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. 2024 Oct 6;15:8658. doi: 10.1038/s41467-024-52666-y

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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. 8 — A MFI of exhaustion markers CD39, LAG3, PD1 and TIM3 expressed by (TOP) CD19 or (BOTTOM) HA ± CD8⁺ GLUT1OE CAR-T cells on day 14. Pooled data of n = 14 donors. P values determined by paired two-tailed t-tests. Error bars represent SEM. B ELISA analysis of IL-2 and IFNγ secretion by CD19 and HA ± GLUT1OE CAR-T cells after 24 hour stimulation with (LEFT) Nalm6 or (RIGHT) Nalm6-GD2 leukemia tumor lines on day 14. Data from n = 6 donors. P values determined by paired two-tailed t-tests. C ELISA analysis of IL-2 and IFNγ secreted by CAR-T cells ± GLUT1OE CAR-T cells after 24 hour stimulation with 143b against (LEFT) CD19 and (RIGHT) HA on day 14. Data from n = 2–4 donors. P values determined by paired two-tailed t-tests. D Intracellular staining of IL-2 and TNFα by (LEFT) CD19 and (RIGHT) HA ± GLUT1OE CAR-T cells after 24 h of stimulation with Nalm6-GD2 leukemia at 1:1 and 1:2 E:T on day 14. Data from n = 4 donors. P values determined by paired two-tailed t-tests. E Serial rechallenge and tumor-GFP killing kinetics data using Incucyte. Pooled data of 4 donors (TOP) CD19 and (BOTTOM) HA ± GLUT1OE CAR-T cells sequentially challenged at 1:2 ratio with Nalm6 leukemia ± GD2. Incucyte p values generated using two way ANOVA. Error bars represent SEM. F Flow cytometry measurements of CD39 and PD-1 of CD8⁺ (TOP) CD19 and (BOTTOM) HA ± GLUT1OE CAR-T cells after each stimulation denoted by arrows in (E). P values determined by paired two-tailed t-tests. G Memory formation data of serially rechallenged CD8⁺ CAR-T cells after (LEFT) 4 stimulations for CD19 ± GLUT1OE with representative flow cytometry of CD62L⁺ cells and (RIGHT) 3 stimulations for HA ± GLUT1OE. EM- effector memory, CM- central memory. P values determined by paired two-tailed t-tests. H Pooled intracellular expression of TCF1 in CD19 and HA ± GLUT1OE CAR-T cells on day 16. Data from n = 3–4 donors. P values determined by paired two-tailed t-tests. Error bars represent SEM. I Pooled surface expression of CD62L in CD19 or HA ± GLUT1OE CAR-T cells on day 16. P values determined by paired two-tailed t-tests. Error bars represent SEM.