PLOS ONERESEARCH ARTICLE

Molecular characterization of feline immune
checkpoint molecules and establishment of
PD-L1 immunohistochemistry for feline
tumorsa1111111111
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OPEN ACCESS
Citation: Maekawa N, Konnai S, Asano Y, Otsuka T,
Aoki E, Takeuchi H, et al. (2023) Molecularcharacterization of feline immune checkpoint
molecules and establishment of PD-L1
immunohistochemistry for feline tumors. PLoS
ONE 18(1): e0281143. https://doi.org/10.1371/
journal.pone.0281143
Editor: Avaniyapuram Kannan Murugan, King
Faisal Specialist Hospital and Research Center,
SAUDI ARABIA
Received: November 4, 2022
Accepted: January 11, 2023
Published: January 26, 2023
Copyright: © 2023 Maekawa et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All data are fully
available without restriction. All relevant data arewithin the manuscript and its Supporting
information file. Nucleotide sequences of feline PDL1 and PD-L2 were submitted to the DDBJ/EMBLBank/GenBank database under accession
numbers LC735019, LC735020, and LC735021.
Funding: This work was supported by the Grant-inAid for Scientific Research (Grant Numbers

Naoya Maekawa1, Satoru Konnai ID1,2,3*, Yumie Asano2, Takumi Otsuka2, Eri Aoki2,
Hiroto Takeuchi2, Yukinari Kato4,5, Mika K. Kaneko4, Shinji Yamada ID4, Yumiko Kagawa6,
Maki Nishimura6, Satoshi Takagi7,8, Tatsuya Deguchi7,9, Hiroshi Ohta7,9,Takayuki Nakagawa10, Yasuhiko Suzuki ID1,3,11,12, Tomohiro Okagawa1, Shiro Murata1,2,
Kazuhiko Ohashi1,2,13
1 Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo,
Japan, 2 Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo,
Japan, 3 Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo,
Japan, 4 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Japan, 5 Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai, Japan, 6 North Lab, Sapporo, Japan, 7 Veterinary Teaching Hospital, Faculty of Veterinary
Medicine, Hokkaido University, Sapporo, Japan, 8 Department of Veterinary Surgery 1, School of Veterinary
Medicine, Azabu University, Sagamihara, Japan, 9 Companion Animal Internal Medicine, Department ofCompanion Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,
Japan, 10 Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Bunkyo, Japan, 11 International Institute for Zoonosis Control, Hokkaido University,
Sapporo, Japan, 12 Global Station for Zoonosis Control, Global Institution for Collaborative Research and
Education (GI-CoRE), Hokkaido University, Sapporo, Japan, 13 International Affairs Office, Faculty of
Veterinary Medicine, Hokkaido University, Sapporo, Japan* konnai@vetmed.hokudai.ac.jp

Abstract
Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has
progressed dramatically in the past decade, partly due to the success of immunotherapies
using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PDligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1
therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1
and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 sharehigh sequence identities (66–83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor–ligand
binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established ananti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PDL1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in afeline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm,
FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1
expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5,

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21K1498301 and 22H02503) from the Japan
Society for the Promotion of Science (JSPS) andby Japan Agency for Medical Research andDevelopment (AMED) under Grant Numbers:
JP223fa627005, JP22ama121008, andJP21am0101078. Funders had no role in the study
design, data collection and analysis, decision to
publish, and manuscript preparation.
Competing interests: SK, NM, T Okagawa, SM,
KO, and Y Suzuki are authors of a patentapplication that covers materials and techniques
described in this paper (JP2022-135595). All otherauthors declare no competing interests. This does
not alter our adherence to PLOS ONE policies on
sharing data and materials.

PD-L1 immunohistochemistry for feline tumors80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results
strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.

Introduction
Tumor treatment in humans has progressed dramatically in the past decade, partly due to thesuccess of molecular-targeted therapies and immunotherapies. Among the emerging therapies,
immune checkpoint inhibitors (ICIs), such as anti-programmed death 1 (PD-1) and anti-PDligand 1 (PD-L1) antibodies, have shown great promise in the treatment of various malignancies including melanoma, non-small-cell lung cancer (NSCLC), and renal-cell cancer [1, 2].
Cats develop spontaneous tumors typically at >8 years of age, and malignant tumors are diagnosed 4.6-fold more often than benign tumors [3]. Feline tumors are treated with surgery,radiation, and chemotherapy; however, despite advances in veterinary care, neoplasia-related
deaths account for 10.8% of the mortality in cats, ranking fourth following trauma (12.2%),
renal disorder (12.1%), and non-specific illness (11.2%) [4]. Therefore, the establishment of
immunotherapies for feline tumors is urgently needed as an additional/alternative option toconventional therapies.
The PD-1/PD-L1 pathway is an important suppressive mechanism for T cell-mediatedimmunity. PD-1 is an immunoinhibitory receptor expressed on T cells that attenuates antigen-specific responses, such as cell proliferation, cytokine secretion, and cytotoxicity [5, 6].
The expression of PD-L1, a PD-1 ligand, can be found in both hematopoietic and nonhematopoietic cell types, whereas the expression of PD-L2, the other ligand, is restricted toimmune cells, such as dendritic cells and macrophages [5]. PD-L1 overexpression is often
reported in various human tumors and its expression is correlated with poor prognosis in cancer patients [7–11]. The blockade of PD-1/PD-L1 using antibodies induces robust antitumor
immune responses by reinvigorating T cell effector functions [6, 12], which has promoted thestandard of care for various malignancies in humans. In cats, limited information is available
on the PD-1/PD-L1 pathway and its association with tumor disease is unclear. Folkl et al. [13]reported the complete feline PD-1 and partial PD-L1 mRNA coding sequences (CDS) andfound upregulation of PD-1/PD-L1 proteins in the lymphocytes of cats chronically infectedwith feline immunodeficiency virus. The complete CDS for feline PD-L2 mRNA has been registered in the NCBI GenBank database by the same authors (accession number:
NM_001290244.1); however, the experimental procedures have not yet been reported, andcharacterization of its protein is yet to be performed. Recently, an immunohistochemical study
using anti-human PD-L1 monoclonal antibody revealed that HER2-positive and triple-negative normal-like feline mammary carcinomas express PD-L1 [14], while PD-L1 expression inother feline tumors remains unknown. Thus, further genetic and functional characterizationof feline PD-L1/PD-L2, as well as the analysis of PD-L1 expression in various feline malignant
tumors, is required to develop effective feline ICI therapies.
PD-L1 is a useful biomarker of the clinical response of tumors to anti-PD-1/PD-L1 antibody therapies in humans. In a clinical study of melanoma, NSCLC, colorectal cancer, renalcell cancer, or castration-resistant prostate cancer, pretreatment tumor cell-surface expressionof PD-L1 (as determined with immunohistochemistry [IHC]) was correlated with the response
to PD-1 blockade, wherein 36% of patients with PD-L1-positive tumors experienced an objective response, while no (0%) patients with PD-L1-negative tumors responded to the treatment

