Fig. 6

Caspase3/7 increases in OPCs that activate OT-1 CD8⁺ T cells. a Fas surface expression on OPCs, either not stimulated (gray) or IFNγ stimulated (blue), as determined by flow cytometry for PDGFRα⁺/CD11b⁻ (FMO; white). Significance was determined by unpaired, two-tailed Student’s t-test (P = 0.0046). Error bars represent standard deviation; 7 independent experiments. b Descriptive images of the cytotoxicity assay using NucLight stained OPCs and a Caspase 3/7 reporter. c Single cell controls of OT-1 CD8s (black circle) and OPCs (black square) cultured separately for a total of 60 hours with caspase 3/7 detection reagent and nuclear staining in OPCs. Representative images for 0 h, 24 h, and 48 h are shown and quantified from 0 to 60 h (h). d Time-lapse phase contrast and fluorescence imaging of OPC-CD8 co-cultures. At 2.0 h CD8s have an elongated/clustering morphology. At 20.0–28.0 h, CD8 cluster with the underlying caspase3/7 active OPC. e Time-lapse imaging of OPC-CD8 co-cultures; no antigen (gray triangle), ovalbumin protein (blue triangle), IFNγ stimulated OPC only (white circle). IFNγ+no antigen (gray circle) and IFNγ+ovalbumin protein (blue circle) [(IFNγ+no antigen vs. IFNγ+ovalbumin mean diff = −11410; 95% CI = −16239–(−)6581)] (h). f Total NucLight positive OPC quantification of the time course of the OPC-OT-1 co-culture. IFNγ stimulated OPCs that were pulsed with ovalbumin protein before the co-culture have a significant increase in number by 46 h. g At the time of CD8⁺ T cell addition to OPCs stimulated with IFNγ+ovalbumin, pan caspase inhibitor (white circle;Q-VD-OPH; 10 μM) [(Vehicle vs. Q-VD-OPH mean diff = 21686; 95% CI = 15771–27601)], granzyme B inhibitor (green circle; 300 nM) [(Vehicle vs. GrB inhibitor mean diff = 16369; 95% CI = 10454–22284)] and FasL decoy receptor (pink circle; DcR3; 300 nM) [(Vehicle vs. DcR3 mean diff = 17751; 95% CI = 11836–23666)] were applied to the co-culture to determine the contribution of CD8 cytotoxicity pathways to OPC caspase activity. Significance of differences between conditions is shown in the legend for each figure. Significance for all quantified data was determined by area under the curve analysis followed by one-way ANOVA and Tukey’s multiple comparisons test (P*≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). All Error bars represent standard deviation from 3 to 5 experiments (unless otherwise specified)