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. 2021 Aug 19;12:5031. doi: 10.1038/s41467-021-25323-x

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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. 5 — a Schematic drawings of constructs. The ERK- or ZAP70-FRET biosensor was co-expressed with WT-CAR or XX3-CAR in Jurkat T cells. In XX3-CAR, the tyrosine in the first and second ITAM motif was mutated to phenylalanine. The CAR-T cells were then dropped onto the 3T3 cells that constitutively express CD19 to monitor the dynamic ZAP70 or ERK kinase activations. b Representative images of ERK biosensor in T cells after attaching to the CD19⁺-3T3 cell monolayer. Scale bar,10 μm. c Time courses of FRET ratio (FRET/ECFP) of ERK FRET biosensor in WT- or XX3-CAR-T cells (n = 8 and 20 in each group). Error bars, mean ± SEM. d Percentage changes of ERK-FRET biosensor in WT- or XX3-CAR-T cells (n = 8 and 20 in each group, unpaired two-tailed Student’s t-test, NS, P > 0.05). Error bars, mean ± SD. e Representative images of ZAP70 biosensor in T cells after attaching to the 3T3 CD19⁺ cell monolayer. Scale bar = 10 μm. f Time courses of FRET ratio (ECFP/FRET) of ZAP70 biosensor in WT- or XX3-CAR-T cells (n = 12 and 10 in each group). Error bars, mean ± SEM. g Percentage changes of ZAP70-FRET biosensor in WT- or XX3-CAR-T cells (n = 12 and 10 in each group, unpaired two-tailed Student’s t-test, *P = 0.017). Error bars, mean ± SD. h–i Flow-cytometry analysis of ECFP/FRET in WT-CAR-T cells (h) or XX3-CAR-T cells (i) before and after CD19⁺ Raji cell stimulation. Each biological replicate had similar results (n = 3). j Percentage of High-FRET ratio cells in different groups of three independent experiments. (n = 3 in each group, One-way ANOVA, ****P < 0.0001, **P = 0.0054). Error bars, mean ± SD. k Normalized FRET ratio of WT- or XX3 CAR-T cells before and after CD19⁺ Raji cell stimulation. (From left to right, n = 1314, 3151, 682, and 3635, respectively, One-way ANOVA, ****P < 0.0001). Error bars, mean ± SEM. l Histogram of FRET ratio in WT- or XX3-CAR-T cells after CD19 ⁺ Raji cell stimulation. Source data including the gating strategies are provided as a Source Data file.