Abstract Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on exhausted T cells during chronic illness. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We reported that the PD-1/PD-L1 pathway is closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Moreover, we found that blocking antibodies targeting PD-1 and PD-L1 restore T-cell functions and may be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway remains unclear for chronic equine diseases, including tumors. In this study, we identified nucleotide sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 monoclonal antibodies (mAbs) against EqPD-L1 using in vitro assays. We also evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a high identity and similarity with homologs from other mammalian species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Importantly, PD-L1 expression was confirmed in EMM tumor tissues by immunohistochemistry. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These results suggest that our anti-PD-L1 mAbs may be useful for investigating the expression and role of the equine PD-1/PD-L1 pathway. Further research is required to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horse.

中文翻译：

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马恶性黑色素瘤中PD-L1的表达和PD-L1阻断的功能作用

摘要程序性死亡1（PD-1）是一种在慢性疾病期间在精疲力竭的T细胞上表达的免疫抑制受体。PD-1及其配体PD-配体1（PD-L1）的相互作用可提供抑制性信号，并损害T细胞的增殖，细胞因子产生和细胞毒性。我们报道了PD-1 / PD-L1通路与牛慢性感染和犬肿瘤中的T细胞衰竭和疾病进展密切相关。此外，我们发现靶向PD-1和PD-L1的封闭抗体可恢复T细胞功能，可用于牛和狗的免疫治疗。但是，PD-1 / PD-L1途径的免疫作用对于包括肿瘤在内的慢性马疾病仍然不清楚。在这个研究中，我们鉴定了马PD-1（EqPD-1）和PD-L1（EqPD-L1）的核苷酸序列，并使用体外测定法研究了抗EqPD-L1的抗牛PD-L1单克隆抗体（mAb）的作用。我们还评估了马恶性黑色素瘤（EMM）肿瘤组织中PD-L1的表达。EqPD-1和EqPD-L1的氨基酸序列与其他哺乳动物物种的同源物具有高度同一性和相似性。抗牛PD-L1 mAb的两个克隆在流式细胞仪中识别EqPD-L1，这些交叉反应性mAb之一阻断了马PD-1 / PD-L1的结合。重要的是，通过免疫组织化学证实了EMM肿瘤组织中PD-L1的表达。培养分析表明，PD-L1阻断增强了马免疫细胞中Th1细胞因子的产生。这些结果表明，我们的抗PD-L1单抗可能可用于研究马PD-1 / PD-L1途径的表达和作用。需要进一步的研究来发现PD-1 / PD-L1在慢性马疾病中的免疫学作用，并阐明在马的免疫治疗中的未来应用。