Cytokine polyfunctionality in stimulated CAR-transduced T cells. (A) Bar plots showing the expression of mass cytometry EMD scores for effector (granzyme B, IFN-γ, TNF-α) and stimulatory (GM-CSF, IL-2) cytokines in stimulated CAR T cells. 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). (B) Bar plots showing the mean cytokine polyfunctionality in stimulated CAR T cells, normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (1) represents the mean polyfunctionality in stimulated CD3⁺ UNTR T cells. (n = 7 HDs, n = 2 independent experiments). (C) Stacked bar plots showing the percentage of stimulated CAR T cells (CD3⁺mCherry⁺) expressing 1 to 4 or ≥5 cytokines per cell as measured by mass cytometry. (D) Circos plots showing all the combinations of the 8 cytokines in stimulated FMC63 (left) and CAT (right) CAR T cells analyzed by mass cytometry. The numbers indicate patterns of cytokine coexpression (from 1-4 or ≥5 cytokines/cell). A specific color code has been assigned to each cytokine. (A-B) Data represent mean ± SEM. Statistical significance was calculated by paired t test; ∗P < .05 and ∗∗∗P < .001. Each experimental condition is indicated by a specific color code (FMC63, blue and CAT, red). GM-CSF, granulocyte-macrophage colony-stimulating factor; SEM, standard error of the mean; TNF-α, tumor necrosis factor α.