Received: 1 July 2021

| Revised: 28 July 2021 | Accepted: 3 August 2021

DOI: 10.1002/iid3.510

ORIGINAL ARTICLE

Programmed death‐ligand 1 expression in swine chronic
infections and enhancement of interleukin‐2 production
via programmed death‐1/programmed death‐ligand 1
blockade
Otgontuya Ganbaatar1 | Satoru Konnai1,2| Tomohiro Okagawa2|

Yutaro Nojima1 | Naoya Maekawa2 | Yoshiki Ichikawa3 |Atsushi Kobayashi3 | Tomoyuki Shibahara4,5 | Yojiro Yanagawa3 |Hidetoshi Higuchi6 | Yukinari Kato7,8
Shiro Murata1,2 | Kazuhiko Ohashi1,2| Yasuhiko Suzuki2,9|1

Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan2

Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan3

Department of Veterinary Clinical Medicine, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan

4

Division of Hygiene Management Research, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO),
Tsukuba, Japan

5

Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan

6

Division of Health and Science, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Japan

7

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan

8

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan

9

Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan

Correspondence
Satoru Konnai, Department of Disease
Control, Faculty of Veterinary Medicine,
Hokkaido University, Sapporo 060‐0818,
Japan.
Email: konnai@vetmed.hokudai.ac.jp
Funding information
AMED, Grant/Award Number:
JP20am0101078; Project of the NARO,
Bio‐oriented Technology Research
Advancement Institution (Research
Program on Development of Innovative
Technology, Grant/Award Number: 26058
BC; Clinical Research Promotion Fund

Abstract
Introduction: Chronic infections lead to the functional exhaustion of T cells.
Exhausted T cells are phenotypically differentiated by the surface expressionof the immunoinhibitory receptor, such as programmed death‐1 (PD‐1). The
inhibitory signal is produced by the interaction between PD‐1 and its PD‐
ligand 1 (PD‐L1) and impairs the effector functions of T cells. However, theexpression dynamics of PD‐L1 and the immunological functions of the PD‐1/
PD‐L1 pathway in chronic diseases of pigs are still poorly understood. In thisstudy, we first analyzed the expression of PD‐L1 in various chronic infections
in pigs, and then evaluated the immune activation by the blocking assay
targeting the swine PD‐1/PD‐L1 pathway.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2021 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.
Immun Inflamm Dis. 2021;9:1573–1583.

wileyonlinelibrary.com/journal/iid3

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2020 by Hokkaido University Veterinary
Teaching Hospital,; JSPS KAKENHI,
Grant/Award Number: 19KK0172

Methods: In the initial experiments, anti‐bovine PD‐L1 monoclonal antibodies (mAbs) were tested for cross‐reactivity with swine PD‐L1. Subsequently, immunohistochemical analysis was conducted using the anti‐PD‐L1
mAb. Finally, we assessed the immune activation of swine peripheral bloodmononuclear cells (PBMCs) by the blockade with anti‐PD‐L1 mAb.
Results: Several anti‐PD‐L1 mAbs tested recognized swine PD‐L1‐expressing
cells. The binding of swine PD‐L1 protein to swine PD‐1 was inhibited bysome of these cross‐reactive mAbs. In addition, immunohistochemical analysis revealed that PD‐L1 was expressed at the site of infection in chronic
infections of pigs. The PD‐L1 blockade increased the production ofinterleukin‐2 from swine PBMCs.
Conclusions: These findings suggest that the PD‐1/PD‐L1 pathway could bealso involved in immunosuppression in chronic infections in pigs. This study
provides a new perspective on therapeutic strategies for chronic diseases inpigs by targeting immunosuppressive pathways.
KEYWORDS

