RNA-seq and mass cytometry analyses of unstimulated UNTR and CAR-transduced T cells. (A) PCA of the top 500 variable genes from RNA-seq analysis across all experimental conditions (n = 6 HDs, n = 2 independent experiments). (B) PCA of mass cytometry EMD scores computed at 24 hours poststimulation in CD3⁺ cells across all experimental conditions (n = 4 HDs, n = 1 independent experiment). (C) Fuzzy clustering analysis of RNA-seq data across all experimental conditions (n = 6 HDs, n = 2 independent experiments). (D) Volcano plot showing differentially expressed genes between unstimulated FMC63 and CAT CAR T cells (top). The dashed horizontal line represents the statistical significance threshold (FDR <0.1). The bar plots show the expression of selected differentially expressed genes (FDR <0.1) in unstimulated UNTR and transduced T cells (bottom). Data represent mean ± SEM (n = 6 HDs, n = 2 independent experiments). (E) Bar plots showing the expression of mass cytometry EMD scores for granzyme B, perforin B, HLA-DR, CD25, NFAT1, pZAP70, and pS6 in unstimulated CAR T cells at 24 hours upon stimulation. The data shown are normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (0) represents the expression of a specific marker in unstimulated CD3⁺ UNTR T cells. Data represent mean ± SEM (n = 7 HDs, n = 2 independent experiments). Statistical significance was calculated by paired t test; ∗P < .05 and ∗∗P < .01. (A-E) Each experimental condition is indicated by a specific color code (unstimulated conditions: UNTR, light gray; FMC63, light blue; and CAT, orange; stimulated conditions: UNTR, gray; FMC63, blue; and CAT, red). FDR, false discovery rate; SEM, standard error of the mean.