RESEARCH ARTICLE
Clinical and Translational Immunology

Enhanced Immunotherapeutic Efficacy of Anti–PD-L1
Antibody in Combination with an EP4 Antagonist*Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan; †Department of Advanced
Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan; ‡Department of Applied Veterinary Sciences,
Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan; §Department of Antibody Drug Development, Tohoku University
Graduate School of Medicine, Sendai 980-8575, Japan; {New Industry Creation Hatchery Center, Tohoku University, Sendai 980-8575, Japan; andkDivision of Bioresources, Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0019, Japan

ABSTRACT
Combination treatment approaches are increasingly considered to overcome resistance to immunotherapy targeting
immunoinhibitory molecules such as programmed death (PD)–1 and PD-ligand 1 (PD-L1). Previous studies have demonstrated thatthe therapeutic efficacy of anti–PD-L1 Abs is enhanced by combination treatment with cyclooxygenase-2 inhibitors, throughdownregulation of the immunosuppressive eicosanoid PGE2, although the underlying mechanism remains unclear. In this study, weshow that serum PGE2 levels are upregulated after anti–PD-L1 Ab administration in a bovine model of immunotherapy and that PGE2
directly inhibits T cell activation via its receptor E prostanoid (EP) 4. Additionally, anti–PD-L1 Ab induces TNF-a production and TNF-a
blockade reduces PGE2 production in the presence of anti–PD-L1 Ab, suggesting that anti–PD-L1 Ab–induced TNF-a impairs T cell
activation by PGE2 upregulation. Our studies examining the therapeutic potential of the dual blockade of PD-L1 and EP4 in bovine
and murine immune cells reveal that the dual blockade of PD-L1 and EP4 significantly enhances Th1 cytokine production in vitro.
Finally, we show that the dual blockade decreases tumor volume and prolongs survival in mice inoculated with the murine lymphoma
cell line EG7. Altogether, these results suggest that TNF-a induced by anti–PD-L1 Ab treatment is associated with T cell dysfunction
via PGE2/EP4 pathway and that the dual blockade of PD-L1 and EP4 should be considered as a novel immunotherapy for cancer.
ImmunoHorizons, 2020, 4: 837–850.

Received for publication October 18, 2020. Accepted for publication November 10, 2020.
Address correspondence and reprint requests to: Dr. Satoru Konnai, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Kita-18
Nishi-9, Kita-ku, Sapporo 060-0818, Japan. E-mail address: konnai@vetmed.hokudai.ac.jp
ORCIDs: 0000-0003-1237-4686 (Z.C.); 0000-0002-1391-9732 (T.O.); 0000-0001-5385-8201 (Y.K.); 0000-0002-1313-1494 (Y. Suzuki).
The microarray data presented in this article have been submitted to ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-9576.
This work was supported by Grants-in-Aid for Scientific Research and a Japan Society for the Promotion of Science (JSPS) Research Fellowship, Research Project for
Improving Animal Disease Prevention Technologies to Combat Antimicrobial Resistance 2017–2021 FY of the Ministry of Agriculture, and grants from the Project of theNARO, Bio-oriented Technology Research Advancement Institution (Research Program on Development of Innovative Technology 26058 BC [to S.K.] and Special
Scheme Project on Regional Developing Strategy Grant 16817557 [to S.K.]). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Y. Sajiki performed most of the experiments, analyzed the data, prepared figures, and wrote the manuscript. S.K. conceived and designed experiments, analyzed the data,
and revised the manuscript. Z.C. and K.T. assisted in the experimental design and the experiments using the murine tumor model and revised the manuscript. T.O. and
N.M. helped with immunological analyses using bovine immune cells, analyzed the data, and assisted in manuscript writing. S.F., Y.K., and Y. Suzuki provided laboratory
materials and reagents. S.M. and K.O. assisted in the experimental design and revised the manuscript. All authors reviewed and approved the final manuscript.
Abbreviations used in this article: BLV, bovine leukemia virus; COX, cyclooxygenase; EP, E prostanoid; FLK, fetal lamb kidney; PD, programmed death; PD-L1, PD-ligand 1;
qPCR, quantitative real-time PCR; TNFRII, TNF receptor type II.
The online version of this article contains supplemental material.
This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license.
Copyright © 2020 The Authors
https://doi.org/10.4049/immunohorizons.2000089
ImmunoHorizons is published by The American Association of Immunologists, Inc.

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Yamato Sajiki,* Satoru Konnai,*,† Zimeng Cai,‡ Kensuke Takada,‡ Tomohiro Okagawa,† Naoya Maekawa,† Sotaro Fujisawa,*
Yukinari Kato,§,{ Yasuhiko Suzuki,†,k Shiro Murata,*,† and Kazuhiko Ohashi*,†

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INTRODUCTION

expression in an in vitro murine model (29). Furthermore,combination treatment with aspirin, a COX inhibitor, andanti–PD-1 Ab has been shown to suppress tumor growth inseveral murine tumor models (32). We have previously shownthat combination treatment with anti–PD-L1 Ab and a COX-2
inhibitor has a signiﬁcantly enhanced therapeutic eﬃcacy in
BLV-infected cattle (12), although the underlying mechanisms have not been fully elucidated.
In the current study, we focused on the function of PGE2,
which is upregulated after the administration of anti–PD-L1 Abs in
BLV-infected cattle. Our analyses using bovine immune cells
revealed that PGE2 directly suppressed the activity of T cells via
EP4. In addition, TNF-a induced by anti–PD-L1 Ab treatment
upregulated PGE2 production from PBMCs. These ﬁndings
revealed that the PGE2/EP4 axis might be a mechanism underlying resistance to anti–PD-L1 Ab treatment. Furthermore, ourinvestigation in a murine lymphoma model revealed the therapeutic potential of combination treatment with anti–PD-L1 Ab andan EP4 antagonist based on the inhibition of tumor growth andprolonged survival in tumor-bearing mice, suggesting that the
combination treatment should be considered as a novel immunotherapeutic approach in cancer.

