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. 2023 Jun 5;6:604. doi: 10.1038/s42003-023-04978-w

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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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Fig. 1 — a Schematic representation of CAR expression under control of activation-dependent promoter. b Physiological expression kinetics of PD1, CD25, CD69, TIGIT, and TIM3 activation markers. Graph presents marker expression over time from at least 3 donors. Cells were restimulated on d6 (arrow) after initial stimulation after d1 . Connecting lines represent mean. c CAR can be successfully knocked-in into PD1 locus. Set of representative dot plots and histogram are shown. Plots pre-gated on living, single CD45+ cells. d CAR expression under PD1 locus is activation-dependent upon polyclonal anti-CD3/CD28 stimulation. Graphs display CAR expression as arbitrary units (A.U.) in rested or restimulated samples from 3 independent experiments. CAR constructs targeted into TRAC locus as well as expressed via lentiviral (LV) random integration were used as a control. Error bars represent mean ± SD. The significance was analyzed using unpaired Student’s t-test. e CAR T cells can be expanded via antigen- specific stimulation. Graph depicts fold expansion upon two rounds of antigen-specific stimulation. Errors bars represent mean ± SD. f Antigen-specific expansion leads to increased CAR T cell content in all samples, whereas polyclonal restimulation enhances CAR T cell content in activation-induced CAR expression setting only. Graphs display indicated marker expression as frequencies in rested or restimulated samples from 3 independent experiments. CAR constructs targeted into PD1 locus as well as expressed via lentiviral (LV) random integration are depicted. Arrows indicate start (d0) and end (d2) of stimulation period. Connecting lines represent mean. The significance was analyzed using paired Student’s t-test.