interleukin‐2, PD‐1, PD‐L1, swine diseases

1 |INTRODUCTION

Programmed death‐ligand‐1 (PD‐L1) is a transmembraneprotein that is associated with the suppression of the adaptive immune system. PD‐L1 is expressed in both immune
and nonimmune cells, such as antigen‐presenting cells(APCs), lymphocytes, epithelial cells, endothelial cells, andtumor cells.1–3 Its receptor, PD‐1, is mainly expressed on T
lymphocytes in peripheral blood and lymphoid tissues.1,4,5
The interaction between PD‐L1 and PD‐1 inhibits T‐cell receptor signaling and suppresses T‐cell effector functions suchas cytokine production and cytotoxicity.1,3–5 During chronic
infection, the expression of PD‐1 and PD‐L1 is upregulated
and causes the dysfunction of antigen‐specific T cells, which
is known as “T‐cell exhaustion.”4,5 It has been well documented that T‐cell exhaustion mediated by PD‐1/PD‐L1
contributes to immune evasion in various types of chronic
infections by human immunodeficiency virus5 and humanhepatitis virus type C.6 Our previous studies have shown that
the PD‐1/PD‐L1 pathway is involved in immune exhaustionand disease progression in chronic infectious diseases ofcattle, such as bovine leukemia virus infection,7–9 Johne's
disease,10,11 anaplasmosis,12 mycoplasmosis,13 canine cancers,14,15 and equine melanoma.16 Furthermore, we established therapeutic antibodies targeting PD‐1 and PD‐L1 andreported their therapeutic efficacy against bovine leukemia
virus infection,17–19 Johne's disease,20 mycoplasmosis,21 andcanine malignant melanoma.22,23
Swine PD‐1 and PD‐L1 molecules have been identified,
and their amino acid sequences were found to share highidentity with human and murine orthologues.24,25 The interaction of swine PD‐1 and PD‐L1 decreases the productionof interferon‐γ (IFN‐γ) and interleukin‐2 (IL‐2) under T‐cellstimulation in swine peripheral blood mononuclear cells(PBMCs),24 indicating the negative regulation of T‐cell response mediated by the PD‐1/PD‐L1 pathway in pigs. During
acute infection of classical swine fever virus (CSFV), theexpression of PD‐1 and PD‐L1 messenger RNA (mRNA) was
upregulated in PBMCs, and it was probably correlated withimmune inhibition.26 In addition, an in vitro model of infection revealed that PD‐L1 expression was increased indendritic cells infected with porcine circovirus 2 (PCV‐2) andporcine reproductive and respiratory syndrome virus(PRRSV).27,28 In this model, gene knockdown of swine PD‐1
decreased apoptosis and increased cell proliferation of swine
lymphocytes.28 Thus, the swine PD‐1/PD‐L1 pathway is also
associated with immune regulation during acute infectionsof pigs.
However, the involvement of the PD‐1/PD‐L1 pathway in the immunosuppression of chronic infections inpigs has not yet been elucidated, because monoclonal
antibody (mAb) against swine PD‐1 and PD‐L1, which
could be powerful tools for elucidating the pathogenesisof swine diseases, have not yet been reported. In thisstudy, we verified the cross‐reactivity of our establishedanti‐bovine PD‐L1 mAbs with swine PD‐L1. Then, we
performed the immunohistochemical analysis in various
swine chronic infections using the anti‐PD‐L1 mAb.
Finally, we evaluated the immune activation of swine
PBMCs by blockade with the anti‐PD‐L1 mAb.

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2 |MATERIALS A ND METHODS

2.1| Isolation of swine PBMCs

Heparin‐treated blood samples were collected from piglets (crossbreed, large white × Landrace × large white ×
Duroc, 1‐ to 6‐months‐old, male or female) raised onconventional farms in Hokkaido, Japan. PBMCs wereseparated from blood samples by density gradient centrifugation using Percoll (GE Healthcare). All experimental procedures were carried out with the approval of
the Animal Experiment Committee of Hokkaido
University (20‐0093). Informed consent was obtained
from all owners.

2.2 | Preparation of swine PD‐1‐ andPD‐L1‐expressing cells
The complementary DNA (cDNA) was synthesized frommRNA of swine PBMCs stimulated with 20 ng/ml phorbol 12‐myristate acetate (PMA; Sigma‐Aldrich) and1 μg/ml ionomycin (Sigma‐Aldrich) for 24 h. The cultivation of PBMCs, total RNA isolation, and cDNA
synthesis was conducted as described previously.16 The
cDNAs encoding swine PD‐1 and PD‐L1 were amplified
by PCR using TaKaRa Ex Taq (Takara Bio) and gene‐
specific primers with restriction enzyme cleavage sites(Table S1), and then subcloned into pEGFP‐N2 (Clontech). The purified plasmids were transfected to COS‐7
cells using Lipofectamine 3000 Reagent (Thermo Fisher
Scientific). After 48 h of cell cultivation, the localizationof PD‐1‐EGFP and PD‐L1‐EGFP in the cells was confirmed using ZOE Fluorescent Cell Imager (Bio‐Rad).

