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

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.

Equine malignant melanoma (EMM) is a common neoplasm among aged gray horses, resulting in dermal tumors at multiple sites [16]. A previous study reported that approximately 80% of aged gray horses developed dermal melanoma and speculated that all gray horses would develop this tumor as they reach old age [17]. Although cellular immune response is critical for eradicating melanoma, but several mechanisms have been suggested to limit antitumor immunity in EMM based on the findings for human malignant melanoma [18]. However, no studies are available on immune evasion mechanisms in EMM, and immune exhaustion mediated by PD-1 and PD-L1 has not been investigated in horses.
Until now, no information was available on cDNA sequences, expression, and function of PD-1/PD-L1 in horses. Furthermore, the role of the PD-1/PD-L1 pathway in EMM remained unclear. Based on the findings of our previous studies, we hypothesized that PD-1 and PD-L1 may provide potential targets for immunotherapy against EMM. Therefore, in this study, we identified cDNA sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1), evaluated the blocking effects of our anti-bovine PD-L1 mAbs against EqPD-L1, and confirmed the expression of PD-L1 on EMM.

Horse blood samples and cell preparation
Heparinized blood samples were collected from Thoroughbred horses in farms and veterinary hospitals in Hokkaido, Japan. Peripheral blood mononuclear cells (PBMCs) were purified using density gradient centrifugation on Percoll (GE Healthcare, Little Chalfont, UK), washed three times with phosphate-buffered saline (PBS), and suspended in PBS. All experimental procedures were conducted following approval from the local committee for animal studies according to the Hokkaido University (20-0093). Informed consent was obtained from all owners.
Total RNA was isolated from cultivated PBMCs using of TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) in accordance with the manufacturer's instructions. Residual DNA was removed from RNA samples by treatment with Deoxyribonuclease I (Thermo Fisher Scientific). cDNA was synthesized from 1 μg of total RNA with PrimeScript Reverse Transcriptase (Takara Bio, Otsu, Japan) and oligo(dT) primer in accordance with the manufacturer's instructions.
Gene-specific primers were designed to amplify EqPD-1 and EqPD-L1 genes, based on the sequences from horses available on GenBank (XM_005610777 and XM_001492842) (S1 Table). EqPD-1 and EqPD-L1 cDNAs were amplified by PCR using TaKaRa Ex Taq (Takara Bio) and specific primers (S1 Table). The PCR products were purified using a FastGene Gel/ PCR Extraction Kit (Nippon Genetics, Tokyo, Japan) and cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA). They were transferred into Escherichia coli HST08 Premium Competent Cells (Takara Bio) and plated onto LB agar plates (Sigma-Aldrich) containing X-gal (Takara Bio) and ampicillin (Sigma-Aldrich). The purified plasmid clones were sequenced using a GenomeLab GeXP Genetic Analysis System (SCIEX, Framingham, MA, USA). The established sequences were aligned and an unrooted neighbor-joining tree was constructed using MEGA software program version 7.0 [32].
Purification of EqPD-1-Ig and EqPD-L1-Ig from the culture supernatants was achieved by affinity chromatography with an Ab-Capcher ExTra (ProteNova, Kagawa, Japan). The buffer was exchanged with PBS by size exclusion chromatography using a PD-10 Desalting Column (GE Healthcare). The purity of EqPD-1-Ig and EqPD-L1-Ig was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in reducing or nonreducing conditions using SuperSep Ace 5%-20% gradient polyacrylamide gel (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 2 × Laemmli Sample Buffer (Bio-Rad). Precision Plus Protein All Blue Standard (Bio-Rad) was used as a molecular-weight size marker. The proteins were visualized with Quick-CBB (FUJIFILM Wako Pure Chemical), and protein concentrations were measured by ultraviolet absorbance at 280 nm with a NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific).
Fresh and stimulated equine PBMCs were analyzed by flow cytometry to analyze the binding ability of anti-bovine PD-L1 mAbs to equine immune cells. Equine PBMCs (4 × 10 6 cells) were stimulated in cultivation with 20 ng/mL of PMA (Sigma-Aldrich) and 1 μg/mL of ionomycin (Sigma-Aldrich) for 24 h, as described above. Fresh and stimulated PBMCs were incubated with PBS supplemented with 10% goat serum (Thermo Fisher Scientific) at room temperature for 15 min to prevent nonspecific reactions. Cells were washed and stained with anti-PD-L1 mAbs (5A2-A1, rat IgG 1 ; 6C11-3A11; rat IgG 2a ) [19,34] at room temperature for 30 min. Rat IgG 1 (R3-34, BD Biosciences) and rat IgG 2a isotype controls (R35-95, BD Biosciences) were used for negative control staining. Cells were washed with PBS containing 1% BSA (Sigma-Aldrich) and labeled with APC-conjugated anti-rat Ig antibody (Southern Biotech) at room temperature for 30 min. After rewashing, cells were immediately analyzed by FACS Verse (BD Biosciences).

