Figure 1.
Generation and phenotypic characterization of CAR T cells from HD-PBMCs. (A) Experimental workflow. PBMCs were isolated from HDs and LV transduced to express CD19 CAR construct (FMC63 or CAT) following overnight activation with CD3/CD28 beads. Six days after transduction, CAR T cells were cultured without (unstimulated) or with target cells (NALM6) at a 1:1 ratio (stimulated). Unstimulated and stimulated cells were analyzed by flow cytometry and sorted for RNA-seq 24 hours poststimulation. Mass cytometry analysis was performed on unstimulated and stimulated cells 24 hours poststimulation. Activated UNTR T cells were used as a control throughout the experiment. (B) (Left) spaghetti plots showing transduction levels of CAR T cells as percentage of mCherry+ (in CD3+) and (right) as MFI of mCherry in unstimulated transduced T cells measured by FACS 7 days posttransduction. Lines connect results from individual donors (n = 12 HDs, n = 3 independent experiments). (C) Spaghetti plot showing the percentage of surface CAR expression (in CD3+) in unstimulated transduced T cells measured by FACS 10 days posttransduction. Lines connect results from individual donors (n = 4 HDs, n = 1 independent experiment). (D) Variation (log2 fold change) of CD4 and CD8 proportion in unstimulated UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 7 days posttransduction. The dotted horizontal line (0) represents the conditions in which CD4 = CD8. Data represent mean ± SEM (n = 12 HDs, n = 3 independent experiments). (E) Bar plots showing the percentage of TCM (CD45RA−CD62L+) (left), TEM (CD45RA−CD62L−) (middle), and TEMRA (CD45RA+CD62L−) (right) in unstimulated CD3+ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 10 days posttransduction. Data represent mean ± SEM (n = 7 HDs, n = 2 independent experiments). (B-E) Statistical significance was calculated by paired t test; ∗P < .05, ∗∗P < .01, and ∗∗∗∗P < .0001. Each experimental condition is indicated by a specific color code (UNTR, light gray; FMC63, light blue; CAT, orange). MFI, mean fluorescent intensity; SEM, standard error of the mean.

Generation and phenotypic characterization of CAR T cells from HD-PBMCs. (A) Experimental workflow. PBMCs were isolated from HDs and LV transduced to express CD19 CAR construct (FMC63 or CAT) following overnight activation with CD3/CD28 beads. Six days after transduction, CAR T cells were cultured without (unstimulated) or with target cells (NALM6) at a 1:1 ratio (stimulated). Unstimulated and stimulated cells were analyzed by flow cytometry and sorted for RNA-seq 24 hours poststimulation. Mass cytometry analysis was performed on unstimulated and stimulated cells 24 hours poststimulation. Activated UNTR T cells were used as a control throughout the experiment. (B) (Left) spaghetti plots showing transduction levels of CAR T cells as percentage of mCherry+ (in CD3+) and (right) as MFI of mCherry in unstimulated transduced T cells measured by FACS 7 days posttransduction. Lines connect results from individual donors (n = 12 HDs, n = 3 independent experiments). (C) Spaghetti plot showing the percentage of surface CAR expression (in CD3+) in unstimulated transduced T cells measured by FACS 10 days posttransduction. Lines connect results from individual donors (n = 4 HDs, n = 1 independent experiment). (D) Variation (log2 fold change) of CD4 and CD8 proportion in unstimulated UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 7 days posttransduction. The dotted horizontal line (0) represents the conditions in which CD4 = CD8. Data represent mean ± SEM (n = 12 HDs, n = 3 independent experiments). (E) Bar plots showing the percentage of TCM (CD45RA−CD62L+) (left), TEM (CD45RA−CD62L−) (middle), and TEMRA (CD45RA+CD62L−) (right) in unstimulated CD3+ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 10 days posttransduction. Data represent mean ± SEM (n = 7 HDs, n = 2 independent experiments). (B-E) Statistical significance was calculated by paired t test; ∗P < .05, ∗∗P < .01, and ∗∗∗∗P < .0001. Each experimental condition is indicated by a specific color code (UNTR, light gray; FMC63, light blue; CAT, orange). MFI, mean fluorescent intensity; SEM, standard error of the mean.

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