Early GrB and caspase-3 activity showed early killing of SPI6^−/− MSCs. (A) Early GrB activity in WT and SPI6^−/− MSCs subjected to CTLs was detected by a GranToxiLux assay and characterized by a population shift from the top left quadrant to the top right quadrant. MSCs were fluorescently labeled (red; FL-4) before coincubation with CTLs in the presence of a fluorogenic GrB substrate. Cleavage of the substrate results in increased green fluorescence (FL-1) in MSCs with GrB activity. (B) The percentage of MSCs with GrB activity (top right quadrant) is shown in the bar graph. SPI6^−/− MSCs showed significantly higher GrB activity at 5 and 20 minutes (6.06% ± 2.56% vs 11.5% ± 3.54% and 41.6% ± 4.32% vs 25.5% ± 2.89%, respectively; P < .02; n = 3 experiments). (C) Caspase-3 was assessed in WT and SPI6^−/− MSCs subjected to CTLs at the same time points as for GrB. Results showed significantly higher caspase-3 activity in SPI6^−/− MSCs than in WT MSCs (B6 MSC/CTL) at 5 and 20 minutes when challenged with CTLs (1.01 ± 0.36 vs 2.14 ± 0.042 and 0.051 ± 0.009 vs 0.831 ± 0.067, respectively; n = 3 experiments; P < .02).