Constitutive expression of SPI6 on MSCs and its role in protecting MSCs from allogeneic CTLs. (A) MSCs revealed constitutive expression of SPI6 as assessed by FACS. SPI6^−/− MSC staining was used as a negative control (blue line) versus the positive stain (black line) in WT MSCs. Ab indicates antibody. (B) Immunostaining was performed on WT MSCs, with intracellular SPI6 shown in green versus blue for DAPI (4′,6-diamidino-2-phenylindole). Immunostained images were taken with a Nikon E-1000 epifluorescence microscope at 200× magnification. (C) MSCs from WT and SPI6^−/− mice were incubated with primed allogeneic CTLs in a killing assay (n = 5 experiments). SPI6^−/− MSCs had 4 times higher cell death rates (expressed as percentage of cell lysis) than WT MSCs (P < .01). (D) MSCs were incubated with increasing E/T ratios, and SPI6^−/− MSCs showed a dose-dependent significant increase in cell death with increasing E/T ratios (P < .01). (E) MSCs were stained for SPI6 before (constitutive) and after (killing assay) incubation with CTLs, and SPI6 expression was analyzed by a dot plot on FACS. Data showed a significant decrease in the expression of SPI6 after exposure to CTLs (n = 3 experiments; P < .01). (F) GrB-i resulted in a significant reduction in the percentage of lysis of SPI6^−/− MSCs at the 2 concentrations used (n = 3 experiments; P < .02 and P < .01, respectively).