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

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.

PD-L1/PD-1 and CTLA-4 Expression in Equine Penile Squamous Cell Carcinomas

“…The WB analysis (Figure 1) indicated that the anti-human PD-L1 and anti-CTLA-4 antibodies tested cross-reacted with the corresponding equine proteins. A single band corresponding to approximately the expected molecular weight of the equine PD-L1 (42.4−50.3 kDa) [35] was detected in the placenta, whereas no positive signal was observed in the liver lane. A single band of approximately 25 kDa corresponding to the expected molecular weight of CTLA-4 (https://www.uniprot.org/uniprot/F6WWE3, accessed on 23 April 2021) was detected in the lymph node but was negative in the placenta lane.…”

Circulating Melanoma-Derived Extracellular Vesicles: Impact on Melanoma Diagnosis, Progression Monitoring, and Treatment Response

“…EV-associated PD-L1 plasma levels also increased with tumour burden in a murine model and in humans, and was higher in metastatic versus non-metastatic melanoma cell lines [ 78 ]. PD-L1 therapies may be applied to canine [ 89 , 90 ] and equine melanoma [ 91 ] patients, and by extension to other species.…”

Programmed death‐ligand 1 expression in swine chronic infections and enhancement of interleukin‐2 production via programmed death‐1/programmed death‐ligand 1 blockade

“…The complementary DNA (cDNA) was synthesized from mRNA of swine PBMCs stimulated with 20 ng/ml phorbol 12‐myristate acetate (PMA; Sigma‐Aldrich) and 1 μg/ml ionomycin (Sigma‐Aldrich) for 24 h. The cultivation of PBMCs, total RNA isolation, and cDNA synthesis was conducted as described previously 16 . The cDNAs encoding swine PD‐1 and PD‐L1 were amplified by PCR using TaKaRa Ex Taq (Takara Bio) and gene‐specific primers with restriction enzyme cleavage sites (Table ), and then subcloned into pEGFP‐N2 (Clontech).…”

“…The protein concentration was measured by ultraviolet absorbance at 280 nm with a NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific). The binding of rPD‐1 and rPD‐L1 to COS‐7 cells expressing swine PD‐L1‐EGFP and PD‐1‐EGFP was investigated using flow cytometry as described previously 16 …”

“…During chronic infection, the expression of PD‐1 and PD‐L1 is upregulated and causes the dysfunction of antigen‐specific T cells, which is known as “T‐cell exhaustion.” 4,5 It has been well documented that T‐cell exhaustion mediated by PD‐1/PD‐L1 contributes to immune evasion in various types of chronic infections by human immunodeficiency virus 5 and human hepatitis virus type C 6 . Our previous studies have shown that the PD‐1/PD‐L1 pathway is involved in immune exhaustion and disease progression in chronic infectious diseases of cattle, such as bovine leukemia virus infection, 7–9 Johne's disease, 10,11 anaplasmosis, 12 mycoplasmosis, 13 canine cancers, 14,15 and equine melanoma 16 . Furthermore, we established therapeutic antibodies targeting PD‐1 and PD‐L1 and reported their therapeutic efficacy against bovine leukemia virus infection, 17–19 Johne's disease, 20 mycoplasmosis, 21 and canine malignant melanoma 22,23 …”