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PD-L1 immunohistochemistry for feline tumors[1]. Another study in NSCLC patients treated with anti-PD-1 antibody revealed that patientswith a proportion score (percentages of neoplastic cells with membranous PD-L1 staining) ofat least 50% had a higher response rate and longer progression-free/overall survival than thosewith a proportion score of less than 50% [15]. Accordingly, the successful application of antiPD-1/PD-L1 antibody drugs requires immunohistochemical assessment of PD-L1 for theselection of eligible patients. Although PD-L1 IHC has some limitations as a predictive biomarker [8] and other biomarkers, such as tumor mutational burden and microsatellite instability, have been proposed [16], the establishment of a sensitive and robust PD-L1 IHC for
feline tumors is of particular importance for informed selection of cat subjects that are most
likely to benefit from anti-PD-1/PD-L1 therapies.
To explore the feasibility of anti-PD-1/PD-L1 therapy for feline tumors, we first determinedthe complete CDS of feline PD-L1 and PD-L2 mRNA and compared the deduced amino acidsequences with known orthologs in other mammalian species. Recombinant proteins of feline
PD-1, PD-L1, and PD-L2 were prepared for molecular characterization, and receptor–ligand
binding was assessed in an expressing cell-based assay. Then, an anti-feline PD-L1 monoclonal
antibody (CL1Mab-7) was established and its applicability for expression analysis was evaluated by flow cytometry using feline macrophage and mammary adenocarcinoma cell lines.
Finally, IHC using CL1Mab-7 was developed to probe PD-L1 expression in various feline
malignant tumors.

Materials and methods
Feline samples
The use of animal-derived samples throughout the study was approved by the Institutional
Animal Care and Use Committee, Faculty of Veterinary Medicine, Hokkaido University(approval #20–0093), which has been fully accredited by the Association for Assessment andAccreditation of Laboratory Animal Care International. No animal was sacrificed for thisstudy. Testis tissue samples were freshly resected from a mixed-breed cat under general anesthesia at the Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Hokkaido University. Peripheral blood samples were obtained from healthy cats (mixed breed) maintained at
the Experimental Animal Facility, Faculty of Veterinary Medicine, Hokkaido University or aveterinary hospital in Sapporo, Japan. Formalin-fixed and paraffin-embedded (FFPE) feline
tissues were obtained from a commercial pathology laboratory (North Lab, Hokkaido, Japan).

Cell culture
Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood samples
by density gradient centrifugation on Percoll (GE Healthcare, Buckinghamshire, UK) and cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10%
fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine,
200 μg/mL streptomycin, 200 U/mL penicillin (Thermo Fisher Scientific), and 5 μg/mL concanavalin A (ConA; Sigma-Aldrich) for 10 h at 37˚C and 5% CO2. ExpiCHO-S cells (Thermo
Fisher Scientific) were cultured on an orbital shaker (125 rpm) at 37˚C and 8% CO2 in ExpiCHO Expression Medium (Thermo Fisher Scientific). The feline macrophage cell line Fcwf-4[Fcwf] (ATCC CRL-2787) was cultured in Eagle’s Minimum Essential Medium (ATCC,
Manassas, VA, USA) containing 10% FBS (Thermo Fisher Scientific) at 37˚C and 5% CO2.
The feline mammary adenocarcinoma cell lines FKNp, FMCp, FMCm, FYMp, FONp, and
FONm [17] were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10%
FBS (Thermo Fisher Scientific), 2 mM L-glutamine, 200 μg/mL streptomycin, and 200 U/mL
penicillin (Thermo Fisher Scientific) at 37˚C and 5% CO2. Cell lines were cultured with 100

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ng/mL recombinant feline interferon-gamma (IFN-γ; Kingfisher Biotech, St. Paul, MN, USA)for 24 h before analysis, where indicated.

Nucleotide sequencing, alignment, and phylogenetic analysis
To determine the complete CDS of feline PD-L1, we designed primers for rapid amplificationof cDNA ends (RACE; fePD-L1_50 GSP1–3, fePD-L1_30 GSP1, and -2) based on a previously
reported partial sequence of feline PD-L1 (GenBank accession number: EU246348.2). Total
RNA was extracted from cat testes using TRI reagent (Molecular Research Center, Cincinnati,
OH, USA), and 30 and 50 unknown sequences were amplified using 30 and 50 RACE System for
Rapid Amplification of cDNA Ends (Thermo Fisher Scientific) and TaKaRa Ex Taq polymerase (Takara Bio, Shiga, Japan). The amplicons were purified, cloned into pGEM-T Easy Vector(Promega, Madison, WI, USA), and sequenced using GenomeLab GeXP Genetic Analysis System (SCIEX, Framingham, MA, USA). Based on the obtained sequence, a primer pair was
designed to amplify the whole CDS of feline PD-L1 (fePD-L1_F and R), and polymerase chain
reaction (PCR) was performed using TaKaRa Ex Taq polymerase (Takara Bio) and cDNA templates synthesized from ConA-stimulated feline PBMCs, as previously described [18]. Nucleotide sequences of the amplicons were determined as described above. Similarly, to determine
the complete CDS of feline PD-L2, a primer pair was designed based on NM_001290244.1(fePD-L2_F and R), PCR was performed using cDNAs of ConA-stimulated feline PBMCs, andthe nucleotide sequences of the amplicons were determined as described above. The CDSs offeline PD-L1 and PD-L2 were translated into deduced amino acid sequences and aligned withknown orthologs in other mammalian species using BioEdit [19]. The signal peptide, transmembrane domain, and other conserved domains were predicted using SignalP 6.0 (https://
services.healthtech.dtu.dk/service.php?SignalP), TMHMM 2.0 (https://services.healthtech.dtu.
dk/service.php?TMHMM-2.0), and CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/
wrpsb.cgi), respectively. Potential N-linked glycosylation sites were predicted using NetNGlyc-1.0 (https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0). The percentages ofidentical and positive matches between amino acid sequences were calculated using Protein
BLAST (blastp; https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). Unrooted neighborjoining phylogenetic trees were inferred using MEGA version 6.06 [20, 21]. Nucleotide
sequences of feline PD-L1 and PD-L2 were submitted to the DDBJ/EMBL-Bank/GenBank
database under accession numbers LC735019, LC735020, and LC735021. Primer sequencesused in this study are listed in Table 1.