2.3 | Preparation of recombinant swine
PD‐1 and PD‐L1
The cDNAs encoding the signal sequences and extracellulardomain fragments of swine PD‐1 and PD‐L1 were amplified
by PCR using gene‐specific primers with restriction enzyme
cleavage sites (Table S1). The amplified products were subcloned with a gene cassette encoding the Fc region of rabbit
IgG at the multi‐cloning site of pCXN2.1(+) (kindly provided
by Dr. T. Yokomizo, Juntendo University, Japan). Recombinant swine PD‐1 and PD‐L1 proteins (rPD‐1 and rPD‐
L1) were produced using the Expi293 Expression System(Thermo Fisher Scientific) and purified from the culture
supernatants by affinity chromatography using Ab‐Capcher
ExTra (ProteNova). The purity of the proteins was evaluatedby sodium dodecyl sulfate‐polyacrylamide gel electrophoresis(SDS‐PAGE) in reducing or nonreducing conditions using

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SuperSep Ace 5%–20% gradient polyacrylamide gel(FUJIFILM Wako Pure Chemical). The protein concentration was measured by ultraviolet absorbance at 280 nm witha NanoDrop 8000 Spectrophotometer (Thermo Fisher
Scientific). The binding of rPD‐1 and rPD‐L1 to COS‐7 cells
expressing swine PD‐L1‐EGFP and PD‐1‐EGFP wasinvestigated using flow cytometry as described previously.16

2.4 | Cross‐reactivity assay against
swine PD‐L1
The cross‐reactivity of anti‐bovine PD‐L1 mAbs to swine
PD‐L1 was examined by flow cytometry as described previously with modifications.16 Briefly, swine PD‐L1‐EGFP‐
expressing cells were incubated with rat anti‐bovine PD‐L1
mAbs (4G12‐C1, 5A2‐A1, 6C11‐3A11, and 6G7‐E1) for 20
min at 25°C. Rat immunoglobulin G 1 (IgG1), IgG2a, and
IgM isotype controls (BD Biosciences) were used as isotype‐matched negative controls. The details of the primary antibodies used in this assay are shown in Table S2. The cells
were washed with PBS containing 1% bovine serum albumin(Sigma‐Aldrich) and then stained with APC‐conjugated goatanti‐rat immunoglobulin antibody (Southern Biotech) for 20
min at 25°C. After washing, the stained cells were examinedby FACS Verse (BD Biosciences). In addition, the binding
ability to swine PBMCs was also evaluated. Briefly, PBMCs
were cultivated with 10 μg/ml swine IFN‐γ (Kingfisher
Biotech) or 20 ng/ml PMA (Sigma‐Aldrich) and 1 μg/ml
ionomycin (Sigma‐Aldrich). To prevent nonspecific reactions, the stimulated PBMCs were incubated with PBS supplemented with 10% goat serum (Thermo Fisher Scientific)
for 15 min at 25°C, and cells were stained as described above.

2.5 | Blockade assay of swine
PD‐1/PD‐L1 binding
To confirm the ability of anti‐PD‐L1 mAbs to interrupt
swine PD‐1/PD‐L1 binding, blocking assays were conducted using swine rPD‐1 and rPD‐L1 as described previously with modifications.16 Briefly, anti‐PD‐L1 mAbs
(4G12‐C1, 5A2‐A1, 6C11‐3A11, and 6G7‐E1) or rat IgG1,
IgG2a, and IgM isotype controls (BD Biosciences)(Table S2) were preincubated with biotinylated rPD‐L1 atvarious concentrations for 30 min at 37°C. The preincubated reagents were added to 96‐well microplates
coated with rPD‐1 (1 μg/ml) and incubated at 37°C for
30 min. The binding of rPD‐L1 was determined using
Neutravidin conjugated with horseradish peroxidase(Thermo Fisher Scientific) and TMB One Component
Substrate (Bethyl Laboratories). The optical density at
450 nm (OD450) was measured with an MTP‐900

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microplate reader (Corona Electric). Three independent
experiments were performed in duplicate. The relative
binding of rPD‐L1 to rPD‐1 was calculated from OD450
of sample preincubated with each antibody, using thatpreincubated without antibody as 100%.

