Rapamycin prevents costimulation blockade (CoB)-resistant rejection in mice and primates, and early human data suggests a similar salutary effect. We have hypothesized that one mechanism by which rapamycin synergizes with CoB may be through its direct effects on allograft endothelial cells (ECs) in addition to its known effect on allospecific T cells. To study this, naïve or rapamycin-conditioned human ECs stimulated with PMA were analyzed using flow cytometry (FACS) to detect the phosphorylation level of mTOR complex-1 (mTORC1) and the downstream molecule S6. The phenotype of ECs including adhesion, costimulation, inhibitory and HLA molecules was also studied by FACS, and EC production of IL-8 was analyzed by intracellular cytokine staining (ICCS). EC-stimulated allospecific T cell responses were evaluated by ICCS and VPD-450 based T cell proliferation with or without a PD1 blocking antibody. Cytokine-treated ECs upregulated adhesion, costimulatory, inhibitory, and HLA class I/II molecules. Rapamycin treated ECs demonstrated reduction of phosphorylation of mTORC1 and S6 without changes in IL-8 production after stimulation. Rapamycin/IFNγ treated ECs showed significant reduction in CD40, HLA-DR/DQ expression (p<0.05) when compared with IFNγ treated-ECs. Rapamycin/TNFa treated ECs showed higher OX40L, CD58, and HLA-ABC expression when compared to TNFa treated-ECs (p<0.05). Rapamycin treated ECs showed higher levels of PD-1L expression when compared with untreated ECs (p<0.05), while CD4⁺ and CD8⁺ cells undergoing proliferation following EC stimulation expressed PD-1. Interestingly, although rapamycin treated ECs significantly inhibited T cell proliferation when compared with untreated ECs (p<0.05), PD-1 blockade did not restore the T cell proliferation to rapamycin-treated ECs, suggesting that the effect was independent of PD-1/PD-1L interactions. Though naïve T cell responses were decreased in rapamycin-treated ECs, the activation of polyfunctional allospecific memory CD8⁺ cells was not suppressed by rapamycin EC treatment. In summary, rapamycin inhibits phosphorylation of mTORC1 and S6 in ECs, and alters the EC surface phenotype. Rapamycin treatment reduces the capacity of ECs to stimulate naïve allospecific T cell proliferation, but does not impair memory CD8 cell activation, and these effects are not altered by PD-1 blockade. Our findings suggest that some of rapamycin's salutary effects may be attributable to direct effects on EC alloimmunogenicity.

CITATION INFORMATION: Gao Q., Bendersky V., Kirk A., Xu H. Rapamycin Impairs Human Endothelial Cell Alloimmunogenicity Independent of PD-1/PD-1 Ligand Inhibitory Signaling Am J Transplant. 2017;17 (suppl 3).

« Back to 2018 American Transplant Congress