In vitro cytolytic activity of CD19.CAR⁺EVs on Raji and SUP-B15 cell lines. (A) Cell viability of 2 cell lines, Raji (i) and SUP-B15 (ii), assessed by MTT assays after incubation for 48 hours with CD19.CAR⁺EVs at the indicated concentrations. Data shown are means ± SD of 3 to 4 replicates. ∗Statistically significant differences, compared with control (0 μg); ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (B) Flow cytometry killing assays were performed using the concentration of 0.015 μg of circulating EVs per target cell to treat both Raji and SUP-B15 cell lines and analyzing their cytolytic activity by measuring the 7-AAD staining of target cells after 24 hours of treatment (Student t test, P = .0187). Values are averages of 3 independent experiments. (C) Flow cytometry killing assays were performed using the concentration of 0.015 μg of preinfusion EVs per target cell to treat both Raji and SUP-B15 cell lines and analyzing their cytolytic activity by measuring the 7-AAD staining of target cells after 24 hours of treatment (Student t test, not significant). (D) The immunogenicity of CD19.CAR⁺EVs was studied by treating heterologous T cells for 24 hours with the same dose of CD19.CAR⁺EVs used for testing their cytolytic abilities (0.015-μg protein EV per target cell). The red histogram represents the CFSE profile of untreated heterologous T cells, whereas the overlaid blue histogram represents the CFSE profile of heterologous T cells treated with CD19.CAR⁺EVs. Data are representative of 2 independent experiments. CFSE, carboxyfluorescein succinimidyl ester.