*Purpose: As semi-professional antigen-presenting cells, microvascular endothelial cells (mECs) play a central role in mediating allograft rejection. Cold storage and ischemia reperfusion injury increases mEC immunogenicity, leading to T-cell alloimmune responses upon reperfusion. Recent studies have demonstrated that altering mitochondrial fusion/fission can change the phenotype of immune cells. However, whether this has an impact on mECs has not been documented. Therefore, in the present study, we sought to modulate mitochondrial fusion/fission in donor mECs prior to cold storage and assessed the impacts on mEC-T cell interaction post-reperfusion.

*Methods: mECs were pre-treated normothermically with M1, a fusion promoter, and Mdivi1, a fission inhibitor, to promote mitochondrial fusion prior to cold storage. Upon reperfusion, they were co-cultured with allogeneic CD8+ T-cells for 7 days. T-cell granzyme B, interferon gamma, Ki-67 as a marker for proliferation, and CD69 as a marker for activation were assessed using this co-culture system. In addition, expression of mEC surface markers was also determined during the early period after reperfusion.

*Results: Pre-treating mECs with M1/Mdivi1 significantly reduced proliferation and activation of co-cultured CD8+ T-cells by day 4, as well as decreased granzyme B and interferon gamma release from CD8+ T-cells by the end of co-cultures. Treatment with M1/Mdivi1 led to a decrease in VCAM-1 and an increase in PD-L1 EC surface expression as early as 24 hours post-reperfusion, and these changes normalized by day 4 post-reperfusion.

*Conclusions: Promoting mitochondrial fusion/inhibiting fission in donor mEC alters the outcome of T-cell alloimmune responses. These findings provide a foundation for translational approaches to pre-treat donor organs and induce allograft tolerance.

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