Veterinary Immunology and Immunopathology 261 (2023) 110609

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Veterinary Immunology and Immunopathology
journal homepage: www.elsevier.com/locate/vetimm

Molecular characterization of immunoinhibitory factors PD-1/PD-L1
in sheepWisa Tiyamanee a, Satoru Konnai a, b, c, *, Tomohiro Okagawa b, Yutaro Nojima a,
Otgontuya Ganbaatar a, Naoya Maekawa b, Rie Hasebe d, Yumiko Kagawa e, Yukinari Kato f, g,
Yasuhiko Suzuki a, c, h, i, Shiro Murata a, b, Kazuhiko Ohashi a, b, ja

Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan
Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, JapandLaboratory of Veterinary Hygiene, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japane
North Lab, Sapporo, Japanf
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japang
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, JapanhDivision of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japani
Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo, Japanj
International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japanbc

A R T I C L E I N F O

A B S T R A C T

Keywords:
Sheep
Immunoinhibitory factors
PD-1
PD-L1

Sheep have been used as a large animal experimental model for studying infectious diseases. However, due to alack of staining antibodies and reagents, immunological studies on sheep have not progressed. The immu­
noinhibitory receptor programmed death-1 (PD-1) is expressed on T lymphocytes. The interaction of PD-1 with
its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, andcytotoxicity of T cells. We previously reported that the PD-1/PD-L1 pathway was closely associated with T-cell
exhaustion and disease progression in bovine chronic infections using anti-bovine PD-L1 monoclonal antibodies(mAbs). Furthermore, we found that blocking antibodies against PD-1 and PD-L1 restore T-cell functions andcould be used in immunotherapy of cattle. However, the immunological role of the PD-1/PD-L1 pathway inchronic diseases of sheep remains unknown. In this study, we identified cDNA sequences of ovine PD-1 and PDL1 and examined the cross-activity of anti-bovine PD-L1 mAbs against ovine PD-L1 as well as the expression of
PD-L1 in ovine listeriosis. The amino acid sequences of ovine PD-1 and PD-L1 share a high degree of identity andsimilarity with homologs from ruminants and other mammalian species. Anti-bovine PD-L1 mAb recognized
ovine PD-L1 on lymphocytes in the flow cytometric assay. Furthermore, an immunohistochemical staining
confirmed the PD-L1 expression on macrophages in the brain lesions of ovine listeriosis. These findings indicatedthat our anti-PD-L1 mAb would be useful for analyzing the ovine PD-1/PD-L1 pathway. Further research isneeded to determine the immunological role of PD-1/PD-L1 in chronic diseases such as BLV infection throughexperimental infection of sheep.

1. Introduction
Programmed death-1 (PD-1) is an inhibitory receptor, also known as
an immune checkpoint molecule, that is expressed on activated T cells
and other lymphocytes (Agata et al., 1996). The binding of PD-1 and its
ligand PD-ligand 1 (PD-L1) leads to the inhibition of T-cell receptor
signaling and the subsequent effector functions of T cells, such as

proliferation, cytokine production, and cytotoxic activity, which iscalled T-cell exhaustion (Carter et al., 2002; Freeman et al., 2000;
Selenko-Gebauer et al., 2003). T-cell exhaustion caused by PD-1/PD-L1
interaction has been observed in human chronic and infectious diseases(Boni et al., 2007; Jurado et al., 2008; Muthumani et al., 2008; Penna
et al., 2007). Many studies have demonstrated that blocking this inter­action can restore T-cell activity when addressing immune exhaustion* Corresponding author at: Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
E-mail address: konnai@vetmed.hokudai.ac.jp (S. Konnai).
https://doi.org/10.1016/j.vetimm.2023.110609
Received 5 March 2023; Received in revised form 2 May 2023; Accepted 10 May 2023
Available online 11 May 2023
0165-2427/© 2023 Elsevier B.V. All rights reserved.

