The Journal of Immunology

Yamato Sajiki,* Satoru Konnai,*,† Tomohiro Okagawa,† Asami Nishimori,*
Naoya Maekawa,† Shinya Goto,* Kei Watari,* Erina Minato,‡ Atsushi Kobayashi,‡
Junko Kohara,x Shinji Yamada,{ Mika K. Kaneko,{ Yukinari Kato,{ Hirofumi Takahashi,‖Nobuhiro Terasaki,‖ Akira Takeda,‖ Keiichi Yamamoto,†,# Mikihiro Toda,†,**
Yasuhiko Suzuki,†,††,‡‡ Shiro Murata,*,† and Kazuhiko Ohashi*,†
Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our
previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes
to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on theassociation of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV
infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression
by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2
concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by
PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes todisease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses
in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo.
Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV
proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number ofanimals are required to support the efficacy of this treatment for clinical application. The Journal of Immunology, 2019, 203:
1313–1324.
rostaglandin E2 is an inflammatory mediator produced bycyclooxygenase (COX) enzymes (COX-1 and COX-2)
from arachidonic acid (1). COX-1 is a constitutive enzyme expressed in many tissues and involves in a multitude ofphysiological processes (2), whereas COX-2 is an inducible enzyme
regulated by inflammatory cytokines via the activation of NF-kB(3). The level of PGE2 is regulated not only by its synthesis but also
by its degradation. 15-HydroxyPG dehydrogenase (15-PGDH),
encoded by the HPGD gene, is a key enzyme for PGE2 inactivation(4, 5). PGE2 suppresses the activity of immune cells, such as T cellsP(especially Th1 cells), NK cells, and dendritic cells, via EP2 andEP4 receptors, thereby contributing to immune evasion during
chronic infection (6, 7). By contrast, by reducing PGE2 synthesis,
COX-2 inhibitors activate immune responses in vitro and in vivo
(8, 9). Thus, COX-2 inhibition is considered a potential immunotherapy for chronic infections.
Bovine leukemia virus (BLV), which is a member of the genus
of Deltaretrovirus, causes enzootic bovine leukosis (EBL) and isclosely related to human T cell leukemia virus type 1 (10). Thisvirus generally infects B cells of cattle, and most BLV-infected*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
Veterinary Clinical Medicine, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060-0818, Japan; xAnimal Research Center, Agriculture Research Department, Hokkaido Research Organization, Shintoku 081-0038, Japan; {Departmentof Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan; ‖Shibecha Branch, Kushiro Central Office, Hokkaido
Higashi Agricultural Mutual Aid Association, Shibecha 088-2311, Japan; #Research and Development Center, Fuso Pharmaceutical Industries, Ltd., Osaka
536-8523, Japan; **New Business and International Business Development, Fuso
Pharmaceutical Industries, Ltd., Osaka 536-8523, Japan; ††Division of Bioresources,
Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0019,
Japan; and ‡‡Global Station for Zoonosis Control, Global Institution for Collaborative
Research and Education, Hokkaido University, Sapporo 001-0019, Japan

Disease Prevention Technologies to Combat Antimicrobial Resistance 2017–2021
FY, and by grants from the Project of the NARO, 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, datacollection and analysis, decision to publish, or preparation of the manuscript.

ORCIDs: 0000-0002-1391-9732 (T.O.); 0000-0003-3936-7515 (A.N.); 0000-00020066-8655 (E.M.); 0000-0001-5385-8201 (Y.K.); 0000-0002-1313-1494 (Y. Suzuki).

Address correspondence and reprint requests to Dr. Satoru Konnai, Faculty of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo, Hokkaido
060-0818, Japan. E-mail address: konnai@vetmed.hokudai.ac.jp
The online version of this article contains supplemental material.
Abbreviations used in this article: AL, aleukemic; BLV, bovine leukemia virus; chAb,
chimeric Ab; COX, cyclooxygenase; CRE, cAMP-response element; EBL, enzootic
bovine leukosis; FLK, fetal lamb kidney, LAG-3, lymphocyte activation gene 3; PD-1,
programmed death 1; PD-L1, programmed death-ligand 1; PL, persistent lymphocytosis; qPCR, quantitative real-time PCR.

Received for publication April 1, 2019. Accepted for publication June 28, 2019.

This article is distributed under The American Association of Immunologists, Inc.,
Reuse Terms and Conditions for Author Choice articles.

This work was supported by Grants-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science, by Research Project for Improving Animal

Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900342

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Prostaglandin E2–Induced Immune Exhaustion andEnhancement of Antiviral Effects by Anti–PD-L1 Antibody
Combined with COX-2 Inhibitor in Bovine Leukemia
Virus Infection

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Materials and Methods
Cell preparation
Blood samples of BLV-infected and uninfected cattle were obtained fromseveral farmers and veterinarians in Hokkaido, Japan. Infection was confirmed by detection of the provirus using nested PCR targeting the virallong terminal repeat (16) and by detection of the anti-BLV Ab using acommercial ELISA (JNC, Tokyo, Japan). The leukocyte numbers in BLVinfected cattle were counted using a Celltac a MEK-6450 automatic
hematology analyzer (Nihon Kohden, Tokyo, Japan), and animals wereclassified as AL or PL as described previously (24). PBMCs derived fromthese blood samples were purified by density gradient centrifugation on
Percoll (GE Healthcare, Buckinghamshire, U.K.) and cultured in RPMI 1640
(Sigma-Aldrich, St. Louis, MO) supplemented with 10% heat-inactivated
FCS (Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin, 100
mg/ml streptomycin, and 2 mM L-glutamine (Thermo Fisher Scientific). All
cell cultures were grown in 96-well plates (Corning, Corning, NY) in 200 mlof medium.
As described previously with slight modifications (18, 21), CD14+ andCD21+ cells were freshly isolated from bovine PBMCs using BD IMagnet

