*Purpose: Endothelial cells (ECs) play a critical role in allorecognition, presentation, and costimulation for initiation of alloimmune rejection. The objective of this study was to test our hypothesis that exogenous mitochondria, a major source of DAMPs, interact with and activate allograft ECs, which fosters costimulation blockade resistant rejection (CoBRR), and to explore the underline mechanisms for mitochondrion-induced EC activation.

*Methods: Human ECs were stimulated by human mitochondria, purified from HeLa cells. Flow cytometry (FACS) and ImageStream FACS were used to detect EC activation, mito-fusion, mitophagy, and major targets involving in EC activation signaling pathway. The phenotype of adhering T cells to ECs was also defined by FACS. The impact of exogenous mitochondria to hearts was evaluated with a Balb/c to B6 heart transplant model.

*Results: Through direct contact, mitochondria induced EC activation as determined by upregulation of ICAM and VCAM expression and MCP-1, IL-8, and IL-6 production (p≤0.01). ECs internalize exogenous mitochondria via scavenger receptors resulting in intracellular adenosine triphosphate elevation, reactive oxygen species production, phosphorylation of nuclear factor kappa-light-chain-enhancer p65 (NF κB), mitophagosome formation, and exogenous-endogenous mitochondria fusion. Increased Bcl-2 expression and phosphorylation at the Ser70 site were observed. Exogenous mitochondrion-induced EC activation was completely inhibited by inhibitors specific for NF κB, and STING but not MyD88 and FPF-1. Increased adhesion of allospecific T cells characterized as effector and terminally differentiated effector memory cells to mitochondrion-activated endothelium was revealed. Infusion of purified mouse mitochondria into Balb/c resulted in upregulation of MHC class I and CD54 expression on cardiac microvascular ECs measured immunohistochemically. In a Balb/c to B6 heart transplant model, exposing donor hearts to exogenous mitochondria promotes B7 Co-BRR, and allograft survival was significantly reduced (mean survival=15.6±4.9 days, p=0.0001) when compared with B6 mice treated with CTLA4-Ig receiving hearts without mitochondrial pre-exposure (mean survival > 65 days).

*Conclusions: Our results indicate that exogenous mitochondria directly initiate EC activation via STING- NF κB signaling pathway, and trigger T cell-mediated costimulation blockade-resistant allograft rejection.

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