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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. 3 — A Unbiased principal component analysis of bulk RNA derived from day 16 CD19 and HA ± GLUT1OE ± 1 µg / mL anti-idiotype stimulation collected at two different time points. Cotransduced cells were magnetically enriched for greater than 95% double positive prior to experiment. Pooled data from two experiments (stimulated and unstimulated) with total n = 6 donors. B UpSets plots showing intersection of genes differentially upregulated (TOP) or downregulated (BOTTOM) upon GLUT1OE in HA-CAR and CD19-CAR T cells unstimulated or at 4 h and 14 h post stimulation. Red boxes highlight shared changes between CD19-CAR and HA-CAR T cells as a consequence of GLUT1OE at baseline. RNAseq data from n = 6 donors on day 16. C GSEA analysis of the NK-like exhaustion signature in unstimulated (LEFT) CD19 and (RIGHT) HA-CAR-T cells, comparing GLUT1OE versus control. D GSEA analysis of (LEFT) glycolysis and (RIGHT) OXPHOS for CD19 ± GLUT1OE after 4 h of anti-idiotype stimulation (1 µg/mL). E (TOP) GSEA analysis of RNA-seq comparing CD19 GLUT1OE vs CD19 showing enrichment of memory and effector T cell signatures over naïve in CD4 and CD8 at every timepoint analyzed (unstimulated, 4 h or 14 h post-stimulation). (BOTTOM) Similar GSEA analysis using as reference KEGG pathways dataset showing wide metabolic reprogramming. The size of the dots correlates with −log10(P-value) by GSEA analysis, with the smallest dots representing non-significant pathways. F Heatmap representing differentially expressed genes with annotations for those significantly upregulated in CD19-GLUT1 ± 4 h of anti-idiotype stimulation.