Immunohistochemical assay of PD-L1
Tumor tissues from four horses bearing EMM were immunohistochemically stained (S2 Table). The tissues were fixed in formalin, embedded into paraffin wax and cut into 4-μmthick sections. The dried sections were deparaffinized in xylene and hydrated through graded alcohols. Melanin was bleached from the sections using 0.25% potassium permanganate and 2% oxalic acid. Antigen retrieval was achieved using 0.01 M citrate buffer (pH 6.0) by microwave heating. Endogenous peroxidase activity was blocked by incubating the sections in methanol containing 0.3% hydrogen peroxide. The sections were incubated with or without anti-PD-L1 mAb (6C11-3A11, rat IgG 2a ) [34] at 4˚C overnight, followed by detection using Vectastain Elite ABC Rat IgG kit (Vector Laboratories, Burlingame, CA, USA). The immunoreaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride. All immunostained sections were examined under an optical microscope.

Immunoactivation assay using equine PBMCs
To determine the effects of inhibiting the PD-1/PD-L1 interaction on equine immune cells, equine PBMCs were cultured with 10 μg/mL of anti-PD-L1 mAb (6C11-3A11, rat IgG 2a ) [34] or rat IgG 2a control (Bio X Cell, West Lebanon, NH, USA) in the presence of 0.1 μg/mL of Staphylococcal enterotoxin B (SEB; Sigma-Aldrich) at 37˚C with 5% CO 2 for 3 days. All cell cultures were grown in 96-well round-bottomed plates (Corning Inc., Corning, NY, USA) containing 4 × 10 5 PBMCs in 200 μl of RPMI 1640 medium (Sigma-Aldrich) which was supplemented with 10% heat-inactivated FBS, 2 mM of L-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Thermo Fisher Scientific). Cytokine concentrations in the culture supernatants were determined using an Equine IFN-γ ELISA Development Kit (Mabtech, Nacka Strand, Sweden) and an Equine IL-2 DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN, USA). Measurements were performed in duplicates in accordance with the manufacturer's protocol.

Statistical analysis
Significant differences were identified using Wilcoxon signed-rank test or Tukey's test. All statistical tests were performed using the MEPHAS (http://www.gen-info.osaka-u.ac.jp/ MEPHAS/) statistical analysis program. Statistical significance was set as p < 0.05.

Immunohistochemical analysis of PD-L1 in EMM
PD-L1 has been shown to be upregulated in many types of tumors in dogs and humans [25,26,35]. Among canine malignant cancers, malignant melanoma has the highest positive rates for PD-L1 expression [26]. Gray horses are susceptible to melanoma and approximately 80% of them develop EMM in their lifetimes [17]. We hypothesized that PD-L1 plays a role in the development of EMM. Therefore, we analyzed the expression of PD-L1 in tumor tissues of EMM by immunohistochemistry. PD-L1 was detected in all EMM samples (n = 4, Fig 6B).
These results indicate that PD-1/PD-L1 blockade enhanced Th1 cytokine production in equine PBMCs, suggesting that the anti-PD-L1 blocking antibody has an application as an immunomodulatory agent for horses.