MATERIALS AND METHODS

Cells
Blood samples derived from BLV-infected and -uninfected cattle
were collected at several farms in Hokkaido, Japan, and BLV
infection was diagnosed as described previously (33). Informed
consent was obtained from all owners of cattle sampled in thecurrent study. PBMCs were separated from the blood samples bydensity gradient centrifugation on Percoll (GE Healthcare, Little
Chalfont, U.K.). For isolation of CD3+ and CD4+ cells, PBMCs from
BLV-uninfected cattle were incubated with anti-bovine CD3 mAb(MM1A; Washington State University mAb Center, Pullman, WA)or anti-bovine CD4 mAb (CC8; Bio-Rad Laboratories, Hercules,
CA) at 4°C for 30 min, followed by incubation with anti-mouse
IgG1 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany)at 4°C for 15 min. CD3+ and CD4+ cells were sorted from the
PBMCs using AutoMACS Pro (Miltenyi Biotec), according to the
manufacturer’s protocol. The purity of cells, conﬁrmed using
FACS Verse (BD Biosciences, San Jose, CA), was routinely .90%.
PBMCs and isolated CD3+ and CD4+ cells were cultured in 200 ml
RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented
with 10% heat-inactivated FCS (Thermo Fisher Scientiﬁc,Waltham, MA), 100 U/ml penicillin (Thermo Fisher Scientiﬁc),
100 mg/ml streptomycin (Thermo Fisher Scientiﬁc), and 2 mM
L-glutamine (Thermo Fisher Scientiﬁc). All bovine cell cultures
were grown in 96-well plates (Corning, Corning, NY) at 37°C
under 5% CO2 atmosphere.
Eight-week-old female BALB/c mice were purchased fromSankyo Labo Service Corporation (Tokyo, Japan) and sacriﬁced by
isoﬂurane inhalation and cervical dislocation. The spleens werecollected, minced with scissors, digested in RPMI 1640 medium
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Programmed death (PD)–1 is an immune checkpoint molecule thatnegatively regulates T cell function via the interaction with its
ligands, PD-ligand 1 (PD-L1) and PD-L2 (1). Upregulation of PD-1
plays a key role in T cell exhaustion, and PD-1/PD-L1 pathway is
involved in the progression of a variety of tumors and chronic
infections (2, 3). In contrast, previous reports have demonstrated
that the inhibition of the PD-1/PD-L1 pathway using speciﬁc Abs
restores the eﬀector functions of exhausted T cells and enhances
antitumor immune responses (4–6). Therefore, the immunotherapy targeting the PD-1/PD-L1 pathway has become a promisingtherapeutic strategy for the treatment of patients with tumors (7,8). Recently, studies have also demonstrated the eﬃcacy of PD-1/
PD-L1 blockade in the ﬁeld of veterinary medicine (9–13). We havepreviously shown the therapeutic potential of anti–PD-1/PD-L1
Abs for the treatment of bovine leukemia virus (BLV)–infected
cattle (9, 10, 12). BLV, an oncogenic deltaretrovirus of cattle that isclosely related to human T cell leukemia virus type 1, infects B cells,and 1–5% of BLV-infected cattle develop fatal lymphoma or
lymphosarcoma after a long latent period (14, 15). After theadministration of anti–PD-1/PD-L1 Abs, BLV proviral loads weresigniﬁcantly reduced in peripheral blood (9, 10, 12), suggesting that
this strategy might contribute to a reduced risk of tumorigenesis
associated with BLV infection.
Although the immunotherapy targeting the PD-1/PD-L1
pathway have been approved for cancer treatment in humans, asigniﬁcant proportion of the patients remains less responsive (16).
A potential strategy to overcome this issue is combining anti–PD1/PD-L1 Abs with other therapies. PGE2 is one of the candidate
targets for combination treatment with anti–PD-1/PD-L1 Abs.
PGE2 is known as an inﬂammatory mediator derived from arachidonic acid by cyclooxygenase (COX)-1, COX-2, and PGE synthases(17). COX-1 is a constitutive enzyme and widely expressed in manytissues, whereas COX-2 is an inducible enzyme whose expression
is regulated by the activation of NF-kB by inﬂammatory cytokinesand growth factors (18, 19). There are four PGE2 receptors,
E prostanoid (EP) 1, EP2, EP3, and EP4 (20). PGE2 inhibits
the activity of immune cells, such as T cells, dendritic cells,
and NK cells, via EP2 and EP4 receptors (21). In addition,
numerous studies have demonstrated the role of COX2/PGE2
in tumor microenvironments (22). The increased expressionof COX-2 in breast and colorectal cancers is associated withpoor prognosis (23, 24). In the tumor microenvironment,
many cell types, including tumor cells and endothelial cells,
produce PGE2 via COX-2 activation, and PGE2 enhances
tumor cell progression via several pathways such as angiogenesis (25–27). In addition, PGE2 aﬀects the immune cells intumor microenvironments. Speciﬁcally, PGE2 regulates theactivity of Th1 cells, whereas it enhances the function of
immunosuppressive cells, such as regulatory T cells (21, 28).
Interestingly, several studies have reported the role of PGE2 as
an inducer of PD-L1 expression. PGE2 upregulates theexpression of PD-L1 in murine and bovine models (29–31),
and treatment with a COX-2 inhibitor reduces PD-L1