Transient expression of recombinant proteins
To prepare feline PD-1 or PD-L1-expressing cells, we constructed expression vectors for feline
PD-1 or PD-L1 fused to the C-terminal enhanced green fluorescent protein (fePD-1–EGFP;
fePD-L1–EGFP) using pEGFP-N2 vector (Clontech, Palo Alto, CA, USA). Nucleotide
sequences encoding feline PD-1 (NM_001145510.1) and PD-L1 were amplified by PCR using
specific primers with restriction enzyme cleavage sites (fePD-1–EGFP_F and R; fePD-L1–
EGFP_F and R, Table 1), and subcloned into the multicloning site of the pEGFP-N2 vector.
The expression plasmids were purified using FastGene Xpress Plasmid PLUS Kit (Nippon
Genetics, Tokyo, Japan) and stored at −20˚C until use. ExpiCHO-S cells were transfected withthe plasmid using ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific) and cultured for 24 h prior to analysis. The subcellular localization of fePD-1–EGFP and fePD-L1–
EGFP was visualized using ZOE Fluorescent Cell Imager (Bio-Rad, Hercules, CA, USA).
To prepare soluble feline PD-1, PD-L1, and PD-L2 proteins, we constructed expression vectors for feline PD-1, PD-L1, or PD-L2 extracellular region fused to the C-terminal rabbit IgG

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Table 1. PCR primer sequences.
Nucleotide sequence (50 –30 )RemarkfePD-L1_5 GSP1CTAGAATCATGAAGTGA

EU246348.2

fePD-L1_50 GSP2

TTGATTCTCAGCGTGCTGGT

EU246348.2

fePD-L1_50 GSP3

GCCATAGCCAATCAAGCAGC

EU246348.2

fePD-L1_3 GSP1

GCTGCTTGATTGGCTATGGC

EU246348.2

fePD-L1_30 GSP2TGCTGAGTTGGTCATCCCAG

EU246348.2

fePD-L1_F

CTCCCCGCCGGCAGAAAA

fePD-L1_R

CTGGTCATGCTTACCCCTGACG

Primer name
00

fePD-L2_F

TCAGATCGGGTACAGAGC

NM_001290244.1

fePD-L2_R

TTCAGAGTCCAGAACATTCTC

NM_001290244.1

fePD-1–EGFP_FCGGAATTCATGGGGACCCCACGGGCGCC

NM_001145510.1, EcoRI
NM_001145510.1, BamHI

fePD-1–EGFP_R

CGGGATCCCGAGGGGCCAAGGGCAGGGTC

fePD-L1–EGFP_FGAAGATCTATGAGGATATTTAGTGTCTT

BglII

fePD-L1–EGFP_R

CGGAATTCCGTCTCCTCAAATTGTAGATEcoRI

fePD-1–Ig_F

CGCGGCTAGCATGGGGACCCCACGGGCGC

NM_001145510.1, NheI
NM_001145510.1, EcoRV

fePD-1–Ig_R

CGCGGATATCCAGCCCCTGGCCTTGGCCG

fePD-L1–Ig_F

CGCGGCTAGCATGAGGATATTTAGTGTCT

NheI

fePD-L1–Ig_R

CGCGGATATCCCTCTCATTTGCTGGAACC

EcoRV

fePD-L2–Ig_F

CGCGGATATCATGTTCCTCCTGCTGATGTT

EcoRV

fePD-L2–Ig_R

CGGGGTACCATTTGAAGGGATCTTGGGTT

KpnI

GenBank accession numbers for the reference sequences are shown. Restriction enzyme recognition sites are underlined.
https://doi.org/10.1371/journal.pone.0281143.t001

Fc region (fePD-1–Ig, fePD-L1–Ig, and fePD-L2–Ig) using pCXN2.1 (+) vector (kindly provided by Dr. T. Yokomizo, Juntendo University, Tokyo, Japan) [22]. Nucleotide sequences
encoding the extracellular regions of feline PD-1 (1–169), PD-L1 (1–237), and PD-L2 (1–219)
were amplified by PCR using specific primers with restriction enzyme cleavage sites (fePD-1–
Ig_F and R, fePD-L1–Ig_F and R, and fePD-L2–Ig_F and R; Table 1), and subcloned into themulticloning site of the pCXN2.1 (+) vector containing a gene cassette encoding the Fc regionof rabbit IgG. The expression plasmids were purified using FastGene Xpress Plasmid PLUS Kit(Nippon Genetics) or NucleoBond Xtra Midi (Takara Bio) and stored at −20˚C until use. ExpiCHO-S cells were transfected with the plasmid using ExpiFectamine CHO Transfection Kit(Thermo Fisher Scientific) and cultured for 10 d. Recombinant fusion proteins were purified
from the culture supernatant by protein A affinity chromatography using Ab-Capcher ExTra(ProteNova, Kagawa, Japan). After elution with Pierce IgG Elution Buffer (Thermo Fisher Scientific), the buffer was replaced with phosphate-buffered saline (PBS; FUJIFILM Wako Pure
Chemical, Osaka, Japan) using PD MidiTrap G25 (Cytiva, Tokyo, Japan). The protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 2×
Laemmli sample buffer (Bio-Rad) prepared with 2-mercaptoethanol (reducing conditions;
FUJIFILM Wako Pure Chemical) or distilled water (non-reducing conditions). Recombinantfusion proteins were incubated at 96˚C for 5 min in sample buffer and separated by electrophoresis using SuperSep Ace 5–20% gradient gel (FUJIFILM Wako Pure Chemical). Precision
Plus Protein Dual Color Standards (Bio-Rad) were used as molecular weight markers. Protein
bands were visualized by Coomassie brilliant blue (CBB) staining using Quick-CBB kit (FUJIFILM Wako Pure Chemical).