Cell Separation System (BD Biosciences, San Jose, CA) or autoMACS Pro(Miltenyi Biotec, Bergisch Gladbach, Germany), and the following Abs:anti-bovine CD14 mAb (CAM36A; Washington State University mAb
Center, Pullman, WA) and anti-bovine CD21 mAb (GB25A; Washington
State University mAb Center). CD14 was used as a marker of monocytes
and CD21 was used as that of B cells. The purity of each cell population
was confirmed using FACSVerse (BD Biosciences). Only highly purified
cells (.90%) were used for expression analysis by quantitative real-time
PCR (qPCR) as described below.

qPCR
Total RNA was extracted from total PBMCs and each subpopulation(CD14+ and CD21+) using TRIzol reagent (Thermo Fisher Scientific)
according to the manufacturer’s instructions. cDNA was synthesized fromthe total RNA using PrimeScript Reverse Transcriptase (Takara Bio,
Otsu, Japan) according to the manufacturer’s instructions. To measure themRNA expression levels of COX2, EP4, EP2 (25), HPGD (26), and IFN-g
in total PBMCs and each subpopulation, qPCR was performed using athermal cycler (LightCycler 480 System II; Roche Diagnostic, Mannheim,
Germany) with SYBR Premix DimerEraser (Takara Bio), following the
manufacturer’s instructions. Internal control genes were b-actin (ACTB)
and GAPDH. All primer sequences for PCR are listed in Table I. To examine whether the expression levels of viral genes gp51, G4, and R3 areincreased by PGE2 signaling, the PBMCs of BLV-infected cattle wereincubated in the presence of PGE2 (1 mM; Cayman Chemical, Ann Arbor,
MI), EP2 agonist [1 mg/ml, Butaprost (free acid), Cayman Chemical] or
EP4 agonist (1 mg/ml, Rivenprost; Cayman Chemical) at 37˚C under 5%CO2 for 3 d and collected, and the expression levels were quantified by
qPCR as described above. To measure the proviral loads of samples from
BLV-infected cattle, qPCR was performed as previously described, withslight modifications (27). Briefly, genomic DNA was extracted from the
PBMCs of BLV-infected cattle using the Wizard Genomic DNA Purification
Kit (Promega, Madison, WI). The DNA concentration was measured by UV
absorbance at 260 nm using a NanoDrop 8000 Spectrophotometer (Thermo
Fisher Scientific). Amplification was conducted in a reaction mixture containing 5 ml of 23 Cycleave PCR Reaction Mix SP (Takara Bio), 0.5 ml ofProbe/Primer Mix for BLV (Takara Bio), 1 ml of template DNA, and 3.5 ml
of RNase-free distilled water (Takara Bio) using a LightCycler 480 System
II. The PCR condition was 95˚C for 10 s, followed by amplification of thetemplate for 55 cycles at 95˚C for 5 s and 64˚C for 30 s. Serial dilutions of
BLV Positive Control (Takara Bio) were used to generate calibration curves
to determine the BLV provirus copy number. Each DNA sample was tested
in triplicate, and the reported values are the mean number of copies per 50 ng
of genomic DNA.

Quantitation of PGE2 by ELISA
The concentration of PGE2 was measured in plasma or serum samples from
BLV-infected and uninfected cattle using the PGE2 Express ELISA Kit(Cayman Chemical) as described previously (22). To assess whether theBLV Ag promotes PGE2 production, PBMCs (4 3 105 cells per well) from
infected and uninfected cattle were incubated with the BLV Ag (fetal lambkidney[FLK]–BLV; 2% heat-inactivated culture supernatant of FLK–BLV
cells) at 37˚C under 5% CO2 for 6 d. Culture supernatants were collected,
and the concentration of PGE2 was measured by ELISA. To compare PGE2
production, the total PBMCs, CD14+ cells and CD21+ cells (5 3 105 cells
per well) were incubated separately at 37˚C under 5% CO2 for 3 d, and theculture supernatant was collected for measurement of PGE2 concentration
by ELISA as described above.

Immunohistochemical assay of PD-L1 and PGE2
Immunohistochemical assays were performed as previously described, withmodifications (22). Briefly, sections of superficial cervical lymph nodes fromhealthy BLV-uninfected cattle (Holstein, female, 75-mo old) and periocularmass lesions from EBL cattle (Holstein, female, 24-mo old) were subjected
to immunohistochemical staining for PGE2 and PD-L1 using an anti-PGE2
polyclonal Ab (ab2318; Abcam, Cambridge, U.K.) and anti–PD-L1 mAb(6C11-3A11, Rat IgG2a; 22). Cattle exhibiting tumors were diagnosed with
EBL as described previously (28, 29). Additionally, the tumor mass lesion
was confirmed as B cell lymphoma by immunohistochemical staining withan anti-CD20 polyclonal Ab (PA5-16701; Thermo Fisher Scientific; data notshown).

In vitro analysis of immunostimulation by COX-2 inhibition
To evaluate the immunostimulatory effects of COX-2 inhibition, PBMCs(4 3 105 cells per well) from BLV-infected cattle were labeled with 0.2 mM
CFSE and incubated with 1 mM meloxicam (Sigma-Aldrich) or DMSO as a