2.6| Immunohistochemical assays

Tissue specimens of pigs were collected for pathological diagnosis at the National Institute of Animal Health, National
Agriculture, and Food Research Organization, Japan. Thesepigs were confirmed to be infected with PRRSV, Mycoplasma
hyopneumoniae, PCV‐2, and Lawsonia intracellularis, respectively, by the immunohistochemistry as shown below
and showed typical clinical signs and pathological lesions.
Tissue samples from a healthy pig were confirmed as being
not infected with these pathogens and were used as a negative control. Detailed information on tissue specimens isshown in Table S3.
Immunohistochemistry was performed to detect the
antigens of PRRSV, M. hyopneumoniae, PCV‐2, andL. intracellularis. Formalin‐fixed, paraffin‐embedded
tissues were cut into 3‐μm‐thick sections and incubatedwith 0.3% hydrogen peroxide in methanol to inhibit theactivity of endogenous peroxidase. Antigen retrieval was
performed as shown in Table S4. The section was incubated with each primary antibody as shown in
Table S4 and was followed by a secondary antibody(Histofine Simple Stain MAX‐PO Mouse or Multi)(Nichirei Bioscience). The sections were then incubatedwith Histofine Simple Stain AEC solution (Nichirei
Bioscience). All immunostained sections were observed
under an optical microscope.
For the immunohistochemical assay of PD‐L1, the tissues were fixed in formalin, embedded in paraffin wax, andcut into 4‐μm‐thick sections. The sections were autoclaved in
10 mM EDTA for antigen retrieval and then incubated with
0.3% hydrogen peroxide in methanol to inhibit the activity ofendogenous peroxidase. The sections were incubated overnight at 4°C with or without anti‐PD‐L1 mAb (6C11‐3A11)(Table S2) and then detected using the Vectastain Elite ABC
Rat IgG kit (Vector Laboratories). The reaction was visualized using 3,3ʹ‐diaminobenzidine tetrahydrochloride. All
immunostained sections were observed under an optical
microscope.

2.7 | Immunoactivation assay
PBMCs were isolated from the peripheral blood of six healthy piglets (crossbreed, large white × Landrace × large
white × Duroc, 7‐week‐old, male or female). To examine theeffects of the inhibition of the PD‐1/PD‐L1 pathway on swine
immune cells, PBMCs (2 × 106 cells/ml) were cultured with
0.1 μg/ml of Staphylococcal Enterotoxin B (Sigma‐Aldrich) in
the presence of 10 μg/ml of anti‐PD‐L1 mAb (4G12‐C1) or
rat IgG2a control (2A3; Bio X Cell) (Table S2) in RPMI 1640
medium (Sigma‐Aldrich) supplemented with 10% heat‐
inactivated fetal bovine serum (Thermo Fisher Scientific),
2 mM ‐L‐glutamine, 100 U/ml penicillin, and 100 μg/ml
streptomycin (Thermo Fisher Scientific) at 37°C, 5% CO2 for
3 days. After the cultivation, IL‐2 levels in the culture supernatant were assessed in duplicate by the Swine IL‐2
DuoSet enzyme‐linked immunosorbent assay (ELISA) development kit (R&D Systems) according to the manufacturer's recommendations.

2.8 | Statistical analysis
Differences were identified using the Wilcoxon signed‐rank
test. All statistical tests were performed using the statisticalanalysis program MEPHAS (http://www.gen‐info.osaka‐u.
ac.jp/MEPHAS/). p <0.05 were considered significant.

3 | R ES U L T S
3.1 | Confirmation of cellular
localization and binding of swine PD‐1 andPD‐L1
We first analyzed the cellular localization of swine PD‐1‐
EGFP and PD‐L1‐EGFP proteins in the overexpressed
COS‐7 cell lines by fluorescence microscopy. Swine PD‐1‐
EGFP and PD‐L1‐EGFP were localized on the cell surfaceof the overexpressed cell lines (Figure 1A). Soluble rPD‐1
and rPD‐L1 were produced in Expi293F cells and purified
from the culture supernatant with protein A resin(Figure 1B). They were dimerized by disulfide bonds(Figure 1B). rPD‐1 and rPD‐L1 bound to the cells expressing swine PD‐L1‐EGFP and PD‐1‐EGFP, respectively (Figure 1C).