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Monoclonal antibody against feline PD-L1
To establish the anti-feline PD-L1 monoclonal antibody CL1Mab-7, BALB/c mice (4-weekold, female; CLEA Japan, Tokyo, Japan) were immunized intraperitoneally with 100 μg
fePD-L1–Ig formulated with Imject Alum Adjuvant (Thermo Fisher Scientific). After the second booster administration, splenocytes were harvested and fused with P3X63Ag8U.1 (ATCC
CRL-1597) cells using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA) to generate ahybridoma pool. Cell cloning was performed by limiting dilution and monoclonal antibodies
were purified from the culture supernatant using Protein G Sepharose 4 Fast Flow (GE Healthcare). Isotype-specific secondary antibodies (Southern Biotech, Birmingham, AL, USA) wereused to identify the CL1Mab-7 antibody (sub)class (mouse IgG1, κ).

Flow cytometry
To examine whether feline PD-1 binds to PD-L1/PD-L2, 2 × 105 fePD-1–EGFP- andfePD-L1–EGFP-expressing cells were incubated for 30 min with 10 μg/mL of either fePD-1–
Ig, fePD-L1–Ig, or fePD-L2–Ig at room temperature (RT), followed by another 30 min incubation with Alexa Fluor 647-conjugated F(ab0 )2-goat anti-rabbit IgG (H+L) secondary antibody(Thermo Fisher Scientific). Rabbit IgG (Southern Biotech) was used as a control protein. Cell
fluorescence was analyzed using BD FACSLyric system (BD Biosciences, Franklin Lakes, NJ,
USA).
To examine PD-L1 expression on cell lines, 2 × 105 cells were incubated for 30 min with
10 μg/mL CL1Mab-7 or mouse IgG1κ isotype-matched control antibody (15H6; Southern Biotech) at RT, followed by another 30 min incubation with Alexa Fluor 647-conjugated F(ab0 )2goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific). Cell fluorescence
was analyzed using BD FACSLyric system (BD Biosciences). For fePD-1–EGFP- andfePD-L1–EGFP-expressing cells, only EGFP-positive cells were gated and subjected to further
analysis.

Immunohistochemistry
We prepared 4-μm-thick sections of FFPE feline squamous cell carcinoma (n = 5), mammary
adenocarcinoma (n = 5), fibrosarcoma (n = 5), and renal cell carcinoma (n = 3) tissue samples,and antigen retrieval was performed twice by microwave treatment for 5 min in Tris-EDTA
buffer (pH 9.0; Aligent Technologies, Santa Clara, CA, USA). Endogenous peroxidase activity
was blocked by incubating sections in methanol containing 0.3% hydrogen peroxide. Sections
were incubated with 10 μg/mL CL1Mab-7 or mouse IgG1κ isotype-matched control antibody(MG1-45; BioLegend, San Diego, CA, USA) at RT for 30 min, followed by another 30 min
incubation with Histofine simple stain MAX PO (MULTI) (Nichirei, Tokyo, Japan). A chromogenic reaction was developed using 3, 30 -diaminobenzidine tetrahydrochloride (Nichirei).
Mayer’s hematoxylin was used as a counterstain.

Results
Characterization of feline PD-L1 and PD-L2
To date, the complete nucleotide sequences of feline PD-L1 and PD-L2 mRNAs have not beenreported in the literature, whereas only a partial sequence of PD-L1 and a complete sequenceof PD-1 have been described [13]. We first determined the mRNA sequences of feline PD-L1
and PD-L2 using cDNAs synthesized from PBMCs of mixed-breed cats. The complete CDS offeline PD-L1 and PD-L2 was 876 and 819 bp in length, encoding 291 and 272 polypeptide
sequences, respectively. The PD-L1 mRNA sequence was identical among four individual cats,

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Table 2. Amino acid sequence similarities of feline PD-L1/PD-L2 with other mammals.
PD-L1 identities (%)

PD-L1 positives (%)

PD-L2 identities (%)

PD-L2 positives (%)

Dog

83

88

NA

NA

Water buffalo

77

84

NA

NA

Cattle

77

84

78

85

Pig

74

86

77

84

Human

73

80

73

81

Rhesus macaque

71

78

76

83

Mouse

67

79

69

80

Rat

67

79

66

79

Percentages of identical and positive (allowing conservative substitution) matches in the amino acid sequence of feline PD-L1 and PD-L2 are calculated in relation toorthologs from other mammalian species by Protein BLAST (blastp). NA: not available.
https://doi.org/10.1371/journal.pone.0281143.t002

whereas the PD-L2 mRNA sequence contained a single nucleotide polymorphism between thetwo cats tested (C520A; silent mutation). The deduced amino acid sequence of feline PD-L1
and PD-L2 showed high sequence identity with orthologs in other mammalian species (66–83%, Table 2). Both feline PD-L1 and PD-L2 were predicted to contain a signal peptide, two
immunoglobulin superfamily (IgSF) domains, and a transmembrane domain (Figs 1a and 2a).
In the phylogenetic analyses, feline PD-L1 and PD-L2 formed a cluster with orthologs in Perissodactyla (e.g., cattle and pig) and Carnivora (dog), and were relatively distant in relation toPrimates (human and rhesus macaque) and Rodentia (mouse and rat; Figs 1b and 2b).

Characterization of feline PD-1, PD-L1, and PD-L2
Next, we expressed recombinant feline PD-1 and PD-L1 as C-terminal EGFP-fusion proteinsand examined their subcellular localization using fluorescence microscopy. In both fePD-1–
EGFP- and fePD-L1–EGFP-expressing cells, EGFP fluorescence was concentrated at the cell
membrane, indicating cell surface expression of the recombinant fusion proteins (Fig 3a). To
examine the binding between PD-1 and PD-L1/PD-L2, the extracellular regions of PD-1,
PD-L1, and PD-L2 were expressed as soluble rabbit IgG Fc fusion proteins. Purified fePD-1–
Ig, fePD-L1–Ig, and fePD-L2–Ig migrated at approximately 60 kDa and 140 kDa under reducing and non-reducing conditions, respectively, suggesting dimer formation by a disulfide
bond at the hinge region of rabbit IgG Fc under non-reducing conditions (Fig 3b). The theoretical molecular weights (as monomers) of fePD-1–Ig, fePD-L1–Ig, and fePD-L2–Ig, calculated from the deduced amino acid sequences, were 42.3, 50.9, and 48.3 kDa, respectively.
Because four, five, and five potential N-glycosylation sites were found in the extracellularregions of feline PD-1, PD-L1, and PD-L2, respectively, these proteins were expected to behighly glycosylated, which influences migration in SDS-PAGE. Then, the bindings of Fcfusion proteins to fePD-1–EGFP- and fePD-L1–EGFP-expressing cells were assessed by flow
cytometry. As expected, both fePD-L1–Ig and fePD-L2–Ig were bound to fePD-1–EGFPexpressing cells, and only fePD-1–Ig was bound to fePD-L1–EGFP-expressing cells (Fig 3c),suggesting receptor–ligand interactions between feline PD-1 and PD-L1/PD-L2.