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cattle are asymptomatic carriers of the virus or aleukemic (AL).
However, ∼30% of the infected cattle show persistent lymphocytosis (PL), characterized by nonmalignant polyclonal
expansion of infected B cells in peripheral blood. Less than 5%of the infected cattle develop EBL, characterized by fatal lymphoma or lymphosarcoma after extended latency periods of 5–10 y(11). BLV infection is highly prevalent in many countries, including
Japan, and several reports have demonstrated that the seroprevalence of BLV in Japanese cattle is increasing because of the lack ofan effective treatment or vaccine (12). Thus, the development of a
new control method for BLV infection is required to guarantee thecontinued supply of livestock and livestock production.
The Th1 response is critical for the control of BLV infection.
During BLV infection, suppression of both CD4+ T cell proliferation and cytotoxic immune response against BLV Ags is associated with disease progression (13, 14). To develop a novel
strategy for effective control of BLV infection, our recent studies
have examined the detailed mechanisms of immune dysfunctionin BLV infection. Upregulation of immunoinhibitory molecules,
such as programmed death 1 (PD-1), programmed death-ligand 1(PD-L1), lymphocyte activation gene 3 (LAG-3), T cell Ig domain and mucin domain-3 (Tim-3), and CTL Ag-4 (CTLA-4),
was shown to suppress BLV-specific Th1 responses and promotedisease progression (15–19). Alternatively, Ab-mediated blockade of the PD-1/PD-L1 pathway significantly reduced the proviral load in BLV-infected cattle (20, 21), suggesting that therapy
targeting immunoinhibitory molecules, such as PD-1 and PD-L1,
may be a novel control strategy against BLV infection. However,
it is still unknown how these molecules are upregulated during
BLV infection.
Our previous report has shown that PGE2 inhibits the Th1 response and induces the expression of PD-L1 in cattle (22). In addition, we showed that PGE2 is associated with the progression ofJohne disease, a bovine chronic infection caused by Mycobacterium
avium subsp. paratuberculosis, and that a COX-2 inhibitor canactivate Th1 responses in cattle with Johne disease. Furthermore,
the combined treatment with an anti–PD-L1 Ab and a COX-2
inhibitor strongly enhanced M. avium subsp. paratuberculosis–
specific Th1 responses in vitro (22).
However, few studies are available on the association of PGE2
with BLV infection (23). Therefore, in this study, we analyzed the
kinetics of PGE2 in BLV infection, the association of PGE2 withdisease progression, and the antiviral effects of a COX-2 inhibitor
in vitro and in vivo. Finally, we also tested whether a combined
blockade of the PGE2 and PD-1/PD-L1 pathways in vitro andin vivo can enhance antiviral effects during BLV infection.

PGE2-INDUCED IMMUNE EXHAUSTION IN BLV INFECTION

The Journal of Immunology
vehicle control (Nacalai Tesque, Kyoto, Japan) at 37˚C under 5% CO2 for 6 din the presence of FLK–BLV. After incubation, the concentrations of IFN-g
and TNF-a in the medium were measured by ELISA, and cell proliferation
was analyzed by FACS based on CFSE staining strength as described
previously (22).

In vitro analysis of immunostimulation by combined COX-2
inhibition and PD-L1 blockade

Evaluation of COX-2 inhibitor administration for reducing
viral load in BLV-infected cattle
To examine the antiviral efficacy of COX-2 inhibition in vivo, three BLVinfected cattle (Table II) were administered 0.5 mg/kg meloxicam(Metacam; Boehringer Ingelheim, Ingelheim, Germany) by s.c. injection
once weekly for a total of nine doses (animals #1 and #2) or a total of fivedoses (animal #3). Animals #1–3 were launched for this experiment andkept separately at the three different locations: in a private dairy farm
(Shibecha, Hokkaido, Japan), a biosafety level I animal facility at the
Animal Research Center, Agricultural Research Department, Hokkaido
Research Organization (Shintoku, Hokkaido, Japan), and an animal
facility at the Faculty of Veterinary Medicine, Hokkaido University(Sapporo, Hokkaido, Japan). These animal experiments were approvedby the Ethics Committee of the Animal Research Center, Agricultural
Research Department, Hokkaido Research Organization, and the Ethics
Committee of the Faculty of Veterinary Medicine, Hokkaido University.
Peripheral blood samples were collected from these cattle at least once
per week after first administration.

Inoculation of the anti–PD-L1 chAb in BLV-infected cattle
To examine the antiviral efficacy of anti–PD-L1 chAb (Boch4G12; 21)
in vivo, two cattle naturally infected with BLV (Table II) were inoculated i.v. with 1 mg/kg purified Boch4G12 (animal #5) or saline (as acontrol, animal #4). These animals were kept in a private dairy farm in
Hokkaido. Peripheral blood samples were collected from both animals
at least once per week after inoculation for measurement of proviralload as described.

Evaluation of combined COX-2 inhibition and PD-L1 blockade
for reducing viral load in infected cattle
To investigate the in vivo antiviral effects of the combined treatmentcompared with monotreatment of anti–PD-L1 chAb, two BLV-infected
cattle (animals #3 and #6) were inoculated with 1 mg/kg Boch4G12 (21),and animal #3 was also coadministered 0.5 mg/kg meloxicam (Metacam)
by s.c. injection three times at weekly intervals (Table II). These animals
were kept in an animal facility at the Faculty of Veterinary Medicine,
Hokkaido University (Sapporo, Hokkaido, Japan). This animal experiment was approved by the Ethics Committee of the Faculty of Veterinary
Medicine, Hokkaido University. Animal #3 was also used for the clinicalstudy of meloxicam treatment alone and inoculated with Boch4G12 after
a long interval (118 d) from the final administration of meloxicam. Peripheral blood samples were then collected from these cattle at least once
per week after the inoculation of Boch4G12, and proviral loads were
evaluated using qPCR as described.

Statistics
Differences were identified using the Dunnett test, Wilcoxon signed-rank
test, and Steel–Dwass test. Correlation was analyzed using the Spearman
correlation. A p value ,0.05 was considered statistically significant. In
clinical tests, we performed the quantitation of proviral load by qPCR in
triplicate. Using the value obtained from the experiments, we performed
statistical analysis using a Dunnett test to test whether the proviral load ateach point was significantly different from that at day 0.

