Figure 2. ECs uptake extracellular mitochondria and upregulate vascular adhesion molecules, causing increased T cell adhesion.

A) Increasing concentrations of purified DsRed expressing murine mitochondrial were incubated with murine ECs and analyzed for mitochondria uptake by FACS. B) Murine ECs incubated with DsRed-mitochondria (30 μg/mL) were imaged with confocal microscopy and demonstrate intracellular localization of the exogenous mitochondria. Nuclei were labeled blue with DAPI. C-E) Upregulation of adhesion molecules, CD54, CD106 and CD62E were assessed by flow cytometry following a 16 hr pulse with mitochondria (n=3 per group). Upregulation of MHC II molecules on ECs following treatment with mitochondria (100 μg/mL). F) Representative flow cytometry plot and D) graph of n=3 per group. Treatment of ECs with mitochondria increases allogeneic T cell adhesion. H) Adherent T cells were quantified using a fluorescent plate reader based on a standard curve of the CFSE-labeled T cells (n=4 per group). I) Mouse ECs (bEnd.3, H-2^d) were treated for 5 hr with third-party mitochondria purified from LMTK fibroblasts (H-2^k), or with murine TNF-α (50 ng/mL), and then incubated with CFSE-labeled 4C-TCR-tg T cells (direct allo-specificity against I-A^d) for 30 mins. Comparisons were performed using a Kruskal-Wallis with Dunn’s multiple comparison test. *A p-value <0.05 was considered significant, ns=not significant.