3.2 | Binding ability of anti‐bovine PD‐
L1 mAbs to swine PD‐L1
Anti‐bovine PD‐L1 mAbs were tested for cross‐reactivity
with swine PD‐L1. All tested mAbs detected swine PD‐
L1‐EGFP in the overexpressed cell line (Figure 2A). In
addition, anti‐PD‐L1 mAbs, except for 5A2‐A1, detected
PD‐L1 in swine PBMCs stimulated with PMA/ionomycin(Figure 2B). The mAb 6C11‐3A11 showed was thestrongest binding intensity to the overexpressing COS‐7|GANBAATAR ET AL.

(A)

EGFP COS-7 cell

(B)

rPD-1
M

R

Swine PD-L1-EGFP
COS-7 cell

Swine PD-1-EGFP
COS-7 cellrPD-L1
M

NR

R

NR

(kDa)

(kDa)

250 -

250 -

M: marker

150 -

150 -

R: reducing

100 75 -100 75 -NR: non-reducing

50 -

50 -

37 -

37 -

25 20 15 -25 20 15 -Rabbit IgG

rPD-1

rPD-1 binding

(C)

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Swine PD-L1-EGFP
COS-7 cells

Swine PD-L1-EGFP

rPD-L1 binding

Rabbit IgG

rPD-L1

Swine PD-1-EGFP
COS-7 cells

Swine PD-1-EGFPF I G U R E 1 Confirmation of binding ability of swine PD‐1 and PD‐L1. (A) COS‐7 cells expressing EGFP (control, left panel), swine
PD‐1‐EGFP (middle panel), or PD‐L1‐EGFP (right panel). The localization of EGFP, PD‐1‐EGFP, and PD‐L1‐EGFP in the cells was
confirmed by fluorescence microscopy. (B) rPD‐1 (left panel) and rPD‐L1 (right panel) were produced using the Expi293 Expression System.
These proteins were purified from culture supernatants and analyzed using SDS‐PAGE. (C) Binding ability of rPD‐1 (upper panel) or rPD‐L1(lower panel) to cells expressing swine PD‐L1‐EGFP or PD‐1‐EGFP. The binding of Ig fusion proteins was analyzed by flow cytometry.
EGFP, enhanced green fluorescent protein; IgG, immunoglobulin G; PD‐1, programmed death‐1; PD‐L1, programmed death‐ligand 1;
rPD‐1, recombinant PD‐1; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

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Normalized %(A) Swine PD-L1-EGFP-expressing COS-7 cells
4G12-C1

5A2-A1

6C11-3A11

6G7-E1

Antibody binding(B) Stimulated swine PBMCs
5A2-A1

6C11-3A11

6G7-E1Count %

4G12-C1

Antibody binding(C) Upregulation of PD-L1 expression on swine PBMCs
IFN-γ

PMA/ionomycin

Count %

No stimulation

PD-L1 expression
F I G U R E 2 Binding ability of anti‐bovine PD‐L1 mAbs against swine PD‐L1. Binding activities of anti‐PD‐L1 mAbs to (A) swine PD‐L1‐
EGFP overexpressed in COS‐7 cells and (B) swine PBMC stimulated with PMA/ionomycin. (C) Expression analysis of swine PD‐L1 on
PBMCs was performed using an anti‐bovine PD‐L1 mAb (6C11‐3A11) by flow cytometry. APC‐conjugated anti‐rat Ig antibody was used tolabel the primary mAbs and analyzed. Rat IgG1, IgG2a, and IgM controls were used as isotype‐matched negative controls. Binding activities
of anti‐PD‐L1 mAbs and the negative controls were shown by black and gray histograms, respectively. EGFP, enhanced green fluorescentprotein; IFN‐γ, interferon‐γ; Ig, immunoglobulin; mAb, monoclonal antibody; PD‐1, programmed death‐1; PD‐L1, programmed death‐ligand 1; PMA, phorbol 12‐myristate acetate; PMBC, peripheral blood mononuclear cell

cells and stimulated PBMCs. Thus, PD‐L1 expression was
further analyzed in swine PBMCs under IFN‐γ stimulation using the mAb 6C11‐3A11. Stimulation with IFN‐γ
strikingly upregulated PD‐L1 expression on swine
PBMCs (Figure 2C).

