Figure 3.
Phenotypic and molecular characterization of stimulated CAR-transduced T cells. (A) Variation (log2 fold change) of CD4 and CD8 proportion in stimulated UNTR T cells and FMC63 and CAT CAR T cells measured by FACS (n = 12 HDs, n = 3 independent experiments). The dotted horizontal line (0) represents the conditions in which CD4 = CD8. (B) Bar plot showing the percentage of TCM (CD45RA−CD62L+) in stimulated CD3+ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 96 hours postantigen stimulation (n = 7 HDs, n = 2 independent experiments). (C) Bar plots showing the expression of T-cell exhaustion markers (PD1, TIM3, and LAG3) as MFI in stimulated CD3+ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 96 hours postantigen stimulation (n = 4 HDs, n = 1 independent experiment). (D) Volcano plot showing differentially expressed genes between FMC63 and CAT CAR T cells upon NALM6 coculture (left). The dashed horizontal line represents the statistical significance threshold (FDR <0.1). Bar plots showing the expression of selected differentially expressed genes (FDR <0.1) in stimulated UNTR T cells and in CAR T cells (n = 6 HDs, n = 2 independent experiments) (right). (E) Bar plots showing the expression of mass cytometry EMD scores for CD25, HLA-DR, NFAT1, FOXP3, pZAP70, pS6, pp38, pCREB, pRB, and CD4 in stimulated 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 stimulated CD3+ UNTR T cells (n = 7 HDs, n = 2 independent experiments). (A-E) Bar plots show mean ± SEM. Each experimental condition is indicated by a specific color code (UNTR, gray; FMC63, blue; and CAT, red). Panels A-C,E show statistical significance calculated by paired t test; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. FDR, false discovery rate; MFI, mean fluorescent intensity; SEM, standard error of the mean.

Phenotypic and molecular characterization of stimulated CAR-transduced T cells. (A) Variation (log2 fold change) of CD4 and CD8 proportion in stimulated UNTR T cells and FMC63 and CAT CAR T cells measured by FACS (n = 12 HDs, n = 3 independent experiments). The dotted horizontal line (0) represents the conditions in which CD4 = CD8. (B) Bar plot showing the percentage of TCM (CD45RA−CD62L+) in stimulated CD3+ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 96 hours postantigen stimulation (n = 7 HDs, n = 2 independent experiments). (C) Bar plots showing the expression of T-cell exhaustion markers (PD1, TIM3, and LAG3) as MFI in stimulated CD3+ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 96 hours postantigen stimulation (n = 4 HDs, n = 1 independent experiment). (D) Volcano plot showing differentially expressed genes between FMC63 and CAT CAR T cells upon NALM6 coculture (left). The dashed horizontal line represents the statistical significance threshold (FDR <0.1). Bar plots showing the expression of selected differentially expressed genes (FDR <0.1) in stimulated UNTR T cells and in CAR T cells (n = 6 HDs, n = 2 independent experiments) (right). (E) Bar plots showing the expression of mass cytometry EMD scores for CD25, HLA-DR, NFAT1, FOXP3, pZAP70, pS6, pp38, pCREB, pRB, and CD4 in stimulated 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 stimulated CD3+ UNTR T cells (n = 7 HDs, n = 2 independent experiments). (A-E) Bar plots show mean ± SEM. Each experimental condition is indicated by a specific color code (UNTR, gray; FMC63, blue; and CAT, red). Panels A-C,E show statistical significance calculated by paired t test; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. FDR, false discovery rate; MFI, mean fluorescent intensity; SEM, standard error of the mean.

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