Discussion
The greater longevity of the horse population has increased the risks of chronic diseases, such as laminitis, pituitary pars intermedia dysfunction, recurrent airway obstruction, osteoarthritis, and neoplasia, and increased multimorbidity in horses [36,37]. However, treatments available for chronic diseases in horses, including malignant tumors, are few. Therefore, new treatment options are being sought. Malignant melanoma is one of the most common cutaneous neoplasia in horses [38]. Surgical treatment is successful in the early stages of the disease, but it is not feasible in cases with A variety of immunotherapies have been developed and tested in clinical trials to treat tumors in humans, and immune checkpoint inhibitors such as anti-PD-1 and anti-PD-L1 antibodies are currently used with notable success for the treatment of multiple human cancers [12][13][14][15]. Blockade therapy using anti-PD-L1 antibody resulted in long-term tumor regression and prolonged progression-free survival in advanced melanoma in humans [12]. Based on these advancements in human medicine, immune checkpoint inhibitors may reasonably be expected to yield equally promising results in the treatment of EMM [18]. However, no studies have been conducted on the PD-1/PD-L1 pathway in horses as yet.
We found that one of the anti-bovine PD-L1 mAbs (6C11-3A11) strongly recognized EqPD-L1, blocked the interaction of EqPD-1/EqPD-L1, and enhanced the Th1 cytokine response in vitro. In contrast, the other cross-reactive mAb (5A2-A1) bound to the EqP-D-L1-overexpressing cell line, but not to equine PBMCs, failed to block the interaction of EqPD-1/EqPD-L1. The difference between the results using these two mAbs would depend on the epitope and binding affinity of the mAbs. The expression level of PD-L1 could be the highest in the overexpressing cells, followed in order by stimulated and fresh PBMCs. The binding affinity of 5A2-A1 would not be enough to detect PD-L1 on equine PBMCs. The mAb 6C11- 3A11 may be used to aid investigation into the expression and immunological function of PD-L1 in future horse studies. Additionally, we discovered that PD-L1 is expressed in EMM tumor tissues. Further studies are required to analyze expression of PD-L1 in other horse tumors and chronic diseases.
The mechanism of PD-L1 upregulation during EMM progression has yet to be elucidated. Generally, PD-L1 expression is regulated by a substantial number of mediators including inflammatory cytokine signaling, oncogenic signaling, microRNAs, genetic alteration of the PD-L1 locus, and post-translational regulators [39]. In gray horses, a gene duplication in intron 6 of STX17 (synataxin 17) contributes to a cis-acting regulatory mutation resulting in a very high incidence of EMM [40]. This gene duplication induces constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and tumorigenesis in EMM [41,42]. The MEK-ERK signaling pathway regulates PD-L1 gene expression via crosstalk with inflammatory cytokine signaling including the IFN-γ-STAT1 pathway [43][44][45]. Hence, the regulatory mechanism of PD-L1 expression in gray horses merits investigation as a natural model of tumorigenesis.  Table. https://doi.org/10.1371/journal.pone.0234218.g006

PLOS ONE
Our results indicate that the PD-1/PD-L1 pathway offers a potential target for immunotherapy against EMM. In future immunotherapy applications, blocking antibodies should be engineered into suitable forms for administration to horses. Chimeric antibodies, for instance, may facilitate clinical trial research into the clinical efficacy of anti-PD-L1 antibody in the treatment of EMM. Further research is required to develop this novel immunotherapy strategy in horses.  Table. GenBank accession number of the nucleotide sequences used in this study. (PPTX)