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days 0, 1, 3, 7, 14, 28, and 56. Serum PGE2 concentrations were measured by ELISA. (B) Statistical significance was determined by the
Mann–Whitney U test.

containing 0.2 mg/ml DNase I (Sigma-Aldrich) and 0.67 U/ml
research-grade Liberase DL (Sigma-Aldrich) for 30 min at 37°C,
and passed through a 100-mm cell strainer (BD Biosciences). Next,
the cells were washed twice with PBS and passed through a 40-mm
cell strainer (BD Biosciences). The isolated splenocytes were thencultured in culture medium as described above.
EG7 murine T cell lymphoma cell line (34) was obtained from
American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium supplemented with 10% heatinactivated FBS (Sigma-Aldrich), 1% penicillin-streptomycin
https://doi.org/10.4049/immunohorizons.2000089(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan),and 0.4 mg/ml G418 (FUJIFILM Wako Pure Chemical Corporation) at 37°C under 5% CO2 atmosphere.

Serum samples
Four BLV-infected cattle (animals 1–4) were i.v. administered
1 mg/kg anti-bovine PD-L1 Ab (Boch4G12, a rat-bovine chimericAb) (9). Serum samples of the cattle obtained on days 0, 1, 3, 7, 14,28, and 56 were stored at 280°C until use in the experiments. All
experimental procedures using cattle were conducted following

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FIGURE 1. Changes in serum PGE2 concentrations after anti–PD-L1 Ab inoculation.
(A and B) BLV-infected cattle (animals 1–4) were administered 1 mg/kg anti–PD-L1 Ab (Boch4G12), and serum samples were collected on

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FIGURE 2. Functional analysis of EP signaling in PBMCs.
(A) Following an hour of incubation with indicated EP antagonists, PBMCs from BLV-uninfected cattle (n = 6) were incubated with PGE2 in thepresence of anti-CD3 mAb and anti-CD28 mAb. After incubation, IFN-g concentrations in culture supernatants were determined by ELISA. (B)
PBMCs from BLV-uninfected cattle (n = 6) were cultured with the EP4 agonist, and the expression of mRNA-encoding IFN-g was quantitated by
qPCR. DMSO was used as a vehicle control. (C) PBMCs from BLV-uninfected cattle (n = 7) were incubated with the EP4 agonist in the presence ofanti-CD3 mAb and anti-CD28 mAb. After incubation, IFN-g concentrations in culture supernatants were determined by ELISA. (Continued)

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TABLE I. Cattle used in this study
Age
Breed
Sex
Body weight
BLV infection
Administration dose

Animal 1

Animal 2

Animal 3

Animal 4

13 mo old
Holsteinfemale295 kg+
1 mg/kg, i.v.

17 mo old
Holsteinfemale522 kg+
1 mg/kg, i.v.

76 mo old
Holsteinfemale799 kg+
1 mg/kg, i.v.

7 mo old
Holsteinmale
267 kg+
1 mg/kg, i.v.

approval from the local committee for animal studies at Hokkaido
University (approval number 17-0024).

ELISA
PGE2 concentrations in sera and culture supernatants were
measured by PG E2 Express ELISA Kit (Cayman Chemical, Ann
Arbor, MI), following the manufacturer’s instructions. Bovine
IFN-g and mouse IL-2 concentrations in culture supernatants
were measured by Bovine IFN-g ELISA Development Kit(Mabtech, Nacka Strand, Sweden) and Mouse IL-2 Matched Ab
Pair Kit (Abcam, Cambridge, U.K.), respectively, according to the
manufacturers’ protocols.
Flow cytometry
For CD69 expression levels, collected cells were incubated in PBS
containing 10% goat serum (Thermo Fisher Scientiﬁc) for 15 min at
25°C to prevent nonspeciﬁc reactions. Next, the cells were stainedfor 20 min at 25°C using the following Abs: FITC-conjugated antibovine CD4 mAb (CC8), PE-conjugated anti-bovine CD8 mAb(CC63; Bio-Rad Laboratories), and Alexa Fluor 647–labeled antibovine CD69 mAb (KTSN7A; Kingﬁsher Biotech, St. Paul, MN).
KTSN7A was prelabeled with a Zenon Alexa Fluor 647 Mouse IgG1

PBMC culture
To examine the individual eﬀects of EP antagonists, PBMCs from
BLV-uninfected cattle were incubated for 1 h with 1 mg/ml of eachof the following EP antagonists from Cayman Chemical: EP1 (SC19220), EP2 (AH6809), EP3 (L-798,106), and EP4 (ONO-AE3208), and then 250 nM PGE2 (Cayman Chemical) was added toeach culture. The PBMCs were stimulated by adding 1 mg/ml antibovine CD3 mAb (MM1A) and 1 mg/ml anti-bovine CD28 mAb(CC220; Bio-Rad Laboratories) to each well. After 24 h, the culture
supernatants were collected to measure IFN-g concentrations by
ELISA.
To examine the eﬀect of the EP4 agonist, PBMCs from BLVuninfected cattle were incubated with 1 mg/ml Rivenprost, an EP4
agonist (Cayman Chemical). The expression of IFN-g after 24 h ofincubation with no additional stimulation was determined by
qPCR as described above, and IFN-g concentrations in culture
supernatants after 24 h of incubation with 1 mg/ml anti-bovine
CD3 mAb (MM1A) and 1 mg/ml anti-bovine CD28 mAb (CC220)
were measured by ELISA as described above. Similarly, IFN-g
production in bovine lymphocyte subsets was measured by ﬂow(D and E) PBMCs from BLV-uninfected cattle (n = 12) were incubated with the EP4 agonist in the presence of anti-CD3 mAb, anti-CD28 mAb, andrecombinant bovine IL-2. After incubation, IFN-g expression levels in CD4+ (D) and CD8+ (E) cells were measured by flow cytometry. (A–E)
Statistical significance was determined by the Steel–Dwass test (A) or the Wilcoxon signed-rank test (B–E).

