DataSheet_1_IFNγ, and to a Lesser Extent TNFα, Provokes a Sustained Endothelial Costimulatory Phenotype.docx

BackgroundVascular 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. MethodsHuman endothelium was stimulated with continuous TNFα or IFNγ for 1-48hr; or primed with TNFα or IFNγ 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 TNFα or IFNγ 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. ResultsTNFα 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. IFNγ 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 TNFα stimulated EC additionally producing IL-6, TL1A and IL-1β. Moreover, endothelium primed for a short period (3hr) with TNFα mostly failed to alter the costimulatory phenotype 24-48hr later, with only somewhat augmented expression of HLA class I. In contrast, brief exposure to IFNγ 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 IFNγ was last present. ConclusionsEndothelia 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 IFNγ elicits prolonged signaling that persists days after initiation and is sufficient to trigger substantial gene expression changes and immune phenotype in vascular endothelium.背景 血管内皮细胞（vascular endothelial cells, EC）可通过协调白细胞从血液中募集并迁移至组织，在局部免疫应答的调控中发挥关键作用。越来越多的研究证据表明，内皮细胞在适应性免疫细胞的活化与共刺激过程中还存在额外功能。然而，这一功能目前仍存在一定争议，内皮细胞共刺激表型增强的完整范围及持久性尚未完全明确。 方法 本研究采用持续肿瘤坏死因子α（tumor necrosis factor α, TNFα）或干扰素γ（interferon γ, IFNγ）刺激人内皮细胞1~48小时；或先用TNFα或IFNγ预刺激3小时，随后移除刺激物并继续培养至多45小时。通过Nanostring技术检测细胞因子、共刺激分子及抗原呈递分子的基因表达水平，并利用已公开的TNFα或IFNγ刺激内皮细胞的数据集进一步佐证实验结果。采用流式细胞术检测细胞表面蛋白的表达情况，通过Luminex液相芯片技术与酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测细胞因子的分泌水平。核心实验结果均在来自4~6种不同血管床的原代人内皮细胞中得到验证。 结果 TNFα主要诱导内皮细胞表达正向免疫检查点分子，包括CD40、4-1BB及ICOSLG，但仅伴随HLA I类分子及免疫蛋白酶体亚基的表达。IFNγ则可促使内皮细胞呈现更具耐受原性的表型，高表达PD-L1、PD-L2，同时上调HLA I类与II类分子及抗原加工相关基因。两种细胞因子均可诱导内皮细胞分泌IL-15及BAFF/BLyS，其中经TNFα刺激的内皮细胞还可额外产生IL-6、TL1A及IL-1β。此外，经TNFα短期预刺激（3小时）的内皮细胞，在刺激移除后24~48小时内，其共刺激表型大多未发生显著改变，仅HLA I类分子的表达有一定程度上调。与之相反，短暂暴露于IFNγ即可触发抗原呈递相关分子、细胞因子及共刺激分子的延迟表达。具体而言，在末次IFNγ暴露两天后，内皮细胞的HLA I类分子、PD-1配体及细胞因子的表达水平显著升高。 结论 来自多种血管床的内皮细胞可表达多种可调控适应性免疫应答的免疫检查点分子与细胞因子。本研究结果进一步证实，IFNγ可触发持续数日的信号转导，该信号在刺激移除后仍可维持，并足以诱导血管内皮细胞发生显著的基因表达改变及免疫表型变化。