Introduction: Vascular endothelial cells (VECs), the initial barrier between host immunity and donor allograft tissue, play a critical role in allograft rejection. We investigated the activation of primary human VECs by mitochondria and their role in activating alloreactive CD8+ T cells.

Methods: Mitochondria were isolated from HeLa cells and their purity was verified by western blot and flow cytometry (FACS). Primary human VECs treated with mitochondria (100 ug/mL for 12-24 hr) or vehicle alone were assessed by measuring surface markers of activation by FACS and cytokine production by ELISA. CD8+ T-cells isolated from healthy donors were co-cultured with activated VECs and assessed for effector cytokine production by FACS analysis following intracellular cytokine staining. Memory CD8+ T cell phenotype was determined by CCR7 and CD45RA expression. Uptake of fluorescently labeled mitochondria by VECs was demonstrated by conventional and 3D-video confocal microscopy.

Results: VECs upregulated surface markers of activation in response to treatment with purified extracellular mitochondria compared with vehicle control; CD54+ (28.4±3.9 vs 1.9±0.5%), CD106+ (14.3±2.0 vs 2.3±1.6%) and CD62E+ (5.6±1.8 vs 2.1±0.7%). Similarly, VECs produced inflammatory cytokines in response to mitochondria compared to vehicle control: MCP-1 (1859±99 vs 0 pg/mL), IL-8 (3925±102 vs 0 pg/mL), and IL-6 (464±40 pg/mL). VEC activation by mitochondria required direct contact, excluding the role of soluble mediators in VEC activation. By 3D video confocal microscopy we observed that VECs internalized fluorescently-labeled mitochondria and that these mitochondria fused with endogenous VEC-mitochondria. The uptake of mitochondria was not associated with annexin V staining, caspase 3 cleavage or cell death. Mitochondrial conditioned VECs augmented allogeneic CD8+ T cell effector memory response as determined by dual TNF-α/IFN-γ production.

Conclusions: Mitochondria uptake leads to VEC activation and augments their ability to simulate alloreactive effector memory T cells. Mitochondrial released in the setting of tissue injury can induce endothelial cell activation, thereby serving as as danger signals that initiate allograft rejection.

CITATION INFORMATION: Brennan T, Feng F, Lin L, Song M, Li Q.-J, Kirk A, Xu H. Uptake of Extracellular Mitochondria Activates Human Endothelial Cells That Stimulate Alloreactive Effector T Cell Responses. Am J Transplant. 2017;17 (suppl 3).

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