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via PD-1/PD-L1 (Barber et al., 2006; Day et al., 2006; Trautmann et al.,2006; Velu et al., 2009). Until now, there have been many therapeuticinterventions approved by the Food and Drug Administration such as
monoclonal antibody therapies targeting PD-1 (nivolumab, pem­
brolizumab, and cemiplimab) and PD-L1 (atezolizumab, durvalumab,
and avelumab). Our previous studies on the PD-1/PD-L1 pathway invarious animals revealed that this pathway mediates T-cell exhaustion
on infectious diseases and cancers in cattle, dog, horse, and pig andanti-PD-1 and anti-PD-L1 monoclonal antibodies (mAbs) can restore
T-cell function in vitro and in vivo (Ikebuchi et al., 2011, 2013; Okagawa
et al., 2017; Nishimori et al., 2017; Maekawa et al., 2014, 2017; Gan­baatar et al., 2020, 2021).
Recently, experimental animal models have become widely used inbiomedical research, including medical and veterinary fields, because invitro models cannot fully mimic the complexity in vivo (Sartoretto et al.,2016). Among the animal models, sheep have been used as an experi­mental model for studying many types of infectious diseases andimmunological disorders in human (Banstola and Reynolds, 2022;
Gaudreault et al., 2022) as well as cattle (Begg et al., 2005; Porta et al.,2019). Sheep are one of the suitable experimental models for biomedical
research due to their availability and societal acceptance as an experi­mental animal (Turner, 2007). Furthermore, sheep have interesting andoutstanding biological features for many types of infectious diseases,
and they reach the late stage of disease earlier than natural hosts (Portaet al., 2019). Thus, the use of sheep as an animal model will provide
more opportunities for greater benefits in biomedical research toaddress the current state of immunological and infectious diseases, as
they can provide faster results. However, a lack of staining antibodies
and reagents affects to limited progress in immunological and infectious
studies using the sheep models (Banstola and Reynolds, 2022). For
example, a series of previous studies has reported the expression levelsof PD-1 and PD-L1 mRNA in lungs and tracheobronchial lymph nodes oflambs infected with respiratory syncytial virus (Sow et al., 2011a,
2011b, 2012). Thus, there is potential interest in analyzing PD-1/PD-L1
to gain a deeper understanding of immune response in sheep, but protein
expression analysis could not be performed due to the lack of the anti­
body reagents.
To fulfill the gap between the need for sheep as experimental models
and the unavailability of reagents, in this study, we first identified thenucleotide sequence of ovine PD-1/PD-L1 mRNA, characterized thesemolecules using alignment and phylogenetic analyses, and then evalu­
ated the possibility of anti-bovine PD-L1 mAbs for detecting andblocking ovine PD-L1.

glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin(Thermo Fisher Scientific) at 37 ◦ C with 5% CO2.
Total RNA was isolated from cultured PBMCs using TRI Reagent(Molecular Research Center, Cincinnati, OH, USA) according to the
manufacturer’s instructions. Excess DNA was removed from RNA sam­ples using deoxyribonuclease I (Thermo Fisher Scientific). cDNA wassynthesized from 1 μg of total RNA using PrimeScript Reverse Tran­
scriptase (Takara Bio, Otsu, Japan) and an oligo (dT) primer, according
to the manufacturer’s instructions.
Gene-specific primers were designed to amplify the OvPD-1 andOvPD-L1 genes, based on the sequences from sheep available on Gen­Bank (XP_027819970 and XP_004004411) (Table 1). OvPD-1 and OvPDL1 cDNAs were amplified from the cDNA templates by PCR using Ex Taq(Takara Bio) and the designed primers. 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). After being introduced to Escherichia coli HST08 Premium
Competent Cells (Takara Bio), they were plated onto LB agar plates(Sigma-Aldrich) containing X-gal (Takara Bio) and ampicillin (SigmaAldrich). The purified plasmid clones were sequenced by a GenomeLab
GeXP Genetic Analysis System (SCIEX, Framingham, MA, USA). The
obtained sequences were aligned, and an unrooted neighbor-joining tree
was created using the MEGA11 software (Molecular Evolutionary Ge­
netics Analysis version 11) (Tamura et al., 2021).
2.3. Preparation of OvPD-1- and OvPD-L1-expressing cells
cDNA encoding OvPD-1 and OvPD-L1 were amplified by PCR using
gene-specific primers with restriction enzyme cleavage sites and subcl­
oned into the multicloning site of pEGFP-N2 (Clontech, Palo Alto, CA,
USA). COS-7 cells were cultured at 37 ◦ C and 5% CO2 in RPMI 1640
medium (Sigma-Aldrich) which was supplemented with 10% heatinactivated FBS (Thermo Fisher Scientific), 2 mM of L-glutamine, 100
U/mL of penicillin, and 100 μg/mL of streptomycin (Thermo Fisher
Scientific). The cells were transfected with pEGPF-N2-OvPD-1 andpEGPF-N2-OvPD-L1 using Lipofectamine 2000 Reagent (Thermo Fisher
Scientific) and cultivated for 48 h after transfection.
2.4. Expression and purification of soluble ovine PD-1 and PD-L1 proteins
The extracellular domain fragments of OvPD-1 and OvPD-L1 with
their leader sequences were amplified by PCR using gene-specificprimers with restriction enzyme cleavage sites (Table 1) to create thesoluble OvPD-1 and OvPD-L1 proteins fused with rabbit IgG Fc region(OvPD-1-Ig and OvPD-L1-Ig) (Table 1). The amplicons were subcloned
into the multicloning site of pCXN2.1(+) (kindly provided by Dr. T.
Yokomizo, Juntendo University, Japan) (Niwa et al., 1991) with the

