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. 2022 Aug 24;609(7925):174–182. doi: 10.1038/s41586-022-05126-w

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© The Author(s) 2022

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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Extended Data Fig. 4 — a, Flow cytometry plots showing gating for cytokines in control (CTRL) and RASA2 KO T cells with (bottom row) and without (top row) PMA stimulation. b, left: Phosphorylated ERK levels (y-axis) measured by flow cytometry 10 min after TCR stimulation with titrated concentrations of anti-CD3/CD28 complexes (1:1 is 25 µl/ml Immunocult, n = 2 T cell donors in duplicates, lines show the 4-parameter logistic fit); middle: Percentage of cells positive for the T cell activation marker CD154 measured 18 h after stimulation with titrated concentrations of anti-CD3/CD28 complexes (1:1 = 25 µl/ml Immunocult, n = 2 T cell donors in duplicate); right: percentage of cells that divided based on CFSE profiles 3 days after stimulation with anti-CD3/CD28 complexes (n = 2 T cell donors in triplicates, lines show the 4-parameter logistic fit, p-value from a two-sample Kolmogorov-Smirnov test). c, Schema of CD19 CAR-T cell production, where the CAR is knocked into the TRAC locus⁴⁶ and RASA2 versus AAS1 is targeted with Cas9 RNPs. d, Flow cytometry plots of CD19-CAR positive T cells after the CAR knockin strategy described in (c). e, TRAC CAR-T cell killing of target Nalm6 cells engineered to express varying CD19 levels (rows) measured by annexin levels in live cell microscopy across increasing CAR-T:Nalm6 ratios (columns) over 48 h (one representative donor out of two, 3 technical replicates, error bars are mean ± SD). f, Summary of cancer cell killing (scaled AUC of annexin levels) shown on the y-axis for a range of effector T cells to target cell ratios. Horizontal lines are the mean (n = 2 human donors, each in triplicates, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, for two-sided unpaired Student’s t-test, shape denotes donor).

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