BB/28 CAR-Tconvs.