Results
PGE2 concentration changes with disease progression in
BLV-infected cattle
Our previous report demonstrated an association between blood
PGE2 concentration and the progression of Johne disease, achronic bacterial infection of cattle (22). Next, to investigate
whether this immune dysfunction is specific to Johne disease orcommonly occurred in bovine chronic infections, we focused on achronic viral infection of cattle, BLV infection, which is known toshow T cell dysfunction during the disease progression (14–16).
To examine whether PGE2 is also associated with the progressionof BLV infection, the plasma PGE2 concentration was comparedamong uninfected, AL, and PL cattle by ELISA. The plasma
concentration of PGE2 was significantly higher in BLV-infected
cattle compared with BLV-uninfected cattle (Fig. 1A). In addition,
the plasma concentration of PGE2 was positively correlated with thenumber of lymphocytes and BLV proviral load (Fig. 1B–D). Plasma
PGE2 concentration was also positively correlated with PD-L1 expression in IgM+ B cells (Fig. 1E). Thus, PGE2 production appears
to increase with disease progression.
To identify the major cell type producing PGE2 in the blood of
BLV-infected cattle, the mRNA expression levels of COX2 were
measured in total PBMCs, isolated CD21+ cells, and isolated
CD14+ cells using qPCR (Table I). Consistent with plasma
PGE2 measurements, COX2 was significantly upregulated in PL
cattle compared with AL and uninfected cattle (Fig. 1F–H). The
production and release of PGE2 were significantly higher in
CD14 + cell cultures than CD21 + cell cultures from infected
cattle (Fig. 1I), suggesting that CD14 + cells are the predominant source of PGE2 in peripheral blood. Immunohistochemical analysis also revealed strong expression of PGE2 andPD-L1 in B cell lymphoma tissues of EBL cattle but very weak
expression in the healthy lymph nodes of BLV-uninfected cattle(Supplemental Fig. 1).
Finally, we measured the mRNA expression of EP4, EP2, and
HPGD in PBMCs from BLV-infected and -uninfected cattle(Table I), and found that the expression of EP4 and EP2 wasupregulated in PL cattle (Fig. 2A, 2B). By contrast, the mRNA
expression of the PGE2 catabolic enzyme gene HPGD was
downregulated in PL cattle and was negatively associated with both
BLV proviral load and serum PGE2 concentration (Fig. 2C–E).
Taken together, PGE2 production and signaling are positively associated with disease progression in BLV-infected cattle. We thenexamined whether elevated PGE2 actually drives disease progression by immune inhibition, particularly suppression of Th1
cell responses, thereby allowing for increased viral replicationand gene expression.
Upregulation of PGE2 production by BLV Ag
The capacity of BLV to evoke PGE2 production was examined
in PBMC cultures from BLV-infected and uninfected cattle using
the viral Ag FLK–BLV. FLK–BLV exposure for 6 d significantly
upregulated PGE2 production by PBMCs from infected AL and
PL cattle (Fig. 3B, 3C) but not by PBMCs from uninfected cattle(Fig. 3A). This induced PGE2 production was inhibited by the
COX-2 inhibitor meloxicam (Fig. 3B, 3C). Collectively, theseresults showed that the COX-2/PGE2 pathway was activated by
the stimulation of BLV Ag in BLV-infected cattle.
Upregulation of gp51, G4, and R3 viral genes by PGE2
A previous report demonstrated that PGE2 can induce the expression of BLV viral genes such as tax (23). To examine PGE2induced viral expression in PBMCs, cells from BLV-infected
cattle were cultured in the presence of PGE2 for 3 d. Indeed, theDownloaded from http://www.jimmunol.org/ at Nankodo Co Ltd Tohoku Univ/Digital Info Sec/Lib/Sendai/Japan on September 13, 2019

To examine the immunostimulatory effects of combined COX-2 inhibition
plus anti–PD-L1 Abs treatment in vitro, PBMCs (4 3 105 cells per well)from BLV-infected cattle were CFSE labeled and incubated with 1 mM
meloxicam and 10 mg/ml anti–PD-L1 Ab in the presence of FLK–BLV for
6 d. DMSO, rat IgG (Sigma-Aldrich), or bovine IgG (Sigma-Aldrich) wasused as negative control. Heat-inactivated culture supernatant of FLK cells
was used as a negative control Ag. Two forms of anti–PD-L1 Ab, namely
anti–PD-L1 mAb (4G12) with the C region of rat IgG2a and Igk (30) andanti–PD-L1 chimeric Ab (chAb; Boch4G12) with the C region of bovine
IgG1 (mutated to reduce Ab-dependent cellular toxicity), and Igl (21)were used to evaluate the combination effects. After 6 d, the concentrationof IFN-g was measured by ELISA, and cell proliferation was analyzed by
FACS analysis of CFSE labeling as described above.

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PGE2-INDUCED IMMUNE EXHAUSTION IN BLV INFECTION

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FIGURE 1. Kinetic analysis of PGE2 in cattle infected with BLV. (A) Plasma PGE2 concentration in cattle uninfected (n = 6) or infected with BLV(AL, n = 7; PL, n = 6) were determined using ELISA. (B–E) Positive correlation between the lymphocyte number (n = 13), proviral load (C: Japanese black
cattle and first filial of Japanese black/Holstein cattle, n = 95; D: Holstein cattle, n = 30) or the percentages of PD-L1+ cells in IgM+ cells and plasma PGE2
concentration in cattle infected with BLV. (F–H) Quantification of COX2 mRNA expression in PBMCs [BLV (2), n = 7; AL, n = 7; PL, n = 7] andsubpopulations of CD21+ [BLV (2), n = 4; AL, n = 7; PL, n = 6) and CD14+ [BLV (2), n = 4; AL, n = 5; PL, n = 6) cells derived from BLV-uninfected and
BLV-infected cattle by qPCR. (I) Quantitation of PGE2 production in PBMCs, CD21+ cells, and CD14+ cells by ELISA (n = 8). Each symbol represents
PGE2 concentration or relative expression level of each animal; pooled data from several experiments were analyzed by the Steel–Dwass test (A and F–I).
Correlation statistic was analyzed using the Spearman correlation (B–E). BLV (2), BLV-uninfected cattle.