PD-L1 expression in feline macrophage and mammary adenocarcinoma
cell lines
To investigate PD-L1 expression in feline tumors, the anti-feline PD-L1 monoclonal antibody
CL1Mab-7 was established by immunizing mice with fePD-L1–Ig. The binding specificity of

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Fig 1. Identification of feline PD-L1 mRNA sequence. (a) Multiple sequence alignment of PD-L1 amino acid sequences in various mammalian
species. The predicted signal peptide (SP), immunoglobulin superfamily (IgSF) domains, and transmembrane (TM) domain are indicated withnumbers representing the positions of amino acid residues. (b) Phylogenetic tree based on the feline PD-L1 amino acid sequence in relation to those ofother mammalian species. The tree was inferred using the neighbor-joining method with a bootstrap of 1,000 replicates. Numbers next to the branches
indicate the bootstrap percentage. The scale bar indicates the divergence time.
https://doi.org/10.1371/journal.pone.0281143.g001

CL1Mab-7 was confirmed by flow cytometry, in which CL1Mab-7 reacted with fePD-L1–
EGFP-expressing cells, while no binding was observed in control EGFP-expressing cells(mock, transfected with an empty vector; Fig 4a). Then, we tested whether CL1Mab-7 could
detect the native PD-L1 protein expressed on the surface of feline cells. Consistent with

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Fig 2. Identification of feline PD-L2 mRNA sequence. (a) Multiple sequence alignment of PD-L2 amino acid sequences in various mammalian
species. The SP, IgSF domains, and TM domain are indicated with numbers representing the positions of amino acid residues. (b) Phylogenetic tree
(inferred as described in Fig 1) of the feline PD-L2 amino acid sequence in relation to those of other mammalian species. Numbers next to the branches
indicate the bootstrap percentage. The scale bar indicates the divergence time.
https://doi.org/10.1371/journal.pone.0281143.g002

previous reports showing that monocytes/macrophages constitutively express PD-L1 [5, 7, 23]
and IFN-γ is a potent inducer of PD-L1 expression [7, 24], CL1Mab-7 showed slight binding
to Fcwf-4 cells, and its binding was increased by treatment with IFN-γ (Fig 4b). We next examined PD-L1 expression on feline mammary adenocarcinoma cell lines using CL1Mab-7 via
flow cytometry. Among the six cell lines, FKNp, FMCm, FYMp, FONp, and FONm expressed
PD-L1 at various levels; IFN-γ stimulation enhanced PD-L1 expression on these five cell lines.
The other cell line, FMCp, did not express PD-L1 even in the presence of IFN-γ (Fig 4c).

PD-L1 expression in feline malignant tumor tissues
Finally, PD-L1 IHC for FFPE feline tumor tissue was established using CL1Mab-7. In contrast
to the isotype-matched control antibody (mouse IgG1), CL1Mab-7 produced specific positive

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Fig 3. Characterization of feline PD-1/PD-L1/PD-L2 proteins. (a) Preparation of fePD-1–EGFP- and fePD-L1–EGFP-expressing cells. Cells were
harvested and observed under a fluorescent microscope at 24 h post transfection. (b) Preparation of fePD-1–Ig, fePD-L1–Ig, and fePD-L2–Ig. The
purified Fc-fusion proteins were separated under reducing (left) or non-reducing (right) conditions by SDS-PAGE. The proteins were visualized by
CBB staining. (c) Binding of Fc-fusion proteins to fePD-1–EGFP- and fePD-L1–EGFP-expressing cells. Cells were incubated with 10 μg/mL of each Fcfusion protein and their binding was detected using anti-rabbit IgG secondary antibody. Rabbit IgG was used as a negative control.
https://doi.org/10.1371/journal.pone.0281143.g003

signals in feline squamous cell carcinoma (Fig 5a and 5b). Among the five squamous cell carcinoma, five mammary adenocarcinoma, five fibrosarcoma, and three renal cell carcinoma samples tested, PD-L1 was detected in tumor cells of five (100%), four (80%), five (100%), andthree (100%) samples, respectively (Fig 5c–5e, Table 3). The normal tubular epithelium wasalso positive for PD-L1 in renal cell carcinoma specimens. Tumor cells were stained intracellularly and showed faint membrane staining in most specimens, with stromal tissues surrounding the tumor cells being mostly negative for PD-L1.

Discussion
ICIs, such as anti-PD-1/PD-L1 antibodies, have become indispensable for the treatment of various malignancies in humans. Recently, ICIs have also been developed for use in veterinary
medicine. In cattle infected with bovine leukemia virus, anti-PD-1/PD-L1 antibody treatment
has been shown to reactivate antigen-specific T cell responses and reduce proviral load [25,26]. The therapeutic potential of anti-PD-L1 antibody has also been suggested for bovine
mycoplasmosis [27] and Johne’s disease [28], implying its broad applicability for chronic infections in cattle. Moreover, in dogs with malignant melanoma and undifferentiated sarcoma,
treatment with anti-PD-L1 antibody exhibited antitumor efficacy with acceptable safety profiles [29, 30]. Because PD-L1 expression is found in several canine malignancies, including
squamous cell carcinoma, mammary adenocarcinoma, transitional cell carcinoma, andhemangiosarcoma [18, 30–34], ICIs may also be effective for these tumors. Based on the similarity in tumorigenesis and the immune system among mammalian species, we hypothesizedthat immune checkpoint blockade could be an effective therapeutic strategy for feline tumor
treatment. We found that the molecular characteristics of feline PD-1/PD-L1/PD-L2 are similar to those of its orthologs in other mammalian species in the aspects of evolutionary history,
membranous expression, and receptor–ligand binding activities. The monoclonal antibody

