Extended Data Fig. 8: E81K editing increases metabolic fitness in BBz CAR T cells.

a.-c. Comparison of gene modules defined by expression of distinct genes (a.) for mock 19BBz versus E81K-modified 19BBz CAR T cells from one healthy donor (b.) and for all CD4+ clusters (c.) determined by scRNA sequencing. Statistical analyses are performed using two-sided unpaired Student’s t-test. Metab. = metabolic, Cytox. = cytotoxic. d. Heatmap demonstrating expression of glycolysis-associated genes in TRBC KO E81K-edited and TRBC KO mock 19BBz CAR T cells after two exposures to Nalm6 target cells based on bulk RNA sequencing (n = 3 technical replicates per group from one healthy donor); rows normalized to mean of control group. e. 2-NBDG uptake of antigen-stimulated CD8+ mock 19BBz and E81K-edited 19BBz CAR T cells (both with TRBC KO) as analyzed by flow cytometry (n = 4 biologically independent donors, values were norml. to mock control of each donor; mean ± SD; one-sample t-test). f. Volcano plot of metabolites in E81K-edited compared to mock 19BBz CAR T cells for three independent donors. (Two-sided unpaired Student’s t-test; Cut offs were log₁₀(Fold Change(FC)) > 0.15 or < 0.15 and p-value < 0.05.) g. Heatmap of log₁₀(Fold Change (FC)) of metabolites in E81K-edited compared to mock 19BBz CAR T cells for each independent donor. The heatmap displays log₁₀(FC), color-coded to indicate magnitude and direction of change. (n = 3 biologically independent donors, two-sided unpaired Student’s t-test; only metabolites with a p-value < 0.01 in at least one donor are shown.) h. Donor-corrected Principal Component Analysis (PCA) of PC1 and PC2 from metabolite profiles of mock 19BBz and E81K-modified 19BBz CAR T cells (both with TRBC KO) from n = 3 biologically independent donors 24 h after second Nalm6 tumor cell stimulation. i. Absolute NADH levels of mock 19BBz or E81K-modified 19BBz CAR T cells (both with TRBC KO) 24 h after second Nalm6 tumor cell stimulation (n = 4 replicates per group, representative of n = 2 biologically independent donors). j. Extracellular acidification rate (ECAR) traces as measured by seahorse mitochondrial stress test for mock 19BBz and E81K-edited 19BBz CAR T cells (both with TRBC KO) after exposure to three repeated stimulations with Nalm6 cells (mean of n = 5 technical replicates for one donor, representative for n = 3 biologically independent donors). k. Average basal ECAR, maximal ECAR, and spare glycolytic capacity levels in seahorse mitochondrial stress test (n = 3 biologically independent donors, mean ± SD, two-sided paired Student’s t-test). l. Mean fluorescent intensity (MFI) of mitochondrial mass content in CD8+ (left panel) and CD4+ (right panel) mock 19BBz and E81K-edited 19BBz CAR T cells after co-culture with Nalm6 cells following two consecutive rounds of cancer target stimulations as detected by MitoTracker orange staining (n = 3 technical replicates, reproduced in n = 2 biologically independent donors).