2. Materials and methods
2.1. Sheep blood samples and cell preparation
Heparinized blood samples from Texel breed sheep were collected at
the Experiment Farm, Field Science Center for Northern Biosphere,
Hokkaido University (Sapporo, Japan). Peripheral blood mononuclear
cells (PBMCs) were isolated using density gradient centrifugation on
Percoll (GE Healthcare, Little Chalfont, UK), washed three times inphosphate-buffered saline (PBS), and suspended in PBS. All experi­mental procedures were conducted with the approval of the Ethics
Committee of the Faculty of Veterinary Medicine, Hokkaido University(approval #20–0093).

Table 1
Primer sequences used in this study.
Primer name

Sequence

Gene cloning
PD-1 F
ATGGGGACCCCGCGGGCGCC
PD-1 RTCAGAGGGGCCAGGAGCAGTGTCCA
PD-L1 FATGAGGATATATAGTGTCTTAACAT
PD-L1 RTTACGTCTCCTCAAAATGTG
Plasmid construction for EGFP-tagged proteinsPD-1 F BglIIGAAGATCTGTGGGGACCCCGCGGGCGCCG
PD-1 R SmaIGACCCGGGGAGGGGCCAGGAGCAGTGTCC
PD-L1 F XhoI
CCGCTCGAGATGAGGATATATAGTGTCT
PD-L1 R SmaI
ATCCCGGGCGTCTCCTCAAAATGTGTAG
Plasmid construction for Ig fusion proteinsPD-1 F NheIATAGCTAGCATGGGGACCCCGCGGGC
PD-1 R EcoRV
AGCTCGATATCGCCGATGACCAGGCTCTG
PD-L1 F NheIGACGCTAGCATGAGGATATATAGTGTCT
PD-L1 R EcoRVGCTCTGATATCCCTCGTTTTTGCTGGAT

2.2. Cloning of cDNA encoding of ovine PD-1 and PD-L1 (OvPD-1 andOvPD-L1)
Ovine PBMCs (4 × 106 cells) were cultured for 24 h with 20 ng/mL ofphorbol 12-myristate acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA)and 1 μg/mL of ionomycin (Sigma-Aldrich) in RPMI 1640 medium(Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM of L-

Underlined; restriction enzyme recognition site2

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gene cassette encoding the Fc region of rabbit IgG. Transient cell lines
expressing OvPD-1-Ig and OvPD-L1-Ig were established using an
Expi293 Expression System (Thermo Fisher Scientific). Briefly,
Expi293F cells were transfected with pCXN2.1(+)-OvPD-1-Ig and
pCXN2.1(+)-OvPD-L1-Ig using Expifectamine (Thermo Fisher Scienti­fic) and cultivated for 7 days in Expi293 medium (Thermo Fisher Sci­
entific) at 37 ◦ C and 125 rpm with 8% CO2.
Purification of OvPD-1-Ig and OvPD-L1-Ig from the culture super­
natants was achieved using affinity chromatography with an AbCapcher ExTra (ProteNova, Kagawa, Japan). The buffer of the purified
fraction was exchanged for PBS by size exclusion chromatography with a
PD-10 Desalting Column (GE Healthcare). The purity of OvPD-1-Ig and
OvPD-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, Hercules, CA, USA). Precision Plus Protein All Blue Standard(Bio-Rad) was used as a molecular-weight size marker. Quick-CBB(FUJIFILM Wako Pure Chemical) was used to visualized the proteins,and protein concentrations were determined using ultraviolet absor­
bance at 280 nm with a NanoDrop 8000 Spectrophotometer (Thermo
Fisher Scientific).

Verse (BD Biosciences).

