The PD-1/PD-L1 pathway is an inhibitory signaling pathway that maintains the balance between the immune response and immunotolerance, and its overactivation in cancer and viral infections inhibits T cell function. The target cells of various viruses, microvascular endothelial cells (MECs) have been shown to be key regulatory points in immune regulation and virion diffusion in vivo during infection with multiple influenza virus subtypes. Furthermore, avian influenza virus (AIV) infection can induce immunosuppression by causing imbalances in immune responses and immune organ damage. Thus, the aim of this study was to investigate whether the H9N2 virus inhibited the immune function of T cells that migrated across MECs by upregulating PD-L1 expression on MECs. The susceptibility of rat pulmonary microvascular endothelial cells (RPMECs) to the H9N2 virus was evaluated by a plaque-forming assay and immunofluorescence staining. Then, we quantified the mRNA and protein levels of PD-L1 in RPMECs induced by H9N2 virus infection using quantitative real-time PCR and flow cytometry. The interaction between the activated T cells and RPMECs infected with the H9N2 virus was revealed using a coculture system. The effect of endothelial-derived PD-L1 on T cell function was investigated by using ELISA and flow cytometry with or without a PD-L1-specific antibody. Surface staining and the plaque-forming assay showed that the H9N2 virus infected and replicated in RPMECs. Both the PD-L1 mRNA level and PD-L1 protein level were upregulated in RPMECs infected with the H9N2 virus. H9N2 virus-induced PD-L1 expression significantly reduced the secretions of IL-2, IFN-γ and granzyme B and perforin expression in T cells. The above data were significantly increased after treatment with an anti-PD-L1 antibody, confirming the above mentioned findings. In addition, the induction of PD-L1 expression decreased the proliferative capacity of the cocultured T cells but did not affect the apoptosis rate of T cells. Taken together, the results suggest that the H9N2 virus is able to inhibit the T cell immune response by upregulating PD-L1 expression in pulmonary microvascular endothelial cells.

中文翻译：

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H9N2禽流感病毒诱导的源自肺内皮的PD-L1抑制T细胞的免疫反应。

PD-1 / PD-L1途径是一种抑制性信号传导途径，可维持免疫应答和免疫耐受之间的平衡，并且它在癌症和病毒感染中的过度活化会抑制T细胞功能。多种病毒的靶细胞，微血管内皮细胞（MEC）已被证明是多种流感病毒亚型感染期间体内免疫调节和病毒体扩散的关键调节点。此外，禽流感病毒（AIV）感染可通过引起免疫反应和免疫器官损害的失衡来诱导免疫抑制。因此，本研究的目的是研究H9N2病毒是否通过上调MEC上的PD-L1表达来抑制跨MEC迁移的T细胞的免疫功能。通过噬斑形成测定和免疫荧光染色评估大鼠肺微血管内皮细胞（RPMECs）对H9N2病毒的敏感性。然后，我们使用实时荧光定量PCR和流式细胞术对H9N2病毒感染引起的RPMECs中PD-L1的mRNA和蛋白水平进行了定量。使用共培养系统揭示了活化的T细胞和感染H9N2病毒的RPMEC之间的相互作用。通过使用ELISA和流式细胞术（有或没有PD-L1特异性抗体）研究了内皮衍生的PD-L1对T细胞功能的影响。表面染色和噬斑形成分析表明，H9N2病毒已感染并在RPMEC中复制。在感染了H9N2病毒的RPMEC中，PD-L1 mRNA水平和PD-L1蛋白水平均被上调。H9N2病毒诱导的PD-L1表达显着降低T细胞中IL-2，IFN-γ和粒酶B的分泌以及穿孔素的表达。用抗PD-L1抗体治疗后，上述数据显着增加，证实了上述发现。另外，PD-L1表达的诱导降低了共培养的T细胞的增殖能力，但不影响T细胞的凋亡率。两者合计，结果表明H9N2病毒能够通过上调肺微血管内皮细胞中PD-L1的表达来抑制T细胞免疫反应。PD-L1表达的诱导降低了共培养T细胞的增殖能力，但不影响T细胞的凋亡率。两者合计，结果表明H9N2病毒能够通过上调肺微血管内皮细胞中PD-L1的表达来抑制T细胞免疫反应。PD-L1表达的诱导降低了共培养T细胞的增殖能力，但不影响T细胞的凋亡率。两者合计，结果表明H9N2病毒能够通过上调肺微血管内皮细胞中PD-L1的表达来抑制T细胞免疫反应。