IFNγ, and to a Lesser Extent TNFα, Provokes a Sustained Endothelial Costimulatory Phenotype

Background Vascular endothelial cells (EC) are critical for regulation of local immune responses, through coordination of leukocyte recruitment from the blood and egress into the tissue. Growing evidence supports an additional role for endothelium in activation and costimulation of adaptive immune cells. However, this function remains somewhat controversial, and the full repertoire and durability of an enhanced endothelial costimulatory phenotype has not been wholly defined. Methods Human endothelium was stimulated with continuous TNF alpha or IFN gamma for 1-48hr; or primed with TNF alpha or IFN gamma for only 3hr, before withdrawal of stimulus for up to 45hr. Gene expression of cytokines, costimulatory molecules and antigen presentation molecules was measured by Nanostring, and publicly available datasets of EC stimulation with TNF alpha or IFN gamma were leveraged to further corroborate the results. Cell surface protein expression was detected by flow cytometry, and secretion of cytokines was assessed by Luminex and ELISA. Key findings were confirmed in primary human endothelial cells from 4-6 different vascular beds. Results TNF alpha triggered mostly positive immune checkpoint molecule expression on endothelium, including CD40, 4-1BB, and ICOSLG but in the context of only HLA class I and immunoproteasome subunits. IFN gamma promoted a more tolerogenic phenotype of high PD-L1 and PD-L2 expression with both HLA class I and class II molecules and antigen processing genes. Both cytokines elicited secretion of IL-15 and BAFF/BLyS, with TNF alpha stimulated EC additionally producing IL-6, TL1A and IL-1 beta. Moreover, endothelium primed for a short period (3hr) with TNF alpha mostly failed to alter the costimulatory phenotype 24-48hr later, with only somewhat augmented expression of HLA class I. In contrast, brief exposure to IFN gamma was sufficient to cause late expression of antigen presentation, cytokines and costimulatory molecules. In particular HLA class I, PD-1 ligand and cytokine expression was markedly high on endothelium two days after IFN gamma was last present. Conclusions Endothelia from multiple vascular beds possess a wide range of other immune checkpoint molecules and cytokines that can shape the adaptive immune response. Our results further demonstrate that IFN gamma elicits prolonged signaling that persists days after initiation and is sufficient to trigger substantial gene expression changes and immune phenotype in vascular endothelium.

Automated summary

The study found that TNF alpha can induce the expression of most immune checkpoint molecules on endothelial cells, while IFN gamma tends to promote a tolerogenic phenotype with high PD-L1 and PD-L2 expression. Both cytokines elicit different cytokine secretion profiles. Brief exposure to IFN gamma is sufficient to induce late expression of antigen presentation, cytokines, and costimulatory molecules on endothelial cells.