Fig. 4: Increased PI3K activity in E81K-edited 19BBz CAR T cells is associated with enhanced effector function and metabolic fitness.

Unmodified (mock) or E81K-modified 19BBz CAR T cells (TRBC KO) from one healthy donor were sorted after two antigen stimulations with Nalm6 tumor cells and subsequently subjected to scRNA-seq analysis. a, UMAP embedding of all analyzed cells. Clusters of interest are encircled in red. b, Cluster distribution within each condition. c, Comparison of selected populations of interest for E81K-modified and unmodified 19BBz CAR T cells as shown by UMAP embedding. d, Level of expression of the indicated exhaustion-related genes across all CD4⁺ clusters. e, The score for genes upregulated in CD19-targeting compared to exhaustion-prone HA-targeting CD4 effector memory CAR T cells⁴⁵ for all identified clusters. f, GSEA of the MSigDB Hallmark glycolysis gene set was performed for all identified clusters (permutation test). g, Joint transcriptomic and metabolomic pathway analysis (using MetaboAnalyst; www.metaboanalyst.ca) showing upregulated metabolic pathways in E81K-modified 19BBz CAR T cells relative to controls after two stimulations with Nalm6 target cells. Top-ranked pathways are labeled. Transcriptomic data were used from experiment shown in a,b. Metabolic analysis is shown in Extended Data Fig. 8e–g (hypergeometric testing). h, Cellular ion counts of NADH across E81K-modified and 19BBz control CAR T cells after second Nalm6 tumor stimulation measured by mass spectrometry (n = 3 biologically independent healthy donors; mean ± s.d.; two-sided paired Student’s t-test). i, OCR traces as measured by seahorse mitochondrial stress test for TRBC-KO control 19BBz (mock) and E81K-edited 19BBz CAR T cells after exposure to three repeated stimulations with Nalm6 cells. Lines mark the addition of oligomycin (2 μM), FCCP (1 μM) and rotenone + antimycin A (R + A; 0.5 μM) (mean of n = 5 technical replicates for one representative T cell donor of three donors). j, Average basal OCR, maximal OCR and spare respiratory capacity levels in seahorse mitochondrial stress test (n = 3 biologically independent T cell donors; mean ± s.d.; two-sided paired Student’s t-test).