Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China.
Beijing Key Laboratory of Traditional Chinese Veterinary Medicine, Beijing University of Agriculture, Beijing, 102206, People's Republic of China.
Virol J. 2020 Jul 6;17(1):92. doi: 10.1186/s12985-020-01341-x.
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.
PD-1/PD-L1 通路是一种抑制性信号通路，它维持着免疫反应和免疫耐受之间的平衡，其在癌症和病毒感染中的过度激活抑制了 T 细胞的功能。已经表明，各种病毒的靶细胞，即微血管内皮细胞（MECs），在感染多种流感病毒亚型时，是体内免疫调节和病毒扩散的关键调节点。此外，禽流感病毒（AIV）感染可通过引起免疫反应失衡和免疫器官损伤导致免疫抑制。因此，本研究旨在探讨 H9N2 病毒是否通过上调 MECs 上的 PD-L1 表达来抑制迁移过 MECs 的 T 细胞的免疫功能。
通过噬斑形成试验和免疫荧光染色评估大鼠肺微血管内皮细胞（RPMECs）对 H9N2 病毒的易感性。然后，我们使用定量实时 PCR 和流式细胞术检测 H9N2 病毒感染诱导的 RPMECs 中 PD-L1 的 mRNA 和蛋白水平。使用共培养系统揭示了 H9N2 病毒感染的 RPMECs 与活化的 T 细胞之间的相互作用。使用 ELISA 和流式细胞术，结合或不结合 PD-L1 特异性抗体，研究内皮细胞衍生的 PD-L1 对 T 细胞功能的影响。
表面染色和噬斑形成试验表明，H9N2 病毒感染并在 RPMECs 中复制。H9N2 病毒感染的 RPMECs 中 PD-L1 mRNA 水平和 PD-L1 蛋白水平均上调。H9N2 病毒诱导的 PD-L1 表达显著降低了 T 细胞中 IL-2、IFN-γ 和颗粒酶 B 和穿孔素的分泌。在用抗 PD-L1 抗体处理后，上述数据显著增加，证实了上述发现。此外，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。
综上所述，研究结果表明，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。
