No abstract

Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on lymphocytes. 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. In our previous studies, we have developed anti-bovine PD-L1 monoclonal antibodies (mAbs) and reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Furthermore, we found that blocking antibodies that target PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway for chronic equine diseases, including tumors, remains unclear. In this study, we identified cDNA sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 mAbs against EqPD-L1 using in vitro assays. In addition, we 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 considerable identity and similarity with homologs from non-primate 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. Of note, immunohistochemistry confirmed the PD-L1 expression in EMM tumor tissues. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These findings showed that our anti-PD-L1 mAbs would be useful for analyzing the equine PD-1/PD-L1 pathway. Further research is warranted to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horses.

Introduction Chronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death‐1 (PD‐1). The inhibitory signal is produced by the interaction between PD‐1 and its PD‐ligand 1 (PD‐L1) and impairs the effector functions of T cells. However, the expression dynamics of PD‐L1 and the immunological functions of the PD‐1/PD‐L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD‐L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD‐1/PD‐L1 pathway. Methods In the initial experiments, anti‐bovine PD‐L1 monoclonal antibodies (mAbs) were tested for cross‐reactivity with swine PD‐L1. Subsequently, immunohistochemical analysis was conducted using the anti‐PD‐L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti‐PD‐L1 mAb. Results Several anti‐PD‐L1 mAbs tested recognized swine PD‐L1‐expressing cells. The binding of swine PD‐L1 protein to swine PD‐1 was inhibited by some of these cross‐reactive mAbs. In addition, immunohistochemical analysis revealed that PD‐L1 was expressed at the site of infection in chronic infections of pigs. The PD‐L1 blockade increased the production of interleukin‐2 from swine PBMCs. Conclusions These findings suggest that the PD‐1/PD‐L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways.

Interactions between programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) cause functional exhaustion of T cells by inducing inhibitory signals, thereby attenuating effector functions of T cells. We have developed an anti-bovine PD-L1 blocking antibody (Ab) and have demonstrated that blockade of the interaction between PD-1 and PD-L1 reactivates T-cell responses in cattle. In the present study, we examined the potential utility of PD-1/PD-L1-targeted immunotherapy in enhancing T-cell responses to vaccination. Calves were inoculated with a hexavalent live-attenuated viral vaccine against bovine respiratory infections in combination with treatment with an anti-PD-L1 Ab. The expression kinetics of PD-1 in T cells and T-cell responses to viral antigens were measured before and after vaccination to evaluate the adjuvant effect of anti-PD-L1 Ab. PD-1 expression was upregulated in vaccinated calves after the administration of a booster vaccination. The activation status of CD4+, CD8+, and γδTCR+ T cells was enhanced by the combination of vaccination and PD-L1 blockade. In addition, IFN-γ responses to viral antigens were increased following combinatorial vaccination with PD-L1 blockade. In conclusion, the blockade of the PD-1/PD-L1 interaction enhances T-cell responses induced by vaccination in cattle, indicating the potential utility of anti-PD-L1 Ab in improving the efficacy of current vaccination programs.

24 Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on exhausted T cells 25 during chronic illness. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory 26 signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We reported 27 that the PD-1/PD-L1 pathway is closely associated with T-cell exhaustion and disease progression 28 in bovine chronic infections and canine tumors. Moreover, we found that blocking antibodies 29 targeting PD-1 and PD-L1 restore T-cell functions and may be used in immunotherapy in cattle 30 and dogs. However, the immunological role of the PD-1/PD-L1 pathway remains unclear for 31 chronic equine diseases, including tumors. In this study, we identified nucleotide sequences of 32 equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 33 monoclonal antibodies (mAbs) against EqPD-L1 using in vitro assays. We also evaluated the 34 expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). 35The amino acid sequences of EqPD-1 and EqPD-L1 share a high identity and similarity with 36 homologs from other mammalian species. Two clones of the anti-bovine PD-L1 mAbs recognized 37 EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine 38 PD-1/PD-L1. Importantly, PD-L1 expression was confirmed in EMM tumor tissues by 39 immunohistochemistry. A cultivation assay revealed that PD-L1 blockade enhanced the 40 production of Th1 cytokines in equine immune cells.41 These results suggest that our anti-PD-L1 mAbs may be useful for investigating the expression 42 and role of the equine PD-1/PD-L1 pathway. Further research is required to discover the 43 immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application 44 in immunotherapy for horse. 45 Keywords: horse, PD-1, PD-L1, IFN-γ, T cells, chronic diseases, melanoma 3 46 Introduction 47 Programmed cell death-1 (PD-1) is an immunoinhibitory receptor, which is expressed on activated 48 and exhausted T cells [1]. Its ligand programmed death ligand 1 (PD-L1) is expressed on immune 49 cells, including antigen-presenting cells, and tumor cells [1]. The interaction of PD-1 and PD-L1 50 suppresses the activation signal mediated by T-cell receptors and inhibits effector functions of T 51 cells, such as cytokine production and cell proliferation [1]. This pathway is invaluable for 52 regulating excessive immune responses. In cancers, however, tumor cells utilize the suppression 53 of T cells mediated by PD-1/PD-L1 to avoid anti-tumor immune responses [1]. In human medicine, 54 the blocking antibodies targeting PD-1 or PD-L1 have been leveraged for treatment of various 55 types of cancers and resulted in remarkable outcomes with 20%-90% response rates in multiple 56 clinical trials [1].57 Equine malignant melanoma (EMM) is a common neoplasm among aged gray horses, which 58 results in dermal tumors at multiple sites [2]. A previous study reported that around 80% of aged 59 gray horses deve...