*Purpose: Beyond simply “recruiting” leukocytes to sites of injury, local stimulation by the donor vascular compartment may prolong alloimmune responses. Moreover, the dynamics of endothelial resolution of inflammation, particularly with respect to costimulatory phenotype, is not well-defined. We characterized the repertoire of cytokines, antigen presentation and costimulatory/coinhibitory molecules produced by inflamed vascular endothelial cells (EC), under chronic stimulation and after short cytokine priming and prolonged withdrawal.

*Methods: Human aortic EC were stimulated with continuous TNFα or IFNγ for 1-48hr, or primed with TNFα or IFNγ for 3hr, before withdrawal of stimulus for up to 45hr. mRNA were measured by Nanostring; protein were measured by flow cytometry, ELISA and Luminex. Key findings were confirmed in primary human EC from 4-6 different vascular beds, and publicly available datasets were leveraged to further corroborate the findings.

*Results: 42 genes encoding antigen presentation, costimulation/coinhibition molecules or cytokines were upregulated in EC by TNFα, IFNγ or both. 35 other cytokines, antigen presentation and costimulatory genes were either not found to be expressed by endothelium or were expressed but not induced by TNFα or IFNγ. TNFα triggered IL6, IL6ST, IL15, TRAIL and BAFF production from EC as an intermediate phase ( >6hr) response, while IFNγ stimulated only TRAIL, BAFF and IL15 after 18hr. EC upregulated CD40, ICOSL, HVEM, PD-L1, PD-L2 and 4-1BB as early as 3hr, and down-regulated B7-H3, in response to TNFα; while IFNγ increased CD40, HVEM, PD-L1 and PD-L2. TNFα augmented expression of HLA I, TAP1 and immunoproteasome components; while IFNγ also increased HLA II, CIITA, and CD74. After only short (3hr) cytokine priming and extended withdrawal from cytokine exposure, TNFα-induced HLA I, IL6ST and PD-L2 were enhanced at 24hr, but other TNFα-induced molecules rapidly contracted. In contrast, HLA I, HLA II and proteasome induction at 48hr was elevated whether IFNγ was chronic or withdrawn. In addition, nearly all IFNγ-induced costimulatory molecules and cytokines persisted up to 45hr after IFNγ withdrawal.

*Conclusions: Although endothelial cells lacked CD28 ligands (CD80, CD86) and did not produce many of the typical cytokines needed for Th skewing, they could be provoked to express PD-1 ligands, CD40 and other cytokines that can bias T cell activation. We were also surprised to find that activated EC elaborated BAFF and IL-15, cytokines which shape B cell and NK cell survival and activation. In addition to different profiles elicited by TNFα to IFNγ, our results show that IFNγ elicits prolonged gene expression changes and immune phenotype alterations that persist days after initiation. That systemic and tissue resident vascular EC possess a wide constellation of costimulatory molecules suggest that adaptive immunity takes shape not only in secondary lymphoid organs but also locally in the periphery.

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