*Purpose: Previous studies have suggested that early adoptive transfer of Teff into Rag KO mice bearing cardiac transplants results in acute rejection. However, if transfer of identical Teff populations is delayed to day >50 post-transplant, the response results in insidious chronic rejection. These findings suggest that the graft itself has potential to dictate the phenotype of rejection independent of alloimmunity. Furthermore, recent reports have identified immune evasive molecules within tissues that are potent to promote/initiate immunoregulation. However, their expression and the mechanisms of locally induced immunomodulation within allografts are not known.

*Methods: We transplanted fully MHC mismatched Balb/c hearts into immunodeficient C57BL/6 Rag2 Il2rg KO mice and harvested CD45^–CD31⁺CD105⁺ graft endothelial cells at different times post-transplant. We also adoptively transferred pooled CD4⁺ T cells alone, CD4⁺ Tregs alone (3.5×10⁵ CD4⁺Foxp3-YFP⁺) or Tregs in combination with CD4⁺ Teffs (3.5×10⁵ CD4⁺Foxp3-YFP⁺ and 1.5×10⁶ CD4⁺Foxp3-YFP^–) on day 2 and/or day 70 post-transplant.

*Results: As expected, we find that transfer of T cells alone on day 2 results in acute rejection (MST 14 days) whereas transfer of identical cells on day 70 results in long-term graft survival (MST >100 days; P<0.0001). To investigate EC-dependent mechanisms underlying this difference, we evaluated transcriptomes from EC at each time point (no adoptive transfer) vs. donor heart ECs (not transplanted). Principal Component Analysis (PCA) revealed three unique clusters with day 70 EC having a most distinct transcriptional profile. While day 2 EC appeared activated with expression of proinflammatory mediators including adhesion molecules and cytokines, day 70 EC appeared more quiescent. Nevertheless, day 70 EC had a distinct phenotype that included high levels of coinhibitory ligands and MHC class I/II antigen presentation associated transcripts. These data suggest that day 70 EC actively promote immunoregulation through alloantigen-dependent interactions in combination with coinhibition. To evaluate if the phenotype of day 70 EC is inducible, we also evaluated transcriptomes from EC harvested from allografts following transfer of Tregs alone or Tregs + Teffs. By PCA, we find that Tregs fail to elicit an identical immunoregulatory phenotype as day 70 EC, with Treg transfer being closest to native EC transcriptomes; Tregs reduce Teff-mediated EC activation but strikingly do not induce the transcriptional profile of day 70 ECs.

*Conclusions: Our findings demonstrate that graft EC have potential to express a novel transcriptome that promotes immunoregulation. Furthermore, this immunomodulatory phenotype is cell intrinsic and independent of immunoregulatory stimuli by the peripheral immune system. Understanding the molecular inducers of this intrinsic program opens the design of a novel class of protolerogenic and immunomodulatory drugs specific for the intragraft microenvironment.

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