2.5. Binding assay of OvPD-1 and OvPD-L1

2.8. Immunohistochemical assay of PD-L1

Binding of OvPD-1-Ig and OvPD-L1-Ig to COS-7 cells expressing
OvPD-L1-EGFP and OvPD-1-EGFP was investigated using flow cytom­
etry. OvPD-L1-EGFP cells or OvPD-1-EGFP cells were incubated at 37 ◦ C
for 30 min with 10 μg/mL of biotinylated OvPD-1-Ig or OvPD-L1-Ig,
respectively. Biotinylated rabbit control IgG (Southern Biotech, Bir­mingham, AL, USA) was used as a negative control. Using a LightningLink Rapid Type A Biotin Conjugation Kit (Innova Biosciences, Cam­bridge, UK), OvPD-1-Ig, OvPD-L1-Ig, and rabbit control IgG were bio­tinylated. Cells were washed with PBS containing 1% bovine serum
albumin (BSA; Sigma-Aldrich) and labeled using APC-conjugatedstreptavidin (BioLegend, San Diego, CA, USA) at 25 ◦ C for 30 min.
After rewashing, the cells were analyzed immediately by FACS Verse(BD Biosciences, San Jose, CA, USA).

Brain tissue was obtained from a sheep that naturally infected with
Listeria monocytogenes in the Experiment Farm, Field Science Center for
Northern Biosphere, Hokkaido University (Sapporo, Japan). This animal
exhibited clinical neurological abnormalities and was diagnosed bypathological analyses as encephalitis due to intracerebral infection with
Listeria monocytogenes. The tissues were fixed in formalin, embedded inparaffin wax, and cut into 4-μm-thick sections. The dried sections weredeparaffinized in xylene before being hydrated with graded alcohols.
Microwave heating for 10 min resulted in antigen retrieval using citratebuffer (0.37 g/mL citric acid and 2.4 g/mL trisodium citrate dihydrate).
Endogenous peroxidase activity was blocked by incubating the sectionsfor 15 min in methanol containing 0.3% hydrogen peroxide. The sec­
tions were incubated at room temperature for 30 min with anti-PD-L1
mAb (6C11, rat IgG2a) (Sajiki et al., 2018), followed by incubation
with Histofine simple stain MAXPO (rat) (Nichirei, Tokyo, Japan) atroom temperature for 30 min. The reaction was visualized with 3-diami­
nobenzidine tetrahydrochloride. Moreover, immunostained sections
were examined by an optical microscope.

2.7. Blockade assay of OvPD-1/OvPD-L1 interaction
Blocking assays were performed in microplates using OvPD-1-Ig and
OvPD-L1-Ig to analyze the ability of anti-PD-L1 mAbs to block PD-1/PDL1 binding. MaxiSorp Immuno Plates (Thermo Fisher Scientific) were
coated with OvPD-1-Ig (1 μg/mL) in carbonate-bicarbonate buffer(Sigma-Aldrich) and blocked using SuperBlock T20 (PBS) Blocking
Buffer (Thermo Fisher Scientific). Biotinylated OvPD-L1-Ig was pre­incubated with anti-PD-L1 mAb 6G7 (rat IgM) (Ikebuchi et al., 2014),
6C11 (rat IgG2a) (Sajiki et al., 2018), 4G12 (rat IgG2a) (Ikebuchi et al.,2014), rat IgM isotype control (Acris Antibodies, Herford, Germany), or
rat IgG2a isotype control (R35–95, BD Biosciences) at various concen­
trations (0, 1.25, 2.5, 5.0, 7.5, and 10 μg/mL) at 37 ◦ C for 30 min. The
preincubated reagents were added to the microplates and incubated at
37 ◦ C for another 30 min. OvPD-L1-Ig binding was detected using
horseradish peroxidase-conjugated Neutravidin (Thermo Fisher Scien­tific) and TMB One Component Substrate (Bethyl Laboratories, Mont­gomery, TX, USA). Further, optical density at 450 nm was measured by amicroplate reader MTP-900 (Corona Electric, Hitachinaka, Japan).
Three independent experiments were carried out in duplicate.