3.3 | Inhibition of swine PD‐1/PD‐L1
binding by anti‐PD‐L1 mAbs
To determine whether anti‐bovine PD‐L1 mAbs are capableof inhibiting the swine PD‐1/PD‐L1 binding, we performed
an ELISA with rPD‐1 and rPD‐L1 in the presence of anti‐PD‐

L1 mAbs. All of the tested anti‐PD‐L1 mAbs blocked thebinding of rPD‐L1 to rPD‐1 (Figure 3). The blocking activity
was strong in the order of 6G7‐E1, 4G12‐C1, 5A2‐A1, and6C11‐3A11.

3.4 | PD‐L1 expression in the tissue ofseveral disease‐infected pigs
In pigs infected with PRRSV, M. hyopneumoniae, PCV‐2,and L. intracellularis, PD‐L1 expression was evaluated at
the site of infection and compared with uninfected tissues
from a healthy pig. The antigens of PRRSV, M. hyopneu-

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rPD-L1 binding to rPD-1 (%)

120

100

Rat IgG1 control
5A2-A180

Rat IgG2a control
4G12-C1
6C11-3A11

60

Rat IgM control
6G7-E1

40

20

0

0

2

4

6

8

10

Antibody concentration (μg/mL)

F I G U R E 3 Blockade of swine PD‐1/PD‐L1 binding by anti‐PD‐L1 mAbs. Preincubated anti‐PD‐L1 mAbs or rat isotype controls withbiotinylated rPD‐L1 were added to 96‐well microplates coated with rPD‐1 and incubated at 37°C for 30 min. Each curve represents therelative binding of rPD‐L1 preincubated with antibodies compared with that preincubated with no antibody. Each point represents the meanof three independent experiments. IgG, immunoglobulin G; mAb, monoclonal antibody; PD‐1, programmed death‐1; PD‐L1, programmed
death‐ligand 1; rPD‐1, recombinant PD‐1

moniae, PCV‐2, and L. intracellularis were detected in thetissues of the infected animals, respectively, but not in thetissues of uninfected control (Figure 4D,F,H,J). In animal
D28530, PRRSV was detected in the cytoplasm of macrophages in the lung interstitium (Figure 4D). In the lung ofanimal D29704, M. hyopneumoniae was detected on the
microvilli of bronchial epithelial cells (Figure 4F). In animal D33827, PCV‐2 was detected in intracytoplasmic inclusion bodies of macrophages in the lymph node
(Figure 4H). In animal D31935, L. intracellularis was detected in the cytoplasm of intestinal epithelial cells(Figure 4J). PD‐L1 was strongly expressed in the lung of
PRRSV‐ or M. hyopneumoniae‐infected pig, the mesentericlymph nodes of PCV‐2‐infected pig, and the ileum of a pig
infected with L. intracellularis (Figure 4E,G,I,K). In theuninfected controls, the mucosal epithelium and submucosal muscular plate of ileum and tracheal and bronchial epithelium and alveolar walls of lungs were stained
very lightly for PD‐L1 (Figure 4A,C).

3.5 | Immune activation in swine
PBMCs by PD‐L1 blockade
Finally, the effect of PD‐L1 blockade on immune activation was evaluated by the change in IL‐2 production inswine PBMCs. Blocking with anti‐PD‐L1 mAb (4G12‐C1)
significantly increased IL‐2 production from all swine
PBMCs, although individual differences were observed(Figure 5).