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Quantitative real-time PCR
Quantitative real-time PCR (qPCR) was performed as described
previously (30). Brieﬂy, total RNA was extracted from cultured
cells using TRI Reagent (Molecular Research Center, Cincinnati,OH) and cDNA was synthesized from the total RNA by PrimeScript Reverse Transcriptase (TaKaRa Bio, Otsu, Japan) following
the manufacturers’ protocols. Next, qPCR was performed usinga LightCycler 480 System II (Roche Diagnostics, Mannheim,
Germany) with SYBR Premix DimerEraser (TaKaRa Bio), following the manufacturers’ protocols. b-actin (ACTB) was used asa reference gene, and relative expression levels were calculatedusing the DD cycle threshold method. The primers were 59-ATA
ACC AGG TCA TTC AAA GG-39 and 59-ATT CTG ACT TCT CTT
CCG CT-39 for bovine IFN-g, 59-ACG TTT TCT CGT GAA GCC
CT-39 and 59-TCT ACC AGA AGG GCG GGA TA-39 for bovine
COX2, and 59-TCT TCC AGC CTT CCT TCC TG-39 and 59-ACC
GTG TTG GCG TAG AGG TC-39 for bovine ACTB.

Labeling Kit (Thermo Fisher Scientiﬁc). The stained cells were
washed twice and analyzed immediately by FACS Verse.
For the intracellular staining of IFN-g and TNF-a, thecollected cells were incubated in PBS containing 10% goat serum
as described above, followed by staining with FITC-conjugatedanti-bovine CD4 mAb (CC8), PerCP/Cy5.5–conjugated antibovine CD8 mAb (CC63), and PE-labeled anti-bovine IgM mAb(IL-A30; Bio-Rad Laboratories) for 20 min at 25°C. IL-A30 was
prelabeled with a Zenon R-PE Mouse IgG1 Labeling Kit (Thermo
Fisher Scientiﬁc). CC63 was conjugated with PerCP/Cy5.5 byusing a Lightning-Link Ab Labeling Kit (Innova Biosciences,
Cambridge, U.K.). After surface staining, the cells were ﬁxed andpermeabilized using FOXP3 Fix/Perm Kit (BioLegend, San Diego,
CA). Next, the cells were stained with biotinylated anti-bovine
IFN-g mAb (MT307; Mabtech) or biotinylated anti-bovine TNF-amAb (CC328; Bio-Rad Laboratories) for 20 min at 25°C. The cells
were then incubated with allophycocyanin-conjugated streptavidin (BioLegend) for 20 min at 25°C. After the ﬁnal staining, thecells were washed twice and analyzed immediately by FACS Verse.

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FIGURE 3. Functional analysis of EP4 signaling in CD3+ cells.
(A–C) CD3+ cells isolated from PBMCs of BLV-uninfected cattle (n = 8) were cultured with the EP4 agonist in the presence of anti-CD3 mAb andanti-CD28 mAb. After incubation, CD69 expression levels in CD4+ (A) and CD8+ (B) cells and IFN-g concentrations in culture supernatants (C) were
measured by flow cytometry and ELISA, respectively. (D) The heat-map for the changes in gene expression levels in CD4+ cells by the EP4 agonist.
(A–C) Statistical significance was determined by the Wilcoxon signed-rank test. MFI, Mean fluorescence intensity.

cytometry by stimulating the cells with 2 mg/ml anti-bovine CD3
mAb (MM1A), 2 mg/ml anti-bovine CD28 mAb (CC220), and10 ng/ml recombinant bovine IL-2 (Kingﬁsher Biotech). Following
19 h of incubation, the cells were incubated with 10 mg/ml brefeldin

A (Sigma-Aldrich) for additional 5 h, and analyzed as describe
above. To examine the eﬀect of anti–PD-L1 Ab and TNF-a on PGE2
production, PBMCs from BLV-uninfected cattle were incubatedwith 10 mg/ml anti-bovine PD-L1 Ab (Boch4G12) or 10 ng/ml
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TABLE II. The change of gene expression in microarray analysis
Fold Change

p Value

IL-2
Chemokine (CC motif) ligand 3
TNF
CSF-2
IL-12ACXC motif chemokine ligand 10
CC motif chemokine ligand 20
Lymphotoxin-a
Chemokine (CC motif) ligand 4
Fibrinogen b-chain
IFN-b 3
CXC motif chemokine ligand 9
IFN-g
cAMP-responsive element modulator
Nuclear receptor subfamily 4 group A member 3
Nuclear receptor subfamily 4 group A member 2
PG-endoperoxide synthase 2
NF-kB subunit 1
CTL–associated protein 4
Programmed cell death 1

22.1255753
22.0820506
22.0492654
22.0447707
21.9293255
21.8574089
21.8484110
21.8189231
21.7715497
21.7014802
21.5604529
21.4785548
21.4232383
2.4109561
1.9490188
1.8775752
1.7771128
1.5010872
1.1734980
1.0657109