The Journal of Immunology

1317

Table I. Sequences of primers used in this study
Primer Sequence (59-39)

IFN-g

Forward: 59-ATA ACC AGG TCA TTC AAA GG-39
Reverse: 59-ATT CTG ACT TCT CTT CCG CT-39
Forward: 59-GTG ACC ATC GCC ACC TAC TT-39
Reverse: 59-CTC ATC GCA CAG ATG ATG CT-39
Forward: 59-CTC TGC TGT CGG GTT TCA TTA-39
Reverse: 59-CTA CCC TCC TCA AAG GTC AAT C-39
Forward: 59-GAA TCT CGA AGC AGG TGT CA-39
Reverse: 59-CCA GCT TTC CAA AGT GGT CT-39
Forward: 59-ACG TTT TCT CGT GAA GCC CT-39
Reverse: 59-TCT ACC AGA AGG GCG GGA TA-39
Forward: 59-ACC TTT CTG TGC CAA GTC-39
Reverse: 59-ATC GGG GCT CGC AAT CAT A-39
Forward: 59-TTC GGC GCC CAG CCA CAT C-39
Reverse: 59-GTC GTT ATC AGG TAA TGG ATC CCG A-39
Forward: 59-GAT CAT CAG ATG GGT CCT GAT GAA C-39
Reverse: 59-GCT GCT GGA TGT GGC TGG AAT GTC-39
Forward: 59-CAC AAC CTG AGC CTG CAC AA-39
Reverse: 59-TCT TGC GGA ACT CAA ACT CAT C-39
Forward: 59-CTG CTG AGG CTC AAG TTA AAA GTG-39
Reverse: 59-CAG CCG GTT GCT GAG GTA G-39
Forward: 59-TCT TCC AGC CTT CCT TCC TG-39
Reverse: 59-ACC GTG TTG GCG TAG AGG TC-39
Forward: 59-GGC GTG AAC CAC GAG AAG TAT AA-39
Reverse: 59-CCC TCC ACG ATG CCA AAG T-39

EP4
EP2
HPGD
COX2
gp51
G4
R3
Foxp3
TGF-b1ACTB
GAPDHmRNA expression levels of env (gp51), G4, and R3 were facilitatedby PGE2 as measured by qPCR (Fig. 4A–C, Table I). To investigate
the transduction mechanism, PBMCs of BLV-infected cattle were
cultured with EP2 or EP4 agonist, both of which induced the expression of these three viral genes (Fig. 4D–F). Thus, the expressionof BLV viral genes is regulated by PGE2 via its endogenous receptors EP2 and EP4.
Activation of BLV-specific Th1 responses by the COX-2
inhibitor meloxicam in vitro
In humans, COX-2 inhibitors activate immune responses in vitro
and in vivo (8, 9), and we previously reported that meloxicam
activates Th1 responses of cattle in vitro (22). To examine whether
meloxicam activates the BLV-specific Th1 responses, PBMCs from
BLV-infected cattle were cultured in the presence of FLK–BLV
plus meloxicam or vehicle, and T cell proliferation was measured
according to CFSE labeling (low is indicative of proliferation) byflow cytometry and Th1 cytokine production by ELISA (Fig. 5).
The gating strategy and representative plots for CFSE staining areshown in Supplemental Fig. 2. The proliferation rates of both CD4+
and CD8+ cell populations were upregulated by meloxicam relative
to vehicle (Fig. 5A, 5B), and the production levels of both IFN-g
and TNF-a were enhanced by meloxicam (Fig. 5C, 5D), consistent
with the activation of BLV-specific Th1 responses.
Activation of BLV-specific T cell responses in vitro bycombined COX-2 inhibition and anti–PD-L1 Ab treatment
We then evaluated the immunomodulatory effects of combined
COX-2 inhibition plus anti–PD-L1 Ab treatment on the BLVspecific Th1 cell response in vitro. The proliferation rates of
CD4+ and CD8+ cells were significantly enhanced by combination
treatment with meloxicam and anti–PD-L1 mAb (4G12) in thepresence of FLK–BLV compared with the other treatment groups(Fig. 5E, 5F). The production of IFN-g was also upregulated by
combined treatment in the presence of FLK–BLV (Fig. 5G), andthe stimulation of FLK, a negative control Ag, had no effect ineither group (Fig. 5E–G). In addition, combined treatment withmeloxicam and anti–PD-L1 chAb Boch4G12 significantly increased the proliferation of CD4+ and CD8+ cells and the production of IFN-g compared with the other treatment groups(Fig. 5H–J). Collectively, these results demonstrate enhancement of BLV-specific Th1 responses by combined COX2 inhibitor
and anti–PD-L1 Ab treatment in vitro.
Antiviral effects of COX-2 inhibition in BLV-infected cattle
Three cattle naturally infected with BLV (animal #1–3) were administered 0.5 mg/kg meloxicam by s.c. injection once a week
(Table II), and BLV proviral load was measured periodically from
PBMCs. Consistent with antiviral efficacy, the proviral load fellsignificantly during the observation periods (Fig. 6A–C). To confirm
whether Th1 cell responses were activated by the administration of
meloxicam, the mRNA expression levels of IFN-g were measured
in PBMCs at several times points (days 0, 1, 7, 21, and 56) by
qPCR (Table I). Expression was significantly higher on days
7, 21, and 56 compared with day 0 (Fig. 6D), indicating that
COX-2 inhibitor administration induced sustained Th1-mediatedantiviral effects in these BLV-infected cattle.
In vivo antiviral effects of anti–PD-L1 chAb,
Boch4G12 inoculation
Our previous report clearly demonstrated the antiviral effects ofanti–PD-L1 chAb Boch4G12 in experimentally BLV-infectedcalf (21). In the current study, we examined the antiviral effects
of anti–PD-L1 chAb Boch4G12 in naturally infected cattle(Table II). Animal #4 was inoculated with saline (as a negative
control) and animal #5 was inoculated with 1 mg/kg Boch4G12,
and peripheral blood samples were then collected periodicallyto measure the BLV proviral load in PBMCs. In animal #5, theproviral load decreased significantly after inoculation, and theviral load in animal #4 continued to rise (Fig. 6E, 6F). Therefore,
Boch4G12 has antiviral effects on naturally infected BLV cattlein vivo.
Antiviral efficacy of combined COX-2 inhibitor and anti–
PD-L1 Ab treatment
Finally, we examined the antiviral efficacy of combined treatmentcompared with Boch4G12 treatment alone in two BLV-infected cattle(animals #3 and #6, Table II) with high proviral load (.2000 copies/
50 ng DNA). Peripheral blood samples were collected from thesecattle at least once per week for the measurement of BLV proviral

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Gene

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PGE2-INDUCED IMMUNE EXHAUSTION IN BLV INFECTION

load following treatment. Proviral load was not reduced in animal#6 by Boch4G12 treatment, but animal #3 exhibited significantly
reduced viral load (nearly 80% at day 14) following combined
treatment (Fig. 6G, 6H). Therefore, these findings demonstrate thepotential of combined COX-2 inhibition plus anti–PD-L1 treatment
as a new control strategy of BLV infection.

