Extended Data Fig. 9: Impact of CD3ζ ITAM mutations in TRAC-1928ζ on T cell differentiation state and effector profile.

a, GSEA of a signature of the top 200 genes upregulated in exhausted CD8 T cells relative to naive or memory CD8 T cells as derived from GSE41867, demonstrating enrichment of exhaustion signature in TRAC-1928ζ versus TRAC-1XX or TRAC-XX3 and in the sorted control T_EFF versus T_N and T_SCM. Experiment was performed in technical triplicates for each CAR construct and in six replicates for each control subset. b, Gene ontology analysis demonstrating significantly enriched gene sets associated with inflammation, cytokine and chemokine signaling in 1928ζ versus XX3, 1XX versus XX3 and 1928ζ versus 1XX (n = 3). Transcriptional analysis was performed after CAR gene integration into the TRAC locus of naive T cells and stimulation with CD19⁺ target cells. Results are shown in order of significance as –log₁₀ (corrected P value). P values were determined by a one-tailed Fisher’s exact test and the Benjamini–Hochberg method was used to correct for multiple hypotheses testing. c, Heat map of selected differentially expressed genes between CAR constructs related to inflammation, cytokine and chemokine activity. d, Flow cytometric analysis of T cell differentiation state on CD8⁺CAR T cells after stimulation with CD19 antigen (representative for n = 2 independent experiments with similar results).