Fig. 7
Antigen concentration and antigen pathway inhibitors modulate cytotoxicity. a Timeline of experimental methodology and treatment times. b Quantification of time-lapse imaging of OVA257–264 peptide (0.5–500 μg range) (top) [(500 μg/mL vs.5.0 μg/mL mean diff = 10320; 95% CI = 1902–18738)] and ovalbumin (5–1000 μg range) (bottom) [(1000 μg/mL vs.50 μg/mL mean diff = 12641; 95% CI = 4199–21083)] concentrations added to OPCs stimulated with IFNγ and antigen prior to CD8 addition. c Proliferation analysis of OPC-CD8 co-cultures treated with antigen cross-presentation pathway inhibitors. No inhibitor (No I) or each inhibitor were added to OPC culture for two hours prior to the addition of OVA257–264 (white) or Ovalbumin (blue), and then left in wells during processing and presentation phase. Chloroquine was added at 100 μM. ONX-0914, PSMB8 inhibitor was added at 30 nM. Cathepsin S inhibitor was added at 10 nM. In all, 48 h after CD8 co-culture was initiated cell proliferation of the Vβ5, CD3, CD8a population of CD8s was analyzed for cell division and total percent divided. Significance for all quantified data was either assessed by two-way ANOVA analysis followed by a Tukey’s multiple comparison analysis [(NoT vs. Chloroquine mean diff = 40.8; 95% CI = 27.1–54.4, NoT vs. ONX-0914 mean diff = 37.6; 95% CI = 24.0–51.3, NoT vs. Cathepsin S inhibitor mean diff = 60.9; 95% CI = 47.2–74.6)]. Error bars in all graphs represent standard deviation from three experiments. d, e Time-lapse images and quantification of from 0 to 60 h with the antigen presentation pathway inhibitors; Chloroquine (pink circle; 300 μM) [(mean diff = 18203; 95% CI = 11864–24542)], Cathepsin S inhibitor (white circle; 30 nM) [(mean diff = 9775; 95% CI = 3436–16114)], PSMB8 inhibitor (green circle; ONX-0914; 30 nM) [(mean diff = 13059; 95% CI = 6720–19398)]. Significance of differences between conditions is shown in the legend for each figure. Significance was determined by area under the curve analysis followed by one-way ANOVA and Tukey’s multiple comparisons test. Error bars represent standard deviation from 3 to 5 experiments. (P*≤0.05, **≤ 0.01, ***≤0.001, ****≤0.0001)
