*Purpose: Current paradigms indicate that endothelial cells (EC) within allografts participate in the initiation and progression of allograft rejection. However, little is known about their function in association with tolerance, and it is generally viewed that intragraft EC are passive and remain in a quiescent state in association with immunoregulation and long-term graft survival. In these studies, we wished to address this paradigm, and test the hypothesis that intragraft EC express novel molecules that promote a local pro-tolerogenic microenvironment.

*Methods: Fully MHC mismatched BALB/c hearts were transplanted into C57BL/6 Rag2^-/-Il2rg^-/- recipients. On day 2 post-transplantation, purified populations of CD4⁺Foxp3-YFP⁺ Tregs or Tregs in combination with CD4⁺Foxp3-YFP^– Teffs were adoptively transferred by tail vein injection to create pro-tolerogenic and effector intragraft microenvironments respectively. Recipients that did not receive T cells served as controls (NoAT). Allografts were harvested on day 21 post-transplantation (when the effects of ischemia-reperfusion injury have resolved) and graft EC were isolated by sorting (CD31⁺CD105⁺) with exclusion of CD45 expressing cells. Full transcriptomic RNA-seq profiling was performed on isolated EC using modified library construction and custom barcodes.

*Results: Initially, we generated differential gene expression data sets in the top 150 genes, and compared EC transcriptomes in Treg (n=6 mice) vs. NoAT microenvironments (n=8 mice). This analysis demonstrated marked differences in gene profiles, and identified a significant (Padj<0.05) regulation of 24 novel transcripts. We also evaluated effector/rejection (n=4 mice) vs. NoAT transcriptomes and found significant (Padj<0.05) regulation in over 500 genes; as expected, these genes were mostly associated with cytokine-mediated signaling, leukocyte migration and chemotaxis. In contrast, gene ontology analysis of the pro-tolerogenic EC signature was associated with reduced leukocyte trafficking as well as changes in metabolism and amino acid biosynthesis.

*Conclusions: Overall, these data demonstrate that the pro-tolerogenic microenvironment within an allograft is associated with novel EC activation responses. Thus, graft EC are not quiescent, and we propose that select gene profiles within EC are necessary for tolerance induction. Gene ontology is suggestive that this EC phenotype inhibits effector T cell trafficking and/or supports local Treg activity. These observations have profound implications for the identification and future development of therapeutics that promote long term graft survival.

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