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Fig 4. PD-L1 expression in feline macrophage and mammary adenocarcinoma cell lines. (a) Binding of the anti-feline PD-L1 monoclonal antibody
CL1Mab-7 to fePD-L1–EGFP-expressing cells. Cells were transfected with EGFP (mock) or fePD-L1–EGFP expression vector and analyzed by flow
cytometry at 24 h post transfection. (b) PD-L1 expression in the feline macrophage cell line Fcwf-4. (c) PD-L1 expression in feline mammary
adenocarcinoma cell lines FKNp, FMCp, FMCm, FYMp, FONp, and FONm. Cells were incubated with 100 ng/mL of recombinant feline IFN-γ for 24
h where indicated. Mouse IgG1 (mIgG1) was used as an isotype-matched negative control antibody.
https://doi.org/10.1371/journal.pone.0281143.g004

against feline PD-L1 CL1Mab-7 successfully detected the inducible expression of PD-L1 on
feline cell lines, and IHC using the same antibody revealed PD-L1 expression in tissue samplesof feline malignant tumors. These results strongly encourage further investigation of the PD-1/
PD-L1 pathway as a therapeutic target for feline tumors.
The high sequence identities and conserved domain structures of PD-1/PD-L1/PD-L2
among mammal species suggest a functional similarity of the PD-1 pathway as a suppressive
mechanism in T cell-mediated immunity. Although immunosuppressive functional motifs(immunoreceptor tyrosine-based inhibitory motif [ITIM] and immunoreceptor tyrosinebased switch motif [ITSM]) are conserved in the cytoplasmic tail of feline PD-1 [13], its signaltransmission and suppression of T cell functions are yet to be investigated experimentally.
Moreover, whether reactivation of T cell responses can be achieved by blocking the PD-1/
PD-L1 pathway is a topic of future research. To date, no blocking antibody has been developed
for feline PD-1/PD-L1, which limits the functional assessment of the PD-1 pathway in cats.
We are now in progress to identify blocking antibodies from the hybridoma pool of antiPD-L1 monoclonal antibodies.

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Fig 5. PD-L1 immunohistochemistry (IHC) for feline malignant tumors. (a) IHC staining of feline squamous cell carcinoma using anti-feline PD-L1
monoclonal antibody CL1Mab-7. (b) IHC staining of feline squamous cell carcinoma using mIgG1. (c) CL1Mab-7 IHC staining of feline mammary
adenocarcinoma. (d) CL1Mab-7 IHC staining of feline fibrosarcoma. (e) CL1Mab-7 IHC staining of feline renal cell carcinoma. Scale bar, 100 μm.
https://doi.org/10.1371/journal.pone.0281143.g005

IFN-γ treatment induced PD-L1 expression in feline macrophage and mammary adenocarcinoma cell lines, consistent with the findings in humans and dogs [7, 18, 24]. IFN-γ signaling
induces interferon-responsive factor-1 (IRF-1) via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, which upregulates the transcription of PD-L1 [24,35]. Although de novo synthesis of PD-L1 may be involved, the detailed mechanism of PD-L1
induction in feline cells remains unclear. In the human tumor microenvironment, various regulatory mechanisms of PD-L1 expression have been reported, including genomic alterations,epigenetic regulation, transcriptional regulation, post-transcriptional regulation, and posttranslational modifications [36, 37]. Interestingly, one out of the six mammary adenocarcinoma cell lines (FMCp, Fig 4c) did not express PD-L1 even after IFN-γ stimulation, suggestinga defect in the expression machinery of PD-L1 or IFN-γ signaling pathway. Because a similar
portion (1/5, 20%) of mammary adenocarcinoma tissues was PD-L1-negative in our IHC
results, investigation of the frequency and types of, for example, genetic mutations inTable 3. Summary of PD-L1 immunohistochemistry in feline malignant tumors.
Tumor typeNo. of positive/tested cases

Positive rate

Squamous cell carcinoma

5/5

100%

Mammary adenocarcinoma

4/5

80%

Fibrosarcoma

5/5

100%

Renal cell carcinoma

3/3

100%

https://doi.org/10.1371/journal.pone.0281143.t003

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mammary adenocarcinoma may provide further insights into the regulatory mechanisms of
PD-L1 in feline tumors.
We observed PD-L1 expression in the vast majority of feline tumor samples tested in thisstudy. IHC using anti-PD-L1 antibody clone 22C3 revealed that 71.3%, 51.2%, and 38.1% of
human squamous cell carcinomas, renal cancers, and breast cancers, respectively, were
PD-L1-positive [38]. The high rates of PD-L1 expression in the current study could be an artifact of a small sample size; therefore, our results need to be verified with a larger sample size of
feline tumors. Fibrosarcoma is a rare soft tissue tumor in humans that, based on limited data,
expresses PD-L1 at a low positive rate (15%) [39]. Fibrosarcoma is more common in cats thanin humans and is often diagnosed as a histologic subtype of feline injection site sarcomas(FISS) [40, 41]. The high frequency of PD-L1 expression in feline fibrosarcoma suggests that,in the unique etiopathogenesis of feline FISS (where chronic inflammation is thought to trigger tumorigenesis [42]), PD-L1 induction by inflammatory signaling (IFNs, IL-6, TNF-α, etc.)[37] may play a pivotal role in the escape from adaptive immune responses. Nonetheless, thedetection sensitivity of PD-L1 IHC using CL1Mab-7 seemed high and sufficient, highlighting
its potential for use in the screening of cats that would respond to anti-PD-1/PD-L1 therapy.
In conclusion, our molecular characterization of feline PD-1/PD-L1/PD-L2 suggests thatthe immunosuppressive functions of the PD-1 pathway are conserved in cats, and PD-L1 IHC
revealed a potential immune evasion mechanism commonly exploited by feline tumors.
Although whether feline T cell-mediated immunity can be reinvigorated by the PD-1/PD-L1
blockade remains to be elucidated, our results support further investigation of ICIs as potential
immunotherapies for feline tumors.