2.6. Cross-reactivity assay of anti-bovine PD-L1 mAbs against OvPD-L1
OvPD-L1-EGFP cells were incubated with four clones of anti-bovine
PD-L1 mAbs (4G12, 5A2, 6C11, 6G7) (Ikebuchi et al., 2014; Sajiki et al.,2018) at room temperature for 20 min to assess the binding ability ofanti-bovine PD-L1 mAbs to OvPD-L1. Rat IgG1 (R3–34, BD Biosciences),
rat IgG2a (R35–95, BD Biosciences), and rat IgM isotype controls (R4–22,
BD Biosciences) were used for negative control staining. Cells were thenwashed with 1% BSA-PBS and labeled with APC-conjugated goat anti-rat
immunoglobulin antibody (Southern Biotech) at room temperature for
20 min. After rewashing, the cells were analyzed immediately by FACS
Verse (BD Biosciences).
Flow cytometry was performed to analyze stimulated ovine PBMCs.
To assess the binding ability of anti-bovine PD-L1 mAbs to ovine im­mune cells, ovine PBMCs (4 × 106 cells) were cultured for 24 h with 20
ng/mL of PMA (Sigma-Aldrich) and 1 μg/mL of ionomycin (SigmaAldrich), as described above. To prevent nonspecific reactions, stimu­
lated PBMCs were incubated at room temperature for 15 min in PBS
supplemented with 10% goat serum (Thermo Fisher Scientific). Cells
were washed and stained with anti-PD-L1 mAbs (5A2, rat IgG1; 4G12,
rat IgG2a; 6C11, rat IgG2a; 6G7, rat IgM) (Ikebuchi et al., 2014; Sajiki
et al., 2018) at room temperature for 30 min. Moreover, rat IgG1 (R3–34,
BD Biosciences) and rat IgG2a isotype control (R35–95, BD Biosciences)were used for negative control stainings. Cells were washed with PBS
containing 1% BSA (Sigma-Aldrich) and labeled with APC-conjugatedanti-rat Ig antibody (Southern Biotech) at room temperature for 30
min. After rewashing, the cells were analyzed immediately by FACS

2.9. Statistical analyses
The Wilcoxon signed-rank test or Turkey’s test was used to identify
significant differences. All statistical tests were performed using the
ASTATSA (https://astatsa.com/) statistical analysis program. The sta­tistical significance level was set as p < 0.05.
3. Results
3.1. Molecular cloning and sequence analysis of ovine PD-1/PD-L1
To begin, we identified cDNA sequences encoding OvPD-1 andOvPD-L1. Figs. 1A and 2A show the putative amino acid sequences of
OvPD-1 and OvPD-L1, respectively. OvPD-1 and OvPD-L1 are type I
transmembrane proteins with a putative signal peptide, an extracellularregion, a transmembrane region, and an intracellular region. A
conserved domain search revealed an immunoglobulin variable (IgV)like domain in the extracellular regions of OvPD-1. Moreover, extra­
cellular regions of OvPD-L1 were found to have Ig domains. The OvPD-1
had two structural motifs in its intracellular region: an immunoreceptor
tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosinebased switch motif (ITSM). Phylogenetic analyses revealed that OvPD-1
3

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Fig. 1. Analysis of ovine PD-1 amino acid sequence. (A) Multiple alignment of the PD-1 predicted amino acid sequences of sheep, cattle, buffalo, pig, dog, cat, horse,
human, monkey, mouse, and rat; GenBank accession numbers are shown in parentheses to the right of the species name. Lines represent the predicted signal peptide,IgV domain, intracellular region, transmembrane region, and intracellular region. ITIM and ITSM are encircled by rectangles. (B) Phylogenetic tree analysis of ovine
PD-1 sequences in relation to other vertebrate species. The bootstrap consensus tree was inferred from 1000 replicates with neighbor-joining method using the
MEGA11 software. The scale indicates the divergence time.

and OvPD-L1 were clustered into the non-primate group (Figs. 1B and
2B). OvPD-1 had 92.2%, 72.7%, 71.3%, 70.8%, 71.0%, 65.6%, and51.7% amino acid similarities to cattle, pig, horse, dog, cat, human, andmouse, respectively (Table 2). Furthermore, OvPD-L1 had 94.1%,
81.7%, 78.9%, 77.5%, 75.6%, 74.2%, and 66.2% amino acid similarities
to cattle, pig, horse, dog, cat, human and mouse, respectively (Table 2).

