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. 2022 Aug 31;609(7926):369–374. doi: 10.1038/s41586-022-05140-y

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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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Fig. 2 — a, CAR expression in cells from two representative healthy donors determined 7 d after electroporation. b, Percentage of CAR⁺ cells detected 7 d after electroporation (n = 20 independent healthy donors). c, Percentages of CAR integration and PD1 indels in total T cells detected 7 d after electroporation in five representative healthy donors. d, Percentage of cells with PD1 expression detected by flow cytometry in CD3⁺CAR⁺ gated cells after 24 h of co-culture with PD-L1-expressing Raji cells (n = 3 independent healthy donors). e, Expansion of CAR⁺ cells after repeated stimulation with PD-L1-expressing Raji cells. Data are shown as the mean ± s.e.m. (n = 3 independent healthy donors). f, Cytokine secretion measured by bead-based immunoassay in the supernatant after co-culture with PD-L1-expressing Raji cells for 24 h. Data are shown as the mean ± s.e.m. (n = 3 independent healthy donors). g, In vitro cytotoxicity against PD-L1-expressing Raji cells determined by LDH assay. Data are shown as the mean ± s.e.m. (n = 3 independent healthy donors). h–j, Immunodeficient mice were injected intravenously with 5 × 10⁵ ffLuc-transduced PD-L1-expressing Raji cells, and 1 × 10⁶ CAR-T cells were administered intravenously after 5 d. h,i, Bioluminescence kinetics (h) and imaging (i) of tumour cell growth following different treatments (n = 4 or 8). Data are shown as the mean ± s.e.m. in h. Imaging on the indicated days after CAR-T cell infusion and the radiance scale (p s^–1 cm^–² sr^–1) are shown in i. j, Kaplan–Meier analysis of survival of the mice in i. Control samples were electroporated the same as PD1-19bbz cells except without sgRNA addition. The mean value is shown in b,d. P values were calculated by one-way ANOVA with Tukey’s multiple-comparisons test (f), two-way ANOVA with Sidak’s multiple-comparisons test (e) or Tukey’s multiple-comparisons test (g), or a log-rank Mantel–Cox test (j). ****P < 0.0001, **P < 0.01; NS, not significant.

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