Supporting information
S1 Fig. The original uncropped and unadjusted images for Fig 3b.
(TIF)

Author Contributions
Conceptualization: Naoya Maekawa, Satoru Konnai, Shiro Murata, Kazuhiko Ohashi.
Data curation: Satoru Konnai.
Formal analysis: Naoya Maekawa, Satoru Konnai, Yumie Asano, Takumi Otsuka, Hiroto
Takeuchi, Yukinari Kato, Shinji Yamada, Yumiko Kagawa, Maki Nishimura, Tomohiro
Okagawa.
Funding acquisition: Naoya Maekawa, Satoru Konnai, Yukinari Kato.
Investigation: Naoya Maekawa, Satoru Konnai, Yumie Asano, Takumi Otsuka, Eri Aoki, Hiroto Takeuchi, Yukinari Kato, Mika K. Kaneko, Shinji Yamada, Yumiko Kagawa, Maki
Nishimura, Tomohiro Okagawa.
Methodology: Naoya Maekawa, Satoru Konnai, Yumie Asano, Takumi Otsuka, Eri Aoki, Hiroto Takeuchi, Yukinari Kato, Mika K. Kaneko, Shinji Yamada, Yumiko Kagawa, Maki
Nishimura, Satoshi Takagi, Tatsuya Deguchi, Hiroshi Ohta, Takayuki Nakagawa, YasuhikoSuzuki, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi.
Project administration: Naoya Maekawa, Satoru Konnai.
Resources: Naoya Maekawa, Satoru Konnai, Yukinari Kato, Mika K. Kaneko, Yumiko
Kagawa, Maki Nishimura, Satoshi Takagi, Tatsuya Deguchi, Hiroshi Ohta, Takayuki Nakagawa, Yasuhiko Suzuki, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi.

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Software: Shinji Yamada.
Supervision: Satoru Konnai, Yasuhiko Suzuki, Tomohiro Okagawa, Shiro Murata, Kazuhiko
Ohashi.
Validation: Naoya Maekawa, Satoru Konnai, Tomohiro Okagawa.
Writing – original draft: Naoya Maekawa, Satoru Konnai, Yumie Asano, Takumi Otsuka, Eri
Aoki, Yumiko Kagawa, Maki Nishimura.
Writing – review & editing: Naoya Maekawa, Satoru Konnai.

References
1.

Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, andimmune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012; 366: 2443–2454. https://doi.
org/10.1056/NEJMoa1200690 PMID: 22658127

2.

Brahmer JR, Tykodi SS, Chow LQM, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PDL1 antibody in patients with advanced cancer. N Engl J Med. 2012; 366: 2455–2465. https://doi.org/10.
1056/NEJMoa1200694 PMID: 22658128

3.

Vascellari M, Baioni E, Ru G, Carminato A, Mutinelli F. Animal tumour registry of two provinces in northern Italy: incidence of spontaneous tumours in dogs and cats. BMC Vet Res. 2009; 5: 39. https://doi.
org/10.1186/1746-6148-5-39 PMID: 19825169

4.

O’Neill DG, Church DB, McGreevy PD, Thomson PC, Brodbelt DC. Longevity and mortality of catsattending primary care veterinary practices in England. J Feline Med Surg. 2015; 17: 125–133. https://doi.org/10.1177/1098612X14536176 PMID: 24925771

5.

Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev
Immunol. 2008; 26: 677–704. https://doi.org/10.1146/annurev.immunol.26.021607.090331 PMID:
18173375

6.

Pauken KE, Wherry EJ. Overcoming T cell exhaustion in infection and cancer. Trends Immunol. 2015;
36: 265–276. https://doi.org/10.1016/j.it.2015.02.008 PMID: 25797516

7.

Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002; 8: 793–800. https://doi.org/10.1038/nm730 PMID: 12091876

8.

Patel SP, Kurzrock R. PD-L1 expression as a predictive biomarker in cancer immunotherapy. Mol Cancer Ther. 2015; 14: 847–856. https://doi.org/10.1158/1535-7163.MCT-14-0983 PMID: 25695955

9.

Thompson RH, Kuntz SM, Leibovich BC, Dong H, Lohse CM, Webster WS, et al. Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up. Cancer Res. 2006;
66: 3381–3385. https://doi.org/10.1158/0008-5472.CAN-05-4303 PMID: 16585157

10.

Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. Immunohistochemical localization of programmed
death-1 ligand-1 (PD-L1) in gastric carcinoma and its clinical significance. Acta Histochem. 2006; 108:
19–24. https://doi.org/10.1016/j.acthis.2006.01.003 PMID: 16530813

11.

Sznol M, Chen L. Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced
human cancer. Clin Cancer Res. 2013; 19: 1021–1034. https://doi.org/10.1158/1078-0432.CCR-122063 PMID: 23460533

12.

Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in theescape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U
S A. 2002; 99: 12293–12297. https://doi.org/10.1073/pnas.192461099 PMID: 12218188

13.

Folkl A, Wen X, Kuczynski E, Clark ME, Bienzle D. Feline programmed death and its ligand: Characterization and changes with feline immunodeficiency virus infection. Vet Immunol Immunopathol. 2010;
134: 107–114. https://doi.org/10.1016/j.vetimm.2009.10.019 PMID: 19931185

14.

Nascimento C, Urbano AC, Gameiro A, Ferreira J, Correia J, Ferreira F. Serum PD-1/PD-L1 levels,
tumor expression and PD-L1 somatic mutations in HER2-positive and triple negative normal-like feline
mammary carcinoma subtypes. Cancers (Basel). 2020; 12: 1386. https://doi.org/10.3390/
cancers12061386 PMID: 32481540

15.

Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015; 372: 2018–2028. https://doi.org/10.1056/
NEJMoa1501824 PMID: 25891174

16.

Li H, van der Merwe PA, Sivakumar S. Biomarkers of response to PD-1 pathway blockade. Br J Cancer.
2022; 126: 1663–1675. https://doi.org/10.1038/s41416-022-01743-4 PMID: 35228677

PLOS ONE | https://doi.org/10.1371/journal.pone.0281143 January 26, 2023

14 / 16

PLOS ONE

PD-L1 immunohistochemistry for feline tumors

17.

Uyama R, Hong SH, Nakagawa T, Yazawa M, Kadosawa T, Mochizuki M, et al. Establishment andcharacterization of eight feline mammary adenocarcinoma cell lines. J Vet Med Sci. 2005; 67: 1273–
1276. https://doi.org/10.1292/jvms.67.1273 PMID: 16397390

18.

Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, et al. Expression of PD-L1 on
canine tumor cells and enhancement of IFN-γ production from tumor-infiltrating cells by PD-L1 blockade. PLoS One. 2014; 9: e98415.

19.

Hall T. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows
95/98/NT. Nucleic Acids Symp Series. 1999; 41: 95–98.

20.

Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol
Biol Evol. 1987; 4: 406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 PMID: 3447015

21.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013; 30: 2725–2729. https://doi.org/10.1093/molbev/mst197 PMID:
24132122

22.

Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel
eukaryotic vector. Gene. 1991; 108: 193–199. https://doi.org/10.1016/0378-1119(91)90434-d PMID:
1660837

23.

Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999; 5: 1365–1369. https://doi.org/10.1038/70932
PMID: 10581077

24.

Lee SJ, Jang BC, Lee SW, Yang YI, Suh SI, Park YM, et al. Interferon regulatory factor-1 is prerequisite
to the constitutive expression and IFN-γ-induced upregulation of B7-H1 (CD274). FEBS Lett. 2006;
580: 755–762.

25.

Nishimori A, Konnai S, Okagawa T, Maekawa N, Ikebuchi R, Goto S, et al. In vitro and in vivo antivirus
activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection. PLoS One. 2017; 12: e0174916.

26.

Okagawa T, Konnai S, Nishimori A, Maekawa N, Ikebuchi R, Goto S, et al. Anti-bovine programmed
death-1 rat-bovine chimeric antibody for immunotherapy of bovine leukemia virus infection in cattle.
Front Immunol. 2017; 8: 650. https://doi.org/10.3389/fimmu.2017.00650 PMID: 28638381

27.

Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, et al. Clinical efficacy of the combined
treatment of anti-PD-L1 rat-bovine chimeric antibody with a COX-2 inhibitor in calves infected with
Mycoplasma bovis. Jpn J Vet Res. 2020; 68: 77–90.

28.

Sajiki Y, Konnai S, Nagata R, Kawaji S, Nakamura H, Fujisawa S, et al. The enhancement of Th1
immune response by anti-PD-L1 antibody in cattle infected with mycobacterium avium subsp. paratuberculosis. J Vet Med Sci. 2021; 83: 162–166.

29.

Maekawa N, Konnai S, Takagi S, Kagawa Y, Okagawa T, Nishimori A, et al. A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma. Sci Rep. 2017; 7: 8951. https://doi.org/10.1038/s41598-017-09444-2 PMID: 28827658

30.

Maekawa N, Konnai S, Nishimura M, Kagawa Y, Takagi S, Hosoya K, et al. PD-L1 immunohistochemistry for canine cancers and clinical benefit of anti-PD-L1 antibody in dogs with pulmonary metastatic oralmalignant melanoma. NPJ Precis Oncol. 2021; 5: 10. https://doi.org/10.1038/s41698-021-00147-6
PMID: 33580183

31.

Maekawa N, Konnai S, Okagawa T, Nishimori A, Ikebuchi R, Izumi Y, et al. Immunohistochemical analysis of PD-L1 expression in canine malignant cancers and PD-1 expression on lymphocytes in canine
oral melanoma. PLoS One. 2016; 11: e0157176. https://doi.org/10.1371/journal.pone.0157176 PMID:
27276060

32.

Gulay KCM, Aoshima K, Maekawa N, Suzuki T, Konnai S, Kobayashi A, et al. Hemangiosarcoma cells
induce M2 polarization and PD-L1 expression in macrophages. Sci Rep. 2022; 12: 2124. https://doi.org/
10.1038/s41598-022-06203-w PMID: 35136176

33.

Kumar SR, Kim DY, Henry CJ, Bryan JN, Robinson KL, Eaton AM. Programmed death ligand 1 isexpressed in canine B cell lymphoma and downregulated by MEK inhibitors. Vet Comp Oncol. 2017;
15: 1527–1536. https://doi.org/10.1111/vco.12297 PMID: 28111882

34.

Shosu K, Sakurai M, Inoue K, Nakagawa T, Sakai H, Morimoto M, et al. Programmed cell death ligand 1
expression in canine cancer. In Vivo. 2016; 30: 195–204. PMID: 27107075

35.

Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, Rodriguez GA, et al. Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Cell Rep. 2017; 19: 1189–1201. https://doi.org/10.1016/j.celrep.2017.04.031 PMID: 28494868

36.

Zerdes I, Matikas A, Bergh J, Rassidakis GZ, Foukakis T. Genetic, transcriptional and post-translational
regulation of the programmed death protein ligand 1 in cancer: biology and clinical correlations. Oncogene. 2018; 37: 4639–4661. https://doi.org/10.1038/s41388-018-0303-3 PMID: 29765155

PLOS ONE | https://doi.org/10.1371/journal.pone.0281143 January 26, 2023

15 / 16

PLOS ONE

PD-L1 immunohistochemistry for feline tumors

37.

Yi M, Niu M, Xu L, Luo S, Wu K. Regulation of PD-L1 expression in the tumor microenvironment. J
Hematol Oncol. 2021; 14: 10. https://doi.org/10.1186/s13045-020-01027-5 PMID: 33413496

38.

O’Malley DP, Yang Y, Boisot S, Sudarsanam S, Wang JF, Chizhevsky V, et al. Immunohistochemical
detection of PD-L1 among diverse human neoplasms in a reference laboratory: observations based
upon 62,896 cases. Mod Pathol. 2019; 32: 929–942. https://doi.org/10.1038/s41379-019-0210-3 PMID:
30760860

39.

Cunningham CR, Dodd L, Esebua M, Layfield LJ. PD-L1 expression in sarcomas: An immunohistochemical study and review of the literature. Ann Diagn Pathol. 2021; 55: 151823. https://doi.org/10.
1016/j.anndiagpath.2021.151823 PMID: 34656856

40.

Doddy FD, Glickman LT, Glickman NW, Janovitz EB. Feline fibrosarcomas at vaccination sites andnon-vaccination sites. J Comp Pathol. 1996; 114: 165–174. https://doi.org/10.1016/s0021-9975(96)80005-3 PMID: 8920216

41.

Carneiro CS, de Queiroz GF, Pinto ACBCF, Dagli MLZ, Matera JM. Feline injection site sarcoma:
immunohistochemical characteristics. J Feline Med Surg. 2019; 21: 314–321. https://doi.org/10.1177/
1098612X18774709 PMID: 29788832

42.

Martano M, Morello E, Buracco P. Feline injection-site sarcoma: Past, present and future perspectives.
Vet J. 2011; 188: 136–141. https://doi.org/10.1016/j.tvjl.2010.04.025 PMID: 20510635

PLOS ONE | https://doi.org/10.1371/journal.pone.0281143 January 26, 2023

16 / 16