4 | D I S C US S I O N
The immune system plays a critical role in the response tochronic infection, but viruses and bacteria have evolved
some strategies to interfere with host immunity. Some of thepathogens evade host immune responses and develop apersistent infection, resulting in the persistent stimulation of
antigens and progressive T‐cell dysfunction.4,5 During
chronic infections, the PD‐1/PD‐L1 pathway has been shown
to mediate T‐cell dysfunction and limit pathogen clearance.
Because of the lack of available treatment, there are still
many chronic infectious diseases with poor prognosis indomestic animals, including pigs. Moreover, the lack of abetter understanding of the immunological pathways leading
to immune evasion can be attributed to this process. To date,
only a few studies have shown that the expression of PD‐1
and PD‐L1 is significantly increased in immune cells of pigs
during acute infection caused by CSFV or PCV‐226,29 or in
the cell culture experiments with PCV‐2 and/or PRRSV.27,28
In this study, we focused on chronic infections of pigs andfound that PD‐L1 expression was also upregulated in infected lesions of pigs affected with PRRS, mycoplasmosis,
PCV‐2 infection, and proliferative enteropathy. Previous
studies have reported the suppression of the T‐cell response
in pigs with these chronic infections.30–33 Further studies areneeded to elucidate detailed mechanisms of the suppressionof T‐cell response mediated by PD‐1/PD‐L1 in these diseases.
Furthermore, IFN‐γ stimulation strongly induced the upregulation of PD‐L1 expression in PBMCs. These results suggest that inflammatory cytokines can regulate the expression

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(A–C)
Uninfected (D34347)(D, E)PRRSV-infected (D28530, lung)(A) PD-L1 (lung)(D) PRRSV(C) PD-L1 (ileum)

100
μm

100
μm

(B) PD-L1 (lymph node)(E) PD-L1

100
μm

100
μm

(F, G)
M. hyopeneumoniae-infected(D29704, lung)(F) M. hyopeneumoniae(G) PD-L1

20
μm

100
μm

20
μm

(H, I)
PCV2-infected(D33827, lymph node)(J, K)
L. intracellularis-infected(D31935, ileum)(H) PCV-2(J) L. intracellularis(I) PD-L1

20
μm

100
μm

(K) PD-L1

50
μm

100
μm

F I G U R E 4 Immunohistochemical analysis of PD‐L1 in swine chronic infections. (A–C, E, G, I, K) Immunohistochemical staining of
PD‐L1 in tissues of pigs with chronic infections (PRRS, mycoplasmosis, PCV2‐infection, Lawsonia intracellularis infection).
Each section was stained using anti‐bovine PD‐L1 mAb (6C11‐3A11). Matched tissue sections stained without a primary antibody as
negative controls are shown in Figure S1. (D, F, H, J) Antigens of PRRSV, Mycoplasma hyopneumoniae, PCV2, and L. intracellularis were
stained with specific antibodies in the tissue sections. mAb, monoclonal antibody; PCV2, porcine circovirus 2;
PD‐L1, programmed death‐ligand 1; PRRS, porcine reproductive and respiratory syndrome; PRRS, PRRS, virus

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GANBAATAR ET AL.

p < 0.05
16

IL-2 production (ng/mL)

14
12
10
8
6
4
2

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chimeric antibodies with the swine IgG subclass. PD‐L1
blockade using the mAb 4G12‐C1 increased the production of IL‐2 from swine PBMCs. IL‐2 is produced mainly
by activated T cells and is vital for the cellular expansion
required for functional immune response. Moreover, theimmune activation effect by the PD‐L1 blockade, such as
the production of other Th1 cytokines and proliferativeand effector activities of antigen‐specific T cells, must be
further analyzed in pigs. The antibacterial and antiviraleffects of the PD‐L1 blockade will be tested in clinicalstudies using infected animals. Thus, to develop a new
therapeutic method for the control of chronic diseases inpigs, the effect of the PD‐L1 blockade must be evaluatedfrom a variety of perspectives.