0.008147
0.004651
0.001460
0.005276
0.012709
0.019228
0.010516
0.019710
0.000768
0.026826
0.043236
0.000302
0.054541
0.001597
0.029402
0.001489
0.042600
0.001316
0.011518
0.355153

bovine rTNF-a (Kingﬁsher Biotech) for 72 or 24 h, respectively.
Bovine IgG (Sigma-Aldrich) was used as a negative control for theanti–PD-L1 Ab Boch4G12, and PBS was used as a vehicle control
for bovine rTNF-a. After incubation, the culture supernatants
were collected to measure PGE2 concentrations by ELISA, andthe cells were collected for the quantiﬁcation of COX2 expression
by qPCR. Additionally, to investigate whether treatment withanti–PD-L1 Ab induces TNF-a production, PBMCs from BLVuninfected cattle were incubated with 10 mg/ml anti-bovine PD-L1
Ab (Boch4G12) in the presence of 2 mg/ml anti-bovine CD3 mAb(MM1A), 2 mg/ml anti-bovine CD28 mAb (CC220), and 10 ng/ml
recombinant bovine IL-2. Following 19 h of incubation, thecultures were incubated with 10 mg/ml brefeldin A for 5 h, after
which the cultured PBMCs were harvested, and TNF-a expression levels were measured by ﬂow cytometry. Furthermore, toexamine whether the blockade of TNF-a reduces PGE2 production in the presence of anti–PD-L1 Ab, PBMCs from BLVuninfected cattle were incubated with 172 nM bovine TNF
receptor type II (TNFRII)–Ig, a decoy receptor for bovine TNF-a(35), in the presence of 10 mg/ml anti-bovine PD-L1 Ab(Boch4G12). Control Ig, which comprised the signal peptide ofbovine TNFRII and the Fc domain of bovine IgG1 (35), was used asa negative control for TNFRII-Ig. Cultures were stimulated byadding 1 mg/ml anti-bovine CD3 mAb (MM1A) and 1 mg/ml antibovine CD28 mAb (CC220) to each well. After 72 h, the culture
supernatants were collected to measure PGE2 concentrations by
ELISA.
To examine the eﬀect of the dual blockade of PD-L1 and EPs incattle, PBMCs from BLV-uninfected or BLV-infected cattle were
cultured with 10 mg/ml anti-bovine PD-L1 Ab (Boch4G12) and
1 mg/ml each EP antagonist. PBMCs from BLV-uninfected cattle
were cultured in the presence of 1 mg/ml anti-bovine CD3 mAb(MM1A) and 1 mg/ml anti-bovine CD28 mAb (CC220) for 72 h,
whereas PBMCs from BLV-infected cattle were cultured in thehttps://doi.org/10.4049/immunohorizons.2000089

presence of a BLV Ag, fetal lamb kidney (FLK)–BLV (2% heatinactivated culture supernatant of FLK-BLV cells), for 144 h. Afterincubation, the culture supernatants were collected, and IFN-g
concentrations were determined by ELISA.

CD3+ cell culture
Isolated CD3+ cells were cultured with 1 mg/ml the EP4 agonist
in the presence of 1 mg/ml anti-bovine CD3 mAb (MM1A) and
1 mg/ml anti-bovine CD28 mAb (CC220) for 72 h. After incubation,
CD69 expression levels and IFN-g concentrations in culture
supernatants were measured by ﬂow cytometry and ELISA,
respectively.
Microarray
Isolated CD4+ cells were cultured with 0.5 mg/ml anti-bovine CD3
mAb (MM1A) and 0.5 mg/ml anti-bovine CD28 mAb (CC220).
Following 18 h of incubation, the cultures were incubated with
1 mg/ml the EP4 agonist or DMSO for 4 h. Microarray analysis was
performed using Agilent Bos taurus (Bovine) Oligo Microarray v2(Design ID: 023647; Agilent Technologies, Santa Clara, CA). After
cell collection, total RNA was extracted using RNeasy Mini Kit(Qiagen, Hilden, Germany) according to the manufacturer’s
instructions. Synthesis and labeling of cRNA were performed
using Low Input Quick Amp Labeling Kit (Agilent Technologies)
according to the manufacturer’s instructions. The Cy3-labeledcRNA was puriﬁed using RNeasy Mini Kit (Qiagen), andhybridization was performed using Gene Expression Hybridization Kit (Agilent Technologies), according to the manufacturers’
instructions. Next, scanning of the hybridized microarray anddata analysis were performed using Agilent DNA Microarray
Scanner (Agilent Technologies), Feature Extraction software(Agilent Technologies), and GeneSpring (Agilent Technologies), according to the manufacturers’ instructions. The microarray data were deposited in ArrayExpress (E-MTAB-9576,

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Gene Name

Gene Symbol
IL2
CCL3
TNF
CSF2
IL12A
CXCL10
CCL20
LTA
CCL4
FGB
IFNB3
CXCL9
IFNG
CREM
NR4A3
NR4A2
PTGS2
NFKB1
CTLA4
PDCD1

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concentrations in culture supernatants (B, n = 6) were measured by qPCR and ELISA, respectively. Bovine IgG was used as a negative control ofanti–PD-L1 Ab (Boch4G12). (C and D) PBMCs from BLV-uninfected cattle (n = 11) were incubated with anti–PD-L1 Ab (Boch4G12) in the presence ofanti-CD3 mAb, anti-CD28 mAb, and recombinant bovine IL-2. After incubation, TNF-a expression levels in CD4+ (C) and CD8+ (D) cells were
measured by flow cytometry. (E and F) PBMCs from BLV-uninfected cattle were cultured with recombinant bovine TNF-a, and COX2 expression
levels (E, n = 10) and PGE2 concentrations in culture supernatants (F, n = 7) were measured by qPCR and ELISA, respectively. PBS was used as avehicle control. (G) PBMCs from BLV-uninfected cattle (n = 6) were cultured with TNFRII-Ig in the presence of anti–PD-L1 Ab (Boch4G12). Cultures
were stimulated by adding anti-CD3 mAb and anti-CD28 mAb. Control Ig was used as a negative control for TNFRII-Ig. PGE2 concentrations in
culture supernatants were determined by ELISA. (A–G) Statistical significance was determined by the Wilcoxon signed-rank test.

https://www.ebi.ac.uk/arrayexpress/). The microarray procedures from RNA extraction to data analysis were conducted at
Hokkaido System Science (Sapporo, Japan).

