*Purpose: Recent studies have highlighted a paradigm whereby the maintenance of normal tissue homeostasis and the prevention of chronic inflammation is dependent on the active process(es) of anti-inflammation/pro-resolution. We previously reported that intracellular DEPTOR serves as a novel regulator of endothelial cell (EC) activation responses through its effect to inhibit mTORC1 and ERK1/2 kinase activity. Furthermore, EC activation is associated with a rapid degradation of intracellular DEPTOR suggesting that it functions to stabilize and maintain quiescence. In these studies, we wish to test the hypothesis that sustained expression of DEPTOR within graft EC is sufficient to augment intragraft immune homeostasis following transplantation.

*Methods: We used VECad^Cre/ERT2, DEPTOR (DEP)^CAGGS and DEP^lox/lox mice to generate inducible EC-specific knockin (OE) and knockout (KO) mice respectively. These transgenics (BL/6 [H-2^b]) were used as donors in a standard fully MHC mismatched cardiac transplantation model (B6 into BALB/c [H-2^d]). Recipients were treated with a short course of anti-CD40L (200mcg on d0 or d0+d2) and graft survival was monitored. For mechanistic studies, EC were isolated from the hearts of transgenic mice and used in in vitro co-culture assays with CD4+ T cells.

*Results: We find that KO cardiac allografts are rejected at an accelerated rate (P<0.005) and graft survival is reduced (MST=38 days, n=11) vs. control WT (DEP^lox/lox) hearts (MST=77 days, n=11). Furthermore, we find that OE allografts survive moderately longer (median prolongation=15 days, P=0.0003, n=10) vs. control DEP^CAGGS mice (n=7). Graft histology, immunohistochemical staining and FACS analysis of d7 and d14 grafts suggest moderate effects of EC expression of DEPTOR on CD45⁺ leukocyte and CD3⁺ T cell infiltration into the graft. Moreover, mRNA cytokine arrays (Qiagen) of d14 allografts suggest additional effects of DEPKO EC on local immune activation (incl. increased IL-2, IL-4, IL-6 and IL-13 production). To further evaluate whether EC-specific DEPTOR regulates the local activation of CD4⁺ T cells, we isolated EC from WT, KO and OE hearts and performed cococulture studies (48-72hrs) with C57BL/6 CD4⁺ T cells (in a 1:1 ratio) in the presence of ConA (1-3mcg/ml). Proliferation of CD4⁺ T cells (by CFSE dilution) was found to be similar following coculture with each EC cell type, but notably KO EC were potent (P=0.003) to costimulate CD4⁺ T cell IFNγ production (by ELISPOT).

*Conclusions: These combined data indicate that EC-specific DEPTOR regulates intragraft mechanisms of allograft rejection. They also lead to the intriguing possibility that DEPTOR-inducible therapeutics have high potential for repurposing to augment long-term graft survival.

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