Discussion
In the advanced stages of BLV infection, BLV-specific Th1 responses are downregulated, leading to further disease progression
and possible EBL (31). Several previous studies have demonstrated that loss of BLV-specific Th1 responses is mediated byupregulation of immunoinhibitory molecules such as PD-1 andPD-L1, as well as by expansion of regulatory T cells (15, 16, 24).
Similarly, our previous study revealed that PGE2 suppresses Th1
responses, such as T cell proliferation and Th1 cytokine production, and induces the expression of PD-L1 (22). In addition, theexpression levels of TGF-b1 and Foxp3, a key cytokine andtranscription factor for the development of regulatory T cells,
were increased in cultured bovine PBMCs by PGE2 treatment(Supplemental Fig. 3). In the current study, we found that the
production of PGE2 increased in parallel with the progression of
BLV infection. The expression of EP4 and EP2, PGE2 receptors
associated with immune dysfunction (32), was also upregulated
in the late stage of BLV infection, and the catabolic enzyme

15-PGDH was downregulated. Thus, PGE2 signaling may bemarkedly enhanced in late stage BLV infection, which, combinedwith the induction of PD-L1 and regulatory T cells via EP4 andEP2, would dramatically reduce BLV-specific Th1 responses.
Monocytes are a major source of PGE2 production (33). In thisstudy, we identified the major cell type producing PGE2 in peripheral blood of BLV-infected cattle by the measurement of
COX2 expression and PGE2 production in CD21+ cells and CD14+
cells. As shown in Fig. 1, COX2 expression was elevated in total
PBMCs, CD21+ cells, and CD14+ cells of PL cattle compared withAL and uninfected cattle. The production capacity of PGE2 washigher in CD14+ cells than in CD21+ cells, suggesting that CD14+
cells are the main source of PGE2. However, in the late stage of
BLV infection, the number of circulating B lymphocytes is dramatically increased (34), and our immunohistochemical staining
results clearly revealed PGE2 production by tumor B cells in cattlewith EBL. Thus, B cells may also contribute to or act as the main
source of PGE2 production in the late stage of BLV infection.
In humans, transcription of COX-2 is induced by inflammatory
cytokines, TLR signaling, and Ag stimulation via NF-kB (35, 36).
In the current study, stimulation of PBMCs from BLV-infected
cattle with BLV Ag induced PGE2 production. Generally, in the
PBMC culture of the infected animals, BLV-specific CD4+ T cells
mainly secrete Th1 cytokines, including TNF-a, by FLK–BLV
stimulation. TNF-a is a key cytokine to induce the COX2 expression

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FIGURE 2. Quantification of EP4, EP2, and HPGD mRNA expression in cattle infected with BLV. (A–C) Quantification of EP4 [BLV (2), n = 7; AL,n = 7; PL, n = 7), EP2 [BLV (2), n = 6; AL, n = 6; PL, n = 7), and HPGD [BLV (2), n = 4; AL, n = 6; PL, n = 6) mRNA expression in PBMCs of BLVuninfected and BLV-infected cattle by qPCR. (D and E) Negative correlation between proviral load or the serum concentration of PGE2 and HPGD expression in PBMCs of cattle infected with BLV (n = 12). Each symbol represents relative expression level of each animal; pooled data from several
experiments were analyzed by the Steel–Dwass test (A–C). Correlation statistic was analyzed using the Spearman correlation (D and E). BLV (2), BLVuninfected cattle.

The Journal of Immunology

1319

via NF-kB (36), suggesting that the BLV Ag upregulates COX2
expression via TNF-a–NF-kB pathway in BLV-infected cattle. In
contrast, T cells from uninfected cattle could not respond to BLV Ag
stimulation because of no acquired immunological memory against
BLV. Our previous paper has demonstrated the same phenomenon
in M. avium subsp. paratuberculosis–infected cattle. We observed
the upregulation of PGE2 production and PD-L1 expression by theculture of PBMCs from M. avium subsp. paratuberculosis–infected
cattle in the presence of M. avium subsp. paratuberculosis Ag, but

not by that of PBMCs from uninfected cattle (22). Therefore, the acquired immunity might be necessary for the phenomena. Furthermore,in this study, the expression of HPGD was downregulated in PL cattle
and negatively correlated with the BLV proviral load and serum PGE2
concentration. Taken together, coordinate regulation of increased PGE2
production and decreased PGE2 degradation leads to elevated levels of
PGE2 which, in turn, suppresses BLV-targeted Th1 responses.
The viral gene gp51 encodes an envelope glycoprotein essential forcell-to-cell infection (37–40), and Tax, R3, and G4 are nonstructural

FIGURE 4. Upregulation on the gene expression of gp51, G4, and R3 by PGE2. (A–F) PBMCs from BLV-infected cattle were incubated with PGE2, EP2
agonist, or EP4 agonist, and the gene expression of gp51, G4, and R3 was quantitated by qPCR (A–C, n = 6; D–F, n = 9). Symbols represent mean fromsingle-culture wells of different individuals; pooled data from several experiments were analyzed by the Wilcoxon signed-rank test (A–C) and the Steel–
Dwass test (D–F).

