Fig. 5

OPCs cross-present ovalbumin and activate CD8⁺ T cells. a Timeline and experimental design. OPCs were cultured in PDGF to inhibit differentiation and stimulated with IFNγ (10 ng/mL) for 12 h prior to Ovalbumin (500 μg/mL) protein addition. Both IFNγ and Ovalbumin protein were incubated with OPCs for a total of 8 h prior to washing the cultures to remove unprocessed Ovalbumin. OT-1 CD8⁺ T cells were isolated by magnetic sorting then stained with Cell Proliferation Dye eFluor 450 (10 μM) prior to initiation of CD8/OPC co-culture. In all, 24–48 h after the start of the co-culture CD8 were analyzed for activation. (b; top left) CD8 percentage, at 24 h after the co-culture was initiated, was determined using Vβ5, CD3, and CD8, and subsequently used as a parent gate for all flow plots in the figure. b CD8 morphology (top), survival (2nd row) (mean diff = −71.6, 95% CI = −107.7 to −35.6), activation status using CD25⁺/CD69⁺ (3rd row) (mean diff = −58.1, 95% CI = −32.7 to −6.6), and proliferation (bottom) (Undivided mean diff = 41.1, 95% CI = 17.8–64.4, 3⁺ Division mean diff = −32.8, 95% CI = −56.1 to −9.5) with quantification (left to each panel) of OT-1s cultured with OPCs; no peptide (white; n = 4), Ovalbumin (light gray; n = 4), IFNγ + no peptide (gray; n = 4), and IFNγ + Ovalbumin (blue; n = 4). c Cytokine and granular protein profiling of OT-1s; IFNγ (1st row) (mean diff = −46.5, 95% CI = −73.4 to −19.36), TNFα (2nd row) (mean diff = −50.1, 95% CI = −72.9 to −27.3), perforin (3rd row) (mean diff = −23.2, 95% CI = −35.6−(−)10.8) and granzyme B (4th row) (mean diff = −32.7, 95% CI = −32.7–(−)6.6). Significance for all quantified data was assessed by one-way ANOVA analysis followed by Tukey’s multiple comparison analysis (P*≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars represent standard deviation for four biological replicates