Figure 5.
Molecular characterization of CAR T cells generated from CD19-depleted PBMCs. (A) UMAP representation of the 4 cell populations (CD8, CD4, CD3 low/neg, and CD19) identified by FlowSOM analysis in 4 representative unstimulated samples analyzed by mass cytometry at 24 hours poststimulation (left) (n = 4 HDs, n = 1 independent experiment). Cell types are indicated by different colors. Percentage of residual B cells detected by mass cytometry in unstimulated samples at 24 hours poststimulation (right) (n = 7 HDs, n = 2 independent experiments). (B) Bar plots showing the percentage of TCM (CD45RA−CD62L+) (left) and TEM (CD45RA−CD62L−) (right) in unstimulated CD19-depleted CD3+ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS (n = 4 HDs, n = 1 independent experiment). (C) Bar plots showing the expression of mass cytometry EMD scores for granzyme B, perforin B, HLA-DR, CD25, NFAT1, pZAP70, and pS6 in unstimulated CD19-depleted 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+ CD19-depleted UNTR T cells. (n = 3 HDs, n = 1 independent experiment). (D) Bar plots showing the expression of mass cytometry EMD scores for CD25, HLA-DR, NFAT1, pZAP70, pS6, pp38, pRB, and CD4 in stimulated CD19-depleted 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 CD19-depleted CD3+ UNTR T cells (n = 3 HDs, n = 1 independent experiment). (E) Bar plots showing the mean polyfunctionality in CD19-depleted CAR T cells at 24 hours upon stimulation. The data shown are normalized to stimulated CD3+ UNTR T cells. The dotted horizontal line (1) represents the mean polyfunctionality in stimulated CD3+ CD19-depleted UNTR T cells (n = 3 HDs, n = 1 independent experiment). (A-E) Each experimental condition is indicated by a specific color code (unstimulated conditions: UNTR, light gray; FMC63, light blue; CAT, orange; stimulated conditions: FMC63, blue; CAT, red). Bar plots show mean ± SEM. Statistical significance was calculated by paired t test; ∗P < .05 and ∗∗P < .01. SEM, standard error of the mean.

Molecular characterization of CAR T cells generated from CD19-depleted PBMCs. (A) UMAP representation of the 4 cell populations (CD8, CD4, CD3 low/neg, and CD19) identified by FlowSOM analysis in 4 representative unstimulated samples analyzed by mass cytometry at 24 hours poststimulation (left) (n = 4 HDs, n = 1 independent experiment). Cell types are indicated by different colors. Percentage of residual B cells detected by mass cytometry in unstimulated samples at 24 hours poststimulation (right) (n = 7 HDs, n = 2 independent experiments). (B) Bar plots showing the percentage of TCM (CD45RA−CD62L+) (left) and TEM (CD45RA−CD62L−) (right) in unstimulated CD19-depleted CD3+ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS (n = 4 HDs, n = 1 independent experiment). (C) Bar plots showing the expression of mass cytometry EMD scores for granzyme B, perforin B, HLA-DR, CD25, NFAT1, pZAP70, and pS6 in unstimulated CD19-depleted 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+ CD19-depleted UNTR T cells. (n = 3 HDs, n = 1 independent experiment). (D) Bar plots showing the expression of mass cytometry EMD scores for CD25, HLA-DR, NFAT1, pZAP70, pS6, pp38, pRB, and CD4 in stimulated CD19-depleted 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 CD19-depleted CD3+ UNTR T cells (n = 3 HDs, n = 1 independent experiment). (E) Bar plots showing the mean polyfunctionality in CD19-depleted CAR T cells at 24 hours upon stimulation. The data shown are normalized to stimulated CD3+ UNTR T cells. The dotted horizontal line (1) represents the mean polyfunctionality in stimulated CD3+ CD19-depleted UNTR T cells (n = 3 HDs, n = 1 independent experiment). (A-E) Each experimental condition is indicated by a specific color code (unstimulated conditions: UNTR, light gray; FMC63, light blue; CAT, orange; stimulated conditions: FMC63, blue; CAT, red). Bar plots show mean ± SEM. Statistical significance was calculated by paired t test; ∗P < .05 and ∗∗P < .01. SEM, standard error of the mean.

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