MKs activate CD8⁺ T cells via OVA crosspresentation. MKs were pulsed with or without OVA (+OVA or –OVA, respectively) and cocultured with OT-1 CD8⁺ T cells at 2 different cell ratio MK:CD8⁺ T cells (effector:target ratios = 1:1 and 1:2.5) for 24, 48, or 72 hours. In addition, B/c MKs were cocultured with OT-1 T cells to verify the MHC class I haplotype specificity of the CD8⁺ T-cell activation. MKs were also cocultured with WT BL/6 CD8⁺ T cells as a negative control with naïve CD8⁺ T cells. CD69 surface expression was assessed to monitor CD8⁺ T-cell activation (A). CD8⁺ T-cell activation was also measured with and without tubulin cytoskeleton or proteasome inhibitors (nocodazole and MG132, respectively) during OVA pulse (B). IL-2 was measured in coculture medium to confirm CD8⁺ T-cell activation (C). CD8⁺ T-cell proliferation was also measured (D). One-way ANOVA with a Tukey correction for multiple testing; n ≥ 5; mean with SD (A-D). Cytotoxic response was quantified in MKs using Annexin V as a marker of apoptosis by flow cytometry (E). Paired t or Wilcoxon test; n = 5, mean with SD. These results were confirmed at 48 hours by quantitative widefield high-content microscopy. Unpaired t test; n = 5; mean with SD (F). A representative image of the coculture used for the quantification shows the staining for Annexin V (green) in CD41⁺ MKs (red) in coculture with CD8⁺ T cells (unlabeled) in the presence or absence of OVA (G). Scale bar represents 50 μm. ****P < .0001; ***P < .001; **P < .01; *P < .05; each n corresponds to an independent MK donor and CD8⁺ T-cell donor coculture.