OvPD-1-Ig and OvPD-L1-Ig to analyze the interactions of the OvPD-1
and OvPD-L1 proteins. OvPD-1-Ig and OvPD-L1-Ig were successfully
purified from culture supernatants and dimerization was confirmed(Fig. 3A). OvPD-1-Ig and OvPD-L1-Ig have predicted molecular weights
of 42.4 kDa and 50.8 kDa under reduced conditions while 84.7 kDa and101.7 kDa under nonreduced condition. Posttranslational modifica­
tions, such as glycosylation, would cause these proteins to have larger
molecular weights than predicted.
Flow cytometry was performed to examine the interactions of OvPD1-Ig or OvPD-L1-Ig with OvPD-L1-EGFP- or OvPD-1-EGFP-expressing

3.2. Interaction of OvPD-1 and OvPD-L1
Expi293 Expression System was used to develop soluble recombinant4

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Fig. 2. Analysis of ovine PD-L1 amino acid sequence. (A) Multiple alignment of the PD-L1 predicted amino acid sequences of sheep, cattle, buffalo, pig, horse, dog,
cat, human, monkey, rat, and mouse; GenBank accession numbers are shown in parentheses to the right of the species name. Lines represent the predicted signal
peptide, Ig domain, extracellular region, transmembrane region and intracellular region. (B) Phylogenetic tree analysis of the ovine PD-L1 sequences in relation toother vertebrate species. The bootstrap consensus tree was inferred from 1000 replicates with neighbor-joining method using the MEGA11 software. The scale
indicates the divergence time.

cells (Fig. 3B and 3C). The flow cytometric assay revealed that OvPD-1Ig binding to OvPD-L1-EGFP-expressing cells is dependent on theexpression level of OvPD-1-EGFP (Fig. 3B). OvPD-L1-Ig also binds to
OvPD-1-EGFP-expressing cells in an expression-dependent manner(Fig. 3C).

bovine PD-L1 mAbs (Ikebuchi et al., 2014; Sajiki et al., 2018) against
OvPD-L1 and found that three of the four tested mAbs (4G12, 6C11, and
6G7) detected OvPD-L1-EGFP overexpressed on COS-7 cells (Fig. 4A). Of
all the tested mAbs, 6C11 had the strongest fluorescent intensity to
OvPD-L1-EGFP-expressing cells (Fig. 4A). We also tested the reactivityof 4G12, 6C11, and 6G7 mAbs against stimulated ovine PBMCs andfound that 6C11 showed the strongest fluorescent intensity to stimulated
PBMCs (Fig. 4B). Furthermore, the flow cytometric assay using the mAb
6C11 detected PD-L1 expressed in fresh ovine PBMCs which did not

3.3. Cross-reactivity of anti-bovine PD-L1 mAbs against OvPD-L1
We evaluated the cross-reactivity of our previously established anti5

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lesion of sheep infected with L. monocytogenes. In ovine listeriosis,
various immune cells accumulate in the brain lesions and macrophages
are involved in the bacterial transfer from the periphery to the central
nervous system and in the pathogenesis of encephalitis (Oevermann
et al., 2009). PD-L1 was clearly detected in macrophages infiltrating theencephalitis lesion of this animal (Fig. 6), confirming the utility of 6C11
mAb for immunohistochemistry of PD-L1 protein in ovine tissue
specimens.

Table 2
Similarities of amino acid sequences of PD-1 and PD-L1 among mammalian
species.
Speciesvs. Sheep (PD-1) (%)

vs. Sheep (PD-L1) (%)

