PD-L1 expression in equine malignant melanoma and functional effects of PD-L1 blockade

Abstract

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...