*Purpose: Endothelial cells (ECs) form a barrier between vascularized organ allografts and host immunity and are known to provoke alloimmune responses. The mTOR inhibitor rapamycin is known to effectively attenuate alloimmunity, a trait that has been attributed to its action on T cells. However, given the ubiquity of the mTOR pathway, we hypothesized that rapamycin could also perhaps act directly on ECs, altering T-cell immunity through a direct effect on donor ECs.

*Methods: Human ECs were studied with and without exposure to rapamycin for cytokine release using a multiplex assay, and T cell responses to ECs were assayed using an intracellular cytokine staining (ICCS) and a VPD450-dilution based T cell proliferation assay. A transwell system was used to evaluate the role of EC-T cell contact in observed responses. RNA-sequencing was performed on naïve and rapamycin-pretreated ECs to assess alterations in transcriptomic profiles. Mouse heart transplants were used to assess effects of rapamycin pre-treatment on donors.

*Results: Unlike untreated ECs, rapamycin-pretreated ECs significantly suppressed T-cell proliferation (p<0.0001). The suppressive effect persisted (p<0.0001) despite transwell separation between ECs and responding T cells in MLR. Rapamycin-pretreated ECs made less IL-6, IL-8, and MCP-1 following TNF-a/IFN-g stimulation, but exogenous IL-6/IL-8/MCP-1 did not reverse the suppressive effect on T-cell proliferation. Rapamycin-pretreated ECs upregulated PD-1 ligand expression, though PD-1 blockade failed to restore T cell proliferation. The effect of rapamycin-treated ECs was partial on polyfunctional allospecific memory CD8+ T cells. Medium collected from control or rapamycin-pretreated ECs was size-filtered based on molecular weights and tested in MLR. We demonstrated that molecule(s) eliciting immunosuppressive effects were above 10 kDa. RNA-sequencing analysis revealed a downregulation of genes in pathways associated with cytokine/chemokines, cell adhesion, NF-kB, and transendothelial migration. Pre-exposing donor hearts to rapamycin improved the effect of B7 costimulation blockade, prolonging the survival of heart allografts in an MHC mismatched murine model.

*Conclusions: mTOR blockade down-regulates genes in ECs involving in pathways associated with immune-recognition, adhesion, presentation, and costimulation, and reduces the capacity of ECs to stimulate allospecific T cell immune-responses independent of PD-1/PD-ligand interaction, acting in a contact-independent manner. Pre-treatment of a donor with rapamycin synergizes with CTLA4Ig, prolonging cardiac allograft survival in MHC-mismatched mice. This finding implies that in addition to its effect on recipient immunity, rapamycin acts directly on the donor organ, lessing its immunogenicity.

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