Monensin and brefeldin

Degranulation, measured by cell surface modulation of CD107a^{25 (link)} and intracellular cytokine production (TNF-α and IFN-γ), was analyzed by flow cytometry of CAR T cells incubated with different target cells. Briefly, CD44v6.CAR T and CD19.CAR T cells from different donors, at day 11–15 after the initial stimulation, were left untreated or stimulated with target cells at the ratio of 1:1. Anti-CD107a Ab (Miltenyi) and monensin and brefeldin (BD Biosciences) were added during the incubation period. As positive control, CAR T cells were stimulated with 10 ng/mL phorbol myristate acetate (PMA; Sigma) and 1 μg/mL ionomycin (IONO; Sigma). After 5 h of incubation, cells were stained with anti-CD3 mAb (BD Bioscience) and Viability Stain 510 (BD Bioscience), fixed, permeabilized (Cytofix/Cytoperm kit, following the manufacturer's instruction; BD Bioscience), and then stained for intracellular cytokines with TNF-α- and IFN-γ (BD Bioscience)-specific Abs. Cells were analyzed by flow cytometry, and viable CD3⁺/CD4⁺ or CD3⁺/CD8⁺ cells were analyzed for CD107a, TNF-α, and IFN-γ expression. The percentage of positive CAR T cells incubated alone (always <5%) was subtracted to the percentage of positive CAR T cells stimulated with the different targets or PMA/IONO.