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FIGURE 3. Upregulation on PGE2 production by FLK–BLV stimulation. (A–C) PBMCs of BLV-uninfected (A) and BLV-infected (B, AL; C, PL) cattle
were incubated with FLK–BLV. The concentration of PGE2 in culture supernatants was determined by ELISA (A, n = 5; B, n = 7; C, n = 7). Symbols
represent mean from single-culture wells of different individuals; pooled data from several experiments were analyzed by the Wilcoxon signed-rank test (A)or the Steel–Dwass test (B and C).

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PGE2-INDUCED IMMUNE EXHAUSTION IN BLV INFECTION

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FIGURE 5. Activation of BLV-specific responses by the combination of meloxicam and anti–PD-L1 Ab. (A–D) PBMCs from BLV-infected cattle (A–C,
n = 12; D, n = 8) were cultured with meloxicam in the presence of FLK–BLV. CD4+ (A) and CD8+ (B) cell proliferations were assayed by flow cytometry.
IFN-g (C) and TNF-a (D) productions were determined by ELISA. (E–G) PBMCs from BLV-infected cattle were incubated with meloxicam and anti–PDL1 mAb in the presence of FLK or FLK–BLV. CD4+ (E: FLK, n = 7; FLK–BLV, n = 15) and CD8+ (F: FLK, n = 8; FLK–BLV, n = 24) cell proliferations
were assayed by flow cytometry. IFN-g production was determined by ELISA (G, FLK, n = 7; FLK–BLV, n = 20). (H–J) PBMCs from BLV-infected cattle
were incubated with meloxicam and anti–PD-L1 chAb in the presence of FLK or FLK–BLV (H and I, n = 16; J, n = 18). CD4+ (H) and CD8+ (I) cellproliferations were assayed by flow cytometry. IFN-g production was determined by ELISA (J). Symbols represent mean from single-culture wells ofdifferent individuals; pooled data from several experiments were analyzed by the Wilcoxon signed-rank test (A–D) and the Steel–Dwass test (E–J). Cont Ig,
rat IgG (for E–G), or bovine IgG (for H–J); aPD-L1 chAb, anti–PD-L1 chAb (Boch4G12); aPD-L1 mAb: anti–PD-L1 mAb (4G12).

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Table II. Cattle used in clinical studies

#1

#2

Age
Breed
Sex
Body weight
Inoculation dose
(Boch4G12)
Administration
dosage (Metacam)

43 mo old
Holstein
Female749 kg

96 mo old
Holstein
Female736 kg47 mo old
Holstein
Female759 kg

0.5 mg/kg,
once aweek, totalof ninetimes, s.c.

0.5 mg/kg,
once aweek, totalof ninetimes, s.c.

0.5 mg/kg, oncea week, total of
five times, s.c.

proteins involved in the activation of viral transcription andpropagation (41–44). A previous report demonstrated that PGE2
promotes Tax expression in PBMCs isolated from BLV-infected
cattle (23), and the current study showed that the expression
levels of gp51, G4, and R4 were also upregulated by PGE2 via
EP2 and EP4 signaling in PBMCs isolated from BLV-infected
cattle. Binding of PGE2 to EP2 and EP4 increases intracellularcAMP production (7), which activates protein kinase A (PKA)
and downstream transcription factors that regulate genes withcAMP-response element (CRE) sites in the promoter. The BLV–
long terminal repeat has a CRE that induces transcription of BLV
gene (45, 46), strongly suggesting that PGE2/EP2 and PGE2/EP4
facilitate BLV viral gene transcription through activation of cAMP/
PKA/CRE signaling.
CD8+ cytotoxic T cells activated by Th1 cytokines play a
critical role in the control of BLV infection. COX-2 inhibitors are
known to activate immune responses, including T cell responses(8, 9). Our previous report demonstrated that a selective COX-2
inhibitor, meloxicam, induced Th1 responses in PBMC culturesfrom both healthy cattle and cattle infected with M. avium subsp.
paratuberculosis (22). In this study, meloxicam also activated
BLV-specific Th1 responses in vitro, and administration of Metacam(a veterinary formulation of meloxicam) activated Th1 responses
and significantly decreased proviral load in BLV-infected cattle.
These findings suggest that inhibition of PGE2 using COX-2 inhibitors is a potential treatment strategy against BLV infection.
Indeed, possible benefits of the COX-2 inhibitor celecoxib on the
immune function were reported in patients with HIV infection(9). However, in the veterinary field, the current study is the first
to investigate the therapeutic effects of COX-2 inhibitors against
chronic infections.
In the current study, BLV proviral load was not dramaticallyreduced by treatment with meloxicam alone, probably because of
the upregulation of immunoinhibitory molecules. Proviral load was
extremely high (.2000 copies/50 ng DNA) in the cattle (animals #1–3) receiving meloxicam alone, and the expression of
immunoinhibitory molecules, such as PD-L1, LAG-3, CTLA-4,
Tim-3, and Foxp3, is substantially higher in BLV-infected cattlewith high proviral loads compared with infected cattle with low
proviral loads (16, 18, 19, 47, 48). Additionally, the proportionsof CD4+ and CD8+ cells coexpressing PD-1 and LAG-3 increase in
the late stage of BLV infection (49). Thus, the dual blockade of
PGE2 and other immunoinhibitory molecules may be required toenhance the antiviral effects in advanced BLV infections.
In a murine model of chronic infection, PGE2 signaling via
EP2 and EP4, impaired survival and the function of CD8+ CTLs,
and CTL function was restored, and viral control was improvedby combined blockade of the PGE2 and PD-1/PD-L1 signaling

#4

#5

#6

#3 (Clinical Studyof the Combined
Treatment)

19 mo old
Holstein
Female433 kgSaline 50 ml, i.v.

13 mo old
Holstein
Female295 kg
1 mg/kg, i.v.

76 mo old
Holstein
Female799 kg
1 mg/kg, i.v.

