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. 2024 Aug 28;25(10):1830–1844. doi: 10.1038/s41590-024-01918-6

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© The Author(s) 2024

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. 3 — a, GSEA for effector- and stemness-associated genes was performed after a transcriptomic comparison of bulk human CD8⁺CD45RA⁻ T cells stimulated with CD3 and CD28 mAbs for 5 d under high and low NaCl conditions. b, RNA velocity analysis after scRNA-seq of human CD8⁺CD45RA⁻ T cells stimulated with CD3 and CD28 mAbs for 3 d under high and low NaCl conditions. The velocities are shown in UMAP embedding. Cells are color coded according to the treatment condition as indicated. c–e, ScRNA-seq and analysis of the module scores of the indicated gene sets (c, effector genes; d, cytokine activity; e, positive regulation of T cell mediated cytotoxicity) for human CD8⁺CD45RA⁻ T cells stimulated with CD3 and CD28 mAbs for 3 d under high and low NaCl conditions (n = 1; Wilcoxon’s rank-sum test). f, Intracellular cytokine staining and flow cytometric analysis of human CD8⁺CD45RA⁻ memory T cells after stimulation for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions. Left, representative experiment; right, cumulative data (n = 13; mean ± s.e.m., two-tailed, paired Student’s t-test). g, ELISA of cell culture supernatants from human CD8⁺ memory T cells stimulated for 5 d with CD3 and CD28 mAbs (n = 6; mean ± s.e.m., two-tailed, paired Student’s t-test). h, Flow cytometric analysis of human CD8⁺CD45RA⁻ T cells performed after stimulation for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions. Left, representative experiment; right, cumulative data. Data present the mean ± s.e.m. (n = 17; two-tailed, paired Student’s t-test). i–k, Intracellular cytokine staining and flow cytometric analysis (i, perforin; j, TNF; k, IL-2) of human CD8⁺CD45RA⁻ T cells after stimulation for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions and restimulation with PMA/ionomycin for 5 h. Left, representative experiment; right, cumulative data. Data present the mean ± s.e.m. (n = 25 (j), n = 18 (k); two-tailed, paired Student’s t-test).