Cattle
Pig
Horse
Dog
Cat
Human
Mouse

92.2
72.7
71.3
70.8
71.0
65.6
51.7

94.1
81.7
78.9
77.5
75.6
74.2
66.2

4. Discussion
As therapeutic intervention for cancer and chronic disease, immu­
notherapy targeting immune checkpoint molecules has been widelyused over the last decade. Many immune checkpoint molecules such as
for example PD-1, CTLA-4, LAG-3, and TIM-3 have been identified andstudied for cancer immunotherapy in human (He and Xu, 2020). In
veterinary field, these molecules have been also studied in various in­
fectious diseases in livestock (Ganbaatar et al., 2020, 2021; Goto et al.,2017; Ikebuchi et al., 2011, 2013; Nakamura et al., 2023; Okagawa
et al., 2012, 2016a,b; Watari et al., 2019). Moreover, many studies
revealed that the PD-1/PD-L1 pathway plays essential roles on T-cell
exhaustion during infections (Buchbinder and Desai, 2016; Jubel et al.,2020). Therefore, this study first focused on PD-1/PD-L1 to gain adeeper understanding of immune checkpoint molecules of sheep.
Furthermore, immunotherapy with anti-PD-1 and anti-PD-L1 mAbs
is effective in the treatment of different types of cancers in human anddogs (Robert et al., 2015; Weber et al., 2015; Brahmer et al., 2015;
Garon et al., 2015; Maekawa et al., 2017, 2021). However, there havebeen numerous reports about limitations in clinical effects and obser­
vation of adverse effects by anti-PD-1 and PD-L1 mAbs due to a lack ofreceive any stimulation (Fig. 4C).
3.4. Inhibition of OvPD-1/OvPD-L1 binding by anti-PD-L1 mAbs
We investigated the blocking activity of anti-bovine PD-L1 mAbs on
the interaction of OvPD-1/OvPD-L1. Although 6C11 had strongest crossreactivity against OvPD-L1, it does not significantly block OvPD-1 andOvPD-L1 binding. In contrast, 4G12 and 6G7 blocked OvPD-1 andOvPD-L1 binding in a dose-dependent manner (Fig. 5).
3.5. Immunohistochemical analysis of PD-L1 in Listeria monocytogenes
PD-L1 expression is upregulated in many types of infectious diseases
in cattle and pigs (Ikebuchi et al., 2011; Sajiki et al., 2019; Okagawa
et al., 2016a, 2016b; Goto et al., 2017, 2020; Ganbaatar et al., 2021). We
hypothesized that PD-L1 expression is also upregulated in infectious
disease of sheep. Therefore, we performed immunohistochemistry using
the mAb 6C11 to analyze the expression of PD-L1 in an encephalitis

Fig. 3. Purification and interaction of OvPD-1 and OvPD-L1. (A) Expression and purification of ovine PD-1 and PD-L1 proteins. OvPD-1-Ig and OvPD-L1-Ig proteins
were expressed by an Expi293 Expression System and purified from culture supernatant using affinity chromatography with an Ab-Capcher ExTra. SDS-PAGE and
CBB staining were performed to evaluate the protein purity. (B) Binding of OvPD-1-Ig to OvPD-L1-EGFP-expressing cells was shown in a dotplot (left) and a his­
togram (right). In the histogram, the red area shows the binding of OvPD-1-Ig, whereas the gray area shows the binding of rabbit IgG (negative control). (C) Bindingof OvPD-L1-Ig to OvPD-1-EGFP-expressing cells was shown in a dotplot (left) and a histogram (right). In the histogram, the red area shows the binding of OvPD-L1-Ig,
whereas the gray area shows the binding of rabbit IgG (negative control).
6

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Fig. 4. Cross-reactivities of anti-bovine PD-L1 mAbs against OvPD-L1. (A–C) Cross-reactivities of four anti-bovine PD-L1 mAbs (5A2; rat IgG1, 4G12 and 6C11; rat
IgG2a, 6G7; rat IgM) against OvPD-L1 were determined by flow cytometry using OvPD-L1-EGFP-expressing cells and ovine PBMCs. Isotype-matched control anti­
bodies were used as negative control. In the histograms, the red area shows the binding of anti-bovine PD-L1 mAb, while the gray area shows the binding of isotype
control. (A) Cross-reactivity of anti-bovine PD-L1 mAbs against OvPD-L1-expressing cells. (B) Cross-reactivity of the mAbs against PBMCs cultured for 24 h under
PMA/ionomycin stimulation. (C) PD-L1 expression in fresh ovine PBMCs detected by flow cytometric analysis using anti-bovine PD-L1 mAb (6C11).

thorough understanding of these molecules (Higano et al., 2009; Kantoff
et al., 2010; Topalian et al., 2012; Weber et al., 2017). We intend tofurther clarify the role of the PD-1/PD-L1 pathway by using sheep as asuitable animal experimental model, but basic information about ovine
PD-1/PD-L1 pathway is still limited. Thus, we first identify and char­acterize the ovine PD-1 and PD-L1 sequences for further immunologicalstudies in sheep.
In this study, we found that cDNA of ovine PD-1 and PD-L1 has highsimilarity of amino acid sequence to other species. Moreover, the ITIM
and ITSM were conserved as the structural motifs of PD-1 that play roles
in negative regulation in T-cell function (Chatterjee et al., 2013;
Chemnitz et al., 2004; Okazaki et al., 2001). Thus, we hypothesized that
ovine PD-1 and PD-L1 would function similarly to other species. Flowcytometric analyses showed that ovine PD-1 and PD-L1 can bind to eachother in an expression-dependent manner, just like other species(Ganbaatar et al., 2020, 2021, Maekawa et al., 2014, 2016; Lin et al.,2008).
The results of cross-reactivities of anti-bovine PD-L1 mAbs against
OvPD-L1 revealed that one of mAbs (6C11) strongly bound to OvPD-L1
in expressing cells and stimulated PBMCs. In comparison to 6C11 mAbs,
other anti-bovine PD-L1 mAbs (4G12 and 6G7) showed moderate crossreactivities. Furthermore, anti-bovine PD-L1 mAbs can interfere thebinding of OvPD-1 and OvPD-L1 (Fig. 5), implying the ability of themAbs to block this interaction.
We observed obvious accumulation of PD-L1+ macrophages in theencephalitis lesion of sheep infected with L. monocytogenes. Macro­phages are observed in the encephalitis lesions of ovine listeriosis fromacute to chronic stages (Oevermann et al., 2009). In addition, infected
macrophages appear to be involved in the bacterial transfer from theperiphery to the central nervous system. Therefore, upregulation of7