52 mo old
Holstein
Female799 kg
1 mg/kg, i.v.
0.5 mg/kg, once aweek, total ofthree times, s.c.

pathways (50). Furthermore, our previous study showed that
PGE2 regulates PD-L1 expression in cattle and that the combined treatment with a COX-2 inhibitor plus an anti–PD-L1 Ab
improves Th1 responses in Johne disease (22). Therefore, in thisstudy, we evaluated the immunostimulative effects of combination treatment and found significantly enhanced BLV-specificTh1 responses in vitro.
Next, we performed the clinical study using BLV-infected cattlewith high proviral load to evaluate antiviral effects of the treatment.
We also examined the effects of monotherapy and combined
therapy on high proviral load in BLV-infected cattle. Monotherapy
with anti–PD-L1 chAb had no impact on BLV-infected cattle withhigh proviral load (animal #6), but combined treatment of animal#3 with the COX-2 inhibitor plus anti–PD-L1 chAb substantiallyreduced BLV proviral load to below 2000 copies/50 ng DNA fromday 7 to day 49 after the first injection. Previous studies havedemonstrated that BLV proviral load is positively correlated withvertical and horizontal transmission risks, and BLV-infected cattlewith a high proviral load (.2000 copies/50 ng DNA) are themajor source of transmission within a herd (26, 51, 52). Therefore,
our findings suggest that the cotargeting of PGE2 and PD-L1 in themost severely infected animals could be a novel method for herd
BLV infection control. In this study, although the number of theinfected animals used in clinical tests was limited, much attention
was paid to obtaining accurate data from these experiments. First,
environmental influence was removed in the clinical tests of thisstudy as much as possible. The clinical test of the single treatmentof anti–PD-L1 chAb (animals #4 and #5) or the combined treatment (animals #3 and #6) was designed to compare the antiviral effect of the treatments between two BLV-infected animals
and was conducted in the same facility at the same day and time,
respectively. In contrast, three animals (animals #1–3) werelaunched for the clinical test of the COX-2 inhibitor and were kept
at the three different locations in Hokkaido. These animals wereadministered the inhibitor at the same time of day on differentdays using the same injection method by a particular veterinarian.
During all of the clinical tests, the tested animals were kept at“loose housing barn” and fed on equivalent grass, hay, and pellet.
Additionally, all of the tested animals were not milked to preventthe influence of sex hormones and other biological changes duringlactation. Second, all of the blood samples were kept at 4˚C immediately after blood collection and shipped to the laboratory of
Hokkaido University because the clinical tests were conducted atthree totally different locations in Hokkaido. Then, PBMCs wereisolated from the blood samples in the laboratory of Hokkaido
University on the day after the blood collection to prevent nonspecific effects on results of the analysis caused by methods of shippingand storage. Finally, blood samples were collected at least once

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Cattle#3 (Clinical Studyof the COX-2
Inhibitor)

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PGE2-INDUCED IMMUNE EXHAUSTION IN BLV INFECTION

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FIGURE 6. Clinical tests using BLV-infected cattle. (A–C) BLV-infected cattle were administered 0.5 mg/kg of meloxicam, s.c. (D) The expression of
IFN-g at days 0, 1, 7, 28, and 56 was quantitated by qPCR in triplicate. (E and F) BLV-infected cattle were inoculated with 1 mg/kg of the purified anti–PDL1 chAb (Boch4G12, animal #5) or saline (control, animal #4), i.v. (G and H) BLV-infected cattle (animals #3 and #6) were inoculated with 1 mg/kg of thepurified Boch4G12, i.v. In addition, animal #3 was administered 0.5 mg/kg meloxicam once a week (total of three times) s.c. Proviral loads of these cattle
were measured by qPCR in triplicate, and data are presented mean 6 SD (A–C and E–H). Statistical significance was determined by the Dunnett test (A–H).
*p , 0.05 versus day 0.

The Journal of Immunology

Acknowledgments
We are grateful to Dr. Hideyuki Takahashi, Dr. Yasuyuki Mori, andDr. Tomio Ibayashi for valuable advice and discussions. We thank Enago
for the English language review.

Disclosures
K.Y. and M.T. are employed by Fuso Pharmaceutical Industries, Ltd.
Y. Sajiki, S.K., T.O., A.N., N.M., S.G., Y. Suzuki, S.M., and K.O. are authorsof a patent application covering materials and techniques described in thispaper (PCT/JP2018/27041). The other authors have no financial conflicts ofinterest.

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Downloaded from http://www.jimmunol.org/ at Nankodo Co Ltd Tohoku Univ/Digital Info Sec/Lib/Sendai/Japan on September 13, 2019per week, and qPCR was performed for the detection of BLV
proviral load in triplicate to reduce experimental error. The resultof these clinical tests requires confirmation in further clinical trials
with larger numbers of the infected cattle from different herds orfarms. To the best of our knowledge, no previous human or veterinary study has investigated the benefits of combining a COX-2 inhibitor with an Ab targeting immune checkpoint molecules in vivo.
Thus, our findings may provide a novel strategy for control ofchronic retrovirus infection in both humans and livestock.
Recently, several studies have demonstrated an association between PGE2 elevation and upregulated expression of other immunoinhibitory molecules, such as PD-1, in human cancer (53, 54). In
addition, dual blockade of PGE2 and PD-1 improved tumor eradication in a mouse model (55). However, the associations between
PGE2 and other immunoinhibitory molecules are generally unclear.
Further study is warranted to investigate these associations using
bovine chronic infection as a disease model.
Although BLV infection is endemic in many countries (12, 56–59), there are no effective therapies or vaccines. Therefore, a novel
strategy is required for effective BLV infection control. Our current
work is a pilot study to assess the potential efficacies of pharmacological COX-2 inhibition and combined COX-2 and PD-1/PD-L1
inhibition. Treatment with the COX-2 inhibitor meloxicam with orwithout anti–PD-L1 Ab demonstrated an antiviral activity in BLVinfected cattle. Additional experiments using larger numbers of
infected cattle are required to confirm the efficacy of these novel
treatments. Finally, better understanding of the molecular mechanisms underlying immune dysfunction by PGE2 and the associations with other immune dysfunctional pathways is essential for thedevelopment of improved antiviral and anti-bacterial treatments.

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34. Nieto Farias, M. V., F. N. Souza, P. A. Lendez, L. Martı́nez-Cuesta,
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