Splenocyte culture
To examine the eﬀects of anti–PD-L1 Ab treatment on PGE2
production in mice, splenocytes were cultured with 10 mg/ml

anti-mouse PD-L1 Ab (10F.9G2; BioXCell, West Lebanon, NH) or
10 mg/ml rat IgG2b isotype control (LTF-2; BioXCell). PGE2
concentrations in supernatants of culture incubated with orwithout 10 mg/ml Con A (Sigma-Aldrich) for 72 h were measured
by ELISA as described above. To examine the eﬀect of the dual
blockade of PD-L1 and EP4 in mice, splenocytes were cultured
with 10 mg/ml anti-mouse PD-L1 Ab (10F.9G2) and 1 mg/ml eachhttps://doi.org/10.4049/immunohorizons.2000089

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FIGURE 4. TNF-a induced by PD-L1 blockade upregulates PGE2 production.
(A and B) PBMCs from BLV-uninfected cattle were cultured with anti–PD-L1 Ab (Boch4G12), and COX2 expression levels (A, n = 8) and PGE2

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(A and B) PBMCs from BLV-uninfected (n = 8) and BLV-infected (n = 7) cattle were cultured with anti–PD-L1 Ab (Boch4G12) and indicated EP
antagonists. PBMCs from BLV-uninfected cattle were stimulated by anti-CD3 mAb and anti-CD28 mAb. PBMCs from BLV-infected cattle were
stimulated by FLK-BLV, a BLV Ag. After incubation, IFN-g concentrations in culture supernatants were measured by ELISA. Statistical significancewas determined by the Steel–Dwass test.

EP antagonist. Cultures were stimulated by adding 1 mg/ml antimouse CD3e mAb (145-2C11; Thermo Fisher Scientiﬁc) to eachwell. After 72 h, the culture supernatants were collected, and IL-2
concentrations were measured by ELISA.

Tumor grafting and tumor growth measurement
Six-week-old male C57BL/6 mice (Japan SLC, Hamamatsu,
Japan) were s.c. inoculated with EG7 (5 3 106 cells per mouse).
The day of EG7 injection was deﬁned as day 0. For anti–PD-L1 Ab
treatment, mice were i.p. injected with anti-mouse PD-L1 mAb(10F.9G2) (10 mg/kg, once a day) on days 7, 10, and 14. For EP4
antagonist treatment, mice were orally administered with ONOAE3-208 (10 mg/kg/d) added to drinking water from day 7 to day
23. Tumor size was monitored at least every other day, starting on
day 5, using a caliper until the length or width exceeded 20 mm.
Tumor volume was calculated according to the following formula:tumor volume (mm3) = (length 3 width2)/2. The animal experiments were performed with the approval of the Institutional
Animal Care and Use Committee of the Graduate School of
Veterinary Medicine at Hokkaido University (approval number:
16-0131). The animals were handled in accordance with the Guide
for the Care and Use of Laboratory Animals, Graduate School of
Veterinary Medicine, Hokkaido University (approved by the
Association for Assessment and Accreditation of Laboratory
Animal Care International).
Statistics
In Fig. 1, diﬀerences were assessed using the Mann–Whitney U
test. In Figs. 2–5 and 6A–C, diﬀerences were assessed using the
Wilcoxon signed-rank test for two-group comparisons and theSteel–Dwass test for multiple-group comparisons. In Fig. 6E andhttps://doi.org/10.4049/immunohorizons.2000089

6F, diﬀerences were assessed using the Tukey test and the logrank test, respectively. In microarray analysis (Table II), diﬀerences were assessed using the paired t test. A p value ,0.05
was considered to indicate statistical signiﬁcance.

RESULTS

Serum PGE2 concentration is increased withanti–PD-L1 immunotherapy
We ﬁrst analyzed the serum samples from BLV-infected cattle thatwere administered the anti–PD-L1 blocking Ab (Table I) andfound that the serum PGE2 concentrations were increased after
the anti–PD-L1 Ab treatment (Fig. 1). Therefore, we speciﬁcallyexamined the role of PGE2 in the blockade of PD-1/PD-L1
interaction in the current study.
T cell activation is suppressed via PGE2/EP4 pathway
We have previously shown that PGE2 suppresses Th1 responses,
such as Th1 cytokine production and T cell proliferation, in cattle(30). In the current study, we further aimed to identify speciﬁc
PGE2 receptors involved in PGE2-mediated immune dysfunction
using EP antagonists and agonists. Bovine PBMCs were preincubated with individual EP antagonists, followed by culturing thecells with PGE2 in the presence of anti-CD3 and anti-CD28 mAbs.
Pretreatment with the EP4 antagonist inhibited the suppressive
eﬀect of PGE2, whereas IFN-g production was suppressed by
PGE2 in PBMCs pretreated with the antagonists of other EPs(EP1–EP3) (Fig. 2A). Additionally, treatment with the EP4 agonist
signiﬁcantly inhibited IFN-g mRNA and its protein expression in
PBMCs (Fig. 2B, 2C). Flow cytometric analysis revealed that the

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FIGURE 5. Functional analysis of the dual blockade of PD-L1 and EP4 in cattle.