0

Rat IgG2a control

Anti-PD-L1 mAb

F I G U R E 5 IL‐2 production via PD‐L1 blockade by anti‐PD‐L1
mAb. PBMCs were isolated from healthy piglets (n = 6) andcultured with anti‐PD‐L1 mAb (4G12‐C1) or rat IgG2a control in thepresence of SEB. After 3 days, culture supernatants were collected
and IL‐2 levels were measured by ELISA in duplicate. Significant
differences between the treatments were identified using the
Wilcoxon signed‐rank test. ELISA, enzyme‐linked immunosorbent
assay; IgG, immunoglobulin G; IL, interleukin; mAb, monoclonal
antibody; PD‐L1, programmed death‐ligand 1; PMBC, peripheral
blood mononuclear cell; SEB, Staphylococcal enterotoxin B

of PD‐L1 in swine immune cells. Unfortunately, our previously established anti‐bovine PD‐1 mAbs8 did not cross‐react with swine PD‐1, thus we were not able to determine
the trend of PD‐1 expression in these swine infections (datanot shown). Analysis of PD‐1 expression during diseaseprogression will be also helpful for understanding the immunopathogenesis of chronic infections in pigs. The relationship between PD‐1 and suppression of T‐cell responses
in these diseases also needs to be clarified and further studies
are needed.
In this study, we compared the cross‐reactivity toswine PD‐L1 and the blockade activity against the interaction of swine PD‐1/PD‐L1 among four clones of anti‐
bovine PD‐L1 mAbs. The mAb 6C11‐3A11 exerted highbinding affinity to swine PD‐L1 and was found to be theoptimal detection antibody for PD‐L1 in pigs. However, it
had limited blockade activity. On the contrary, the mAbs
6G7‐E1 and 4G12‐C1 showed moderate binding affinity
to swine PD‐L1, but their blockade activities were robust.
The difference among these three mAbs would dependon the epitope of the mAbs. The mAbs 4G12‐C1 and 6G7‐
E1 may interact with the region of swine PD‐L1 which isessential for the interaction with swine PD‐1. The subclass of the mAb 4G12‐C1 is IgG2a, and this clone isconsidered to be suitable for the future development of

AC KNOW LEDGM ENTS
We are grateful to Dr. Hideyuki Takahashi, Dr. Yasuyuki
Mori, and Dr. Tomio Ibayashi for valuable advice anddiscussions. We would like to thank Enago (https://www.enago.jp) for the English language review. This
study was supported by JSPS KAKENHI grant number
19KK0172 (to Satoru Konnai), grants from the Project of
the NARO, Bio‐oriented Technology Research Advancement Institution (Research Program on Development of
Innovative Technology 26058 BC (to Satoru Konnai),
Clinical Research Promotion Fund 2020 by Hokkaido
University Veterinary Teaching Hospital (to Tomohiro
Okagawa) and AMED under grant number
JP20am0101078 (to Yukinari Kato). The funders had norole in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
C O NF L I C T O F I N T E R E S T S
The authors declare that there are no conflict of interests.
A U T H O R C O N TR I B U T I O N S
Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa,
Shiro Murata, and Kazuhiko Ohashi designed the work.
Otgontuya Ganbaatar, Tomohiro Okagawa, Yutaro Nojima, and Yoshiki Ichikawa performed the experiments.
Atsushi Kobayashi, Tomoyuki Shibahara, Yojiro Yanagawa, Hidetoshi Higuchi, Yukinari Kato, and YasuhikoSuzuki provided intellectual input, field samples, laboratory materials, reagents, and/or analytic tools. Otgontuya
Ganbaatar, Satoru Konnai, Tomohiro Okagawa, andAtsushi Kobayashi acquired, analyzed, and interpreted thedata. Otgontuya Ganbaatar, Satoru Konnai, and Tomohiro
Okagawa wrote the manuscript. Satoru Konnai, Tomohiro
Okagawa, Naoya Maekawa, Atsushi Kobayashi, Tomoyuki
Shibahara, Yojiro Yanagawa, Hidetoshi Higuchi, Yukinari
Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
revised the manuscript. All authors read and approved thefinal manuscript.

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DATA A VAILABILITY S TATEMENT
The data that support the findings of this study areavailable from the corresponding author upon reasonable
request.

15.

ORCID
Yukinari Kato16.

http://orcid.org/0000-0001-5385-8201

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SU PPOR T ING INF OR MATI ON
Additional Supporting Information may be found online
in the supporting information tab for this article.

How to cite this article: Ganbaatar O, Konnai S,
Okagawa T, et al. Programmed death‐ligand 1
expression in swine chronic infections andenhancement of interleukin‐2 production via
programmed death‐1/programmed death‐ligand 1
blockade. Immun Inflamm Dis. 2021;9:1573‐1583.
https://doi.org/10.1002/iid3.510