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Veterinary Immunology and Immunopathology 261 (2023) 110609

Fig. 5. Inhibition of ovine PD-1/PD-L1 binding by anti-PDL1 mAbs. The blocking effect of anti-bovine PD-L1 mAbs on
the binding of OvPD-L1-Ig to OvPD-1-Ig. Biotinylated
OvPD-L1-Ig was preincubated with various concentrationsof anti-PD-L1 mAb (6G7, 6C11, and 4G12) and then incu­
bated in microwell plates coated with OvPD-1-Ig. Rat IgG2aand IgM control antibodies were used as isotype-matchednegative controls. Each value represents the relative bind­ing of ovine PD-L1-Ig preincubated with blocking anti­
bodies compared to that preincubated without the blocking
antibody. Each value is the average determined from three
independent experiments. Significant differences between
each treatment were identified using Turkey’s test. Asterisk(*) indicates p < 0.01.

Fig. 6. Immunohistochemistry analysis of PD-L1 in Listeria monocytogenes infection in sheep. Immunohistochemical staining of PD-L1 in brain lesion of sheep withlisteriosis using an anti-bovine PD-L1 mAbs (6C11) were performed. PD-L1 positive macrophages are shown with white arrows.

PD-L1 may be involved in immune evasion of infected macrophages
during ovine listeriosis.
However, this study did not examine T-cell response are suppressedby the PD-1/PD-L1 signaling in sheep. Further investigations are
required in the expression kinetics of PD-1 and PD-L1 and their associ­ation with T-cell exhaustion in infectious diseases of sheep. According toprevious studies, blocking PD-1/PD-L1 binding with mAbs restores Tcell function and improves disease prognosis in humans, dogs, and cattle(John et al., 2013; Maekawa et al., 2017, 2021; Nishimori et al., 2017;
Okagawa et al., 2017; Sajiki et al., 2020). Thus, additional experiments
are needed to analyze the restoration of T-cell functions by PD-1/PD-L1
blockade in sheep, including the measurement of T-cell cytokines.
Lack of available reagents has limited immunological analysis of
ruminants to a very limited extent. It would be very beneficial to develop
detection antibodies that could be used for the analysis of such animals.
This study provides an opportunity for immunological analysis focusedon the PD-1/PD-L1 pathway in sheep. The achievements of this study
will lead to progress in immunological analysis of diseases in sheep andincrease their usefulness as model animals.

Grants JP223fa627005 [to S.K.], JP21am0101078 [to Y.K.] andJP22ama121008 [to Y.K.]. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of themanuscript.
Declaration of Competing Interest
All authors declare that they have no competing interests.
Acknowledgments
We are grateful to Dr. Hideyuki Takahashi, Dr. Yasuyuki Mori, andDr. Tomio Ibayashi for their valuable advice and discussions. We would
like to thank Enago (http://www.enago.jp) for the English language
review.
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Funding
This work was supported by JSPS KAKENHI grant number
19KK0172, 22H02503, 22K19232 [to S.K.], 19K15993, and 22K15005[to T.O.], grants from the Project of the NARO, Bio-oriented Technology
Research Advancement Institution (Research Program on Developmentof Innovative Technology 26058 BC [to S.K.] and Special Scheme Project
on Regional Developing Strategy, Grant 16817557 [to S.K.]), grants
from Regulatory Research Projects for Food Safety, Animal Health andPlant Protection (JPJ008617.17935709 [to S.K.]), and supported by theJapan Agency for Medical Research and Development (AMED) under8

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