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FIGURE 6. Functional analysis of the dual blockade of PD-L1 and EP4 in mice.
(A and B) Murine splenocytes (A, n = 6; B, n = 8) were cultured with anti–PD-L1 Ab (10F.9G2). Cultures were stimulated with or without Con A, andPGE2 concentrations in culture supernatants were measured by ELISA. Statistical significance was determined by the Wilcoxon signed-rank test. (C)Murine splenocytes (n = 6) were cultured with anti–PD-L1 Ab (10F.9G2) and indicated EP antagonists in the presence of anti-mouse CD3e mAb. IL-2
concentrations in culture supernatants were determined by ELISA. Statistical significance was determined by the Steel–Dwass test. (D–F) Evaluationof the antitumor effects of dual blockade in the EG7 mouse model. (D) Experimental design. (E) Tumor growth in each group. (Continued)

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PD-L1 blockade–mediated induction of TNF-a upregulates
PGE2 production
As shown in Fig. 1, the serum PGE2 concentrations were increased
after anti–PD-L1 Ab treatment. To examine whether treatment
with the anti–PD-L1 Ab induces PGE2 production in vitro, bovine
PBMCs were cultured with anti–PD-L1 Ab (Boch4G12), whichsigniﬁcantly induced the COX2 expression and PGE2 production
in vitro (Fig. 4A, 4B). Blockade of the PD-1/PD-L1 pathway
using speciﬁc Abs reactivates exhausted T cells, leading to theenhancement of Th1 cytokine production from T cells (5, 6). In thecurrent study, ﬂow cytometric analysis revealed that anti–PD-L1
Ab (Boch4G12) signiﬁcantly increased the TNF-a expression
levels in both CD4+ and CD8+ cells (Fig. 4C, 4D, Supplemental Fig.
1A, 1C). Previous reports have clearly demonstrated that TNF-a
induces NF-kB activation, which is essential for COX-2 upregulation (36–38). Therefore, we examined whether anti–PD-L1
Ab–induced TNF-a is involved in the observed PGE2 upregulation. Treatment with bovine rTNF-a signiﬁcantly induced both
the COX2 expression and PGE2 production in bovine PBMCs (Fig.
4E, 4F). Interestingly, the blockade of TNF-a using the decoy
receptor TNFRII-Ig (35) reduced PGE2 production in the presence of anti–PD-L1 Ab (Fig. 4G). Collectively, these results suggestthat TNF-a induced by PD-L1 blockade upregulates PGE2
production, contributing to the impaired eﬃcacy of anti–PD-L1
Ab treatment via the PGE2/EP4 signaling.
Th1 cytokine production is enhanced by the dual blockade of
PD-L1 and EP4
To examine whether the inhibition of EP4 enhances the eﬃcacy
of anti–PD-L1 Ab in vitro, bovine PBMCs were cultured withindividual EP antagonists in the presence of anti–PD-L1 Ab(Boch4G12). As shown in Fig. 5A, the dual blockade of PD-L1 andEP4 increased IFN-g production compared with other treatment847

groups. Additionally, the dual blockade of PD-L1 and EP4
signiﬁcantly enhanced the BLV-speciﬁc IFN-g production from
PBMCs of BLV-infected cattle (Fig. 5B). Taken together, theseresults suggest that combination with an EP4 antagonist might be anovel strategy to enhance the eﬃcacy of anti–PD-L1 Ab treatment
in cattle.

Antitumor effects are enhanced by the dual blockade of
PD-L1 and EP4
Our studies in bovine immune cells revealed the novel mechanismof anti–PD-L1 Ab resistance and the potential of enhancing Th1
cytokine production by the dual blockade of PD-L1 and EP4. We
then used murine splenocytes to examine whether the dual
blockade enhances Th1 immune responses in other animal models.
As shown in Fig. 6A and 6B, anti–PD-L1 Ab induced PGE2
production from murine splenocytes stimulated with or without
Con A (Fig. 6A, 6B). Additionally, treatment with the EP4antagonist increased IL-2 production from murine splenocytes
in the presence of anti–PD-L1 Ab (10F.9G2) (Fig. 6C), suggestingthat the dual blockade enhanced Th1 responses not only in cattle
but also in mice. Finally, based on these results, we used a mouse
lymphoma model to evaluate the potential antitumor eﬀects of thedual blockade as a potent immunotherapy in cancers resistant toanti–PD-1/PD-L1 Ab alone. C57BL/6 mice were inoculated with alymphoma cell line, EG7, and the EG7-bearing mice wereadministered anti–PD-L1 Ab (10F.9G2) i.p. and the EP4 antagonist
orally (Fig. 6D). Compared with the animals treated with the EP4antagonist or the anti–PD-L1 Ab alone, the growth of EG7 cells wasinhibited in those administered the combination treatment (Fig.
6E). Additionally, the survival of the combination treatment groupwas signiﬁcantly prolonged compared with that of the untreatedgroup (Fig. 6F). Taken together, these data suggest that the dual
blockade of PD-L1 and EP4 is a promising strategy as a novel
immunotherapy.

DISCUSSION
Numerous studies have recently elucidated the mechanisms ofresistance to cancer immunotherapy (39–41). For instance,
Koyama et al. (39) demonstrated that therapeutic PD-1 blockade
upregulated the expression of alternative immune checkpoint
molecules, which caused resistance to the PD-1/PD-L1 blockade.
Treatments including anti–PD-1/PD-L1 Abs in combination withother medicines to overcome resistance are garnering increasing
attention. Previous studies have shown that combination treatment with anti–PD-1/PD-L1 Abs with COX inhibitors enhances
the therapeutic eﬃcacy in murine and bovine models (12, 32).
However, the mechanisms underlying the observed therapeutic

Data are presented as means, and the error bars indicate SEs. Statistical significance was determined by the Tukey test. (F) The Kaplan–Meier Curvefor survival in all groups. Statistical significance was determined by the log-rank test. (D–F) Data are representative of two independent experiments,
each performed with five to eight mice per group.

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EP4 agonist decreased the percentage of IFN-g+ cells in both theCD4+ and CD8+ cell populations (Fig. 2D, 2E, Supplemental Fig. 1A,
1B). Furthermore, to examine whether PGE2 directly suppresses
the activity of bovine T cells, isolated CD3+ T cells were cultured
with the EP4 agonist, and the expression levels of CD69, anactivation marker, in these cells and IFN-g production in culture
supernatants were assayed by ﬂow cytometry and ELISA,
respectively. Treatment with the EP4 agonist signiﬁcantly reduced
the CD69 expression levels in CD4+ and CD8+ cells and IFN-g
production (Fig. 3A–C, Supplemental Fig. 1C, 1D). Microarray
analysis revealed that the EP4 agonist treatment downregulated
the expression of Th1-related cytokine genes, such as IL-2, IFN-g,
TNF-a, and IL-12, in CD4+ cells (Fig. 3D, Table II). Taken together,
these data suggest that PGE2 induced by PD-L1 blockade directly
inhibits T cell activation via the EP4 signaling.

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eﬀect of these combination approaches remain unclear. In thecurrent study, we identify a novel mechanism of resistance related
to PGE2 using a bovine model (Supplemental Fig. 2). It was
revealed that the anti–PD-L1 Ab treatment induced the productionof Th1 cytokines, such as TNF-a, and that TNF-a–induced PGE2
suppressed the activation of T cells via EP4. This might partiallyexplain a reason that the combined treatment enhances theeﬃcacy of anti–PD-1/PD-L1 Abs. In addition, our study clearly
showed the therapeutic potential of combination treatment withanti–PD-L1 Abs and EP4 antagonists in bovine and murine models.
To the best of our knowledge, this is the ﬁrst study to demonstrate
the therapeutic eﬃcacy of a dual blockade strategy using ananti–PD-L1 Ab and an EP4 antagonist in an in vivo model. Futurestudies in other murine tumor models are warranted to further
investigate the eﬃcacy of the dual blockade.
Among the four PGE2 receptors EP1–4 (20), EP2 and EP4 are
involved in PGE2-associated immune dysfunction (21). In thecurrent study, the blockade of EP4, but not EP2, inhibited the
suppression of IFN-g production by PGE2. EP4 is a high-aﬃnity
receptor for PGE2, whereas EP2 requires signiﬁcantly higher
PGE2 concentrations for eﬀective signaling (21). Thus, theobserved diﬀerences in the results following the blockade of EP2
and EP4 might be due to the diﬀerence in the aﬃnity of each
receptor. The contribution of EP2 to immunosuppression shouldcarefully be investigated in other preclinical models in whichhigher PGE2 levels are expected during disease progression.
Anti–PD-1/PD-L1 Abs reactivate exhausted T cells, leading to
the production of Th1 cytokines, such as IFN-g and TNF-a (5, 6).
TNF-a not only plays a critical role in cellular immunity against
cancer, but also has a direct cytotoxic eﬀect on tumor cells by
inducing apoptosis (42, 43). Although known as an antitumor
cytokine, TNF-a paradoxically promotes tumor progression insome circumstances (44–46). For example, serum TNF-a concentration is correlated with the progression of several cancer
types, such as renal cell carcinoma and prostate cancer (47, 48).
Additionally, the blockade of TNF-a using Abs inhibits tumor
growth (49). Furthermore, recent studies have shown that theblockade of TNF-a improves the eﬃcacy of PD-1 blockade (50, 51);
however, the underlying detailed mechanism has not been fully
elucidated. In the current study, we demonstrated that TNF-a wasinvolved in PGE2 upregulation under anti–PD-L1 Ab treatment,
and that the dual blockade of PD-1/PD-L1 and EP4 enhanced theeﬃcacy of immunotherapy. Our strategy might be more eﬀective
than the dual blockade of PD-1/PD-L1 and TNF-a because theantitumor eﬀects of TNF-a are not inhibited. Further studies arenecessary to compare the eﬃcacy between the two strategies.
PGE2/EP4 signaling increases cAMP production (52). One
study has previously shown that the PGE2/EP4/cAMP upregulates the expression of T cell Ig and mucin domain-3 (TIM-3), an
immunoinhibitory molecule, in a human T cell line (53). Additionally, several reports have investigated that TIM-3 expression is
induced after the PD-1/PD-L1 blockade, leading to the resistanceto PD-1/PD-L1 blockade (39, 50). Therefore, PGE2 upregulation
after the PD-1/PD-L1 blockade might also contribute to resistance
via the upregulation of other immunoinhibitory molecules.

PD-L1 blockade–TNF-a–PGE2–EP4 axis is a novel mechanismof resistance to anti–PD-1/PD-L1 immunotherapy. A novel
combined approach targeting PD-L1 and EP4 as immunotherapy
may overcome resistance to the anti–PD-1/PD-L1 Ab therapy.
Further studies using diﬀerent murine tumor models as well asother animal models will herald new avenues for cancer treatment.
DISCLOSURESThe authors have no ﬁnancial conﬂicts of interest.

We thank Dr. Hideyuki Takahashi, Dr. Yasuyuki Mori, and Dr. Tomio Ibayashi
for valuable advice and discussions. We thank Enago (www.enago.jp) for the
English language review.

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ACKNOWLEDGMENTS

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