MICROBIAL IMMUNITY AND VACCINES

Prostaglandin E2-Induced Immune Suppression via Cytotoxic
T-Lymphocyte Antigen 4 in Paratuberculosis
Yamato Sajiki,a Satoru Konnai,a,b Kei Watari,a Tomohiro Okagawa,b Akina Tanaka,a Satoko Kawaji,c Reiko Nagata,cNaoya Maekawa,b Yasuhiko Suzuki,b,d Yukinari Kato,e,f Shiro Murata,a,b Yasuyuki Mori,c Kazuhiko Ohashia,bDepartment of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japana

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

Bacterial and Parasitic Disease Research Division, National Institute of Animal Health, Tsukuba, Japan
Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japand

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

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, JapanfParatuberculosis is a chronic enteritis of ruminants caused by the facultative intracellular pathogen Mycobacterium avium subsp. paratuberculosis. The Th1
response inhibits the proliferation of M. avium subsp. paratuberculosis during theearly subclinical stage. However, we have previously shown that immune inhibitory
molecules, such as prostaglandin E2 (PGE2), suppress M. avium subsp. paratuberculosis-speciﬁc Th1 responses as the disease progresses. To date, the mechanism underlying immunosuppression during M. avium subsp. paratuberculosis infection has notbeen elucidated. Therefore, in the present study, we investigated the function of cytotoxic T-lymphocyte antigen 4 (CTLA-4) expressed by peripheral blood mononuclear
cells (PBMCs) from cattle with paratuberculosis because CTLA-4 expression is known
to be elevated in T cells under an M. avium subsp. paratuberculosis experimental
infection. M. avium subsp. paratuberculosis antigen induced CTLA-4 expression in T
cells from cattle experimentally infected with M. avium subsp. paratuberculosis.
Interestingly, both PGE2 and an E prostanoid 4 agonist also induced CTLA-4 expression in T cells. In addition, a functional assay with a bovine CTLA-4-immunogobulin
fusion protein (CTLA-4-Ig) indicated that CTLA-4 inhibited gamma interferon (IFN-g)
production in M. avium subsp. paratuberculosis-stimulated PBMCs, while blockade byanti-bovine CTLA-4 monoclonal antibody increased the secretion of IFN-g and tumor
necrosis factor alpha production in these PBMCs. These preliminary ﬁndings showthat PGE2 has immunosuppressive effects via CTLA-4 to M. avium subsp. paratuberculosis. Therefore, it is necessary to clarify in the future whether CTLA-4-mediatedimmunosuppression facilitates disease progression of paratuberculosis in cattle.
KEYWORDS CTLA-4, PGE2, EP4, Johne’s disease, cattleJ

ohne’s disease, which is also known as paratuberculosis, is a chronic enteric diseaseof ruminants caused by the facultative intracellular pathogen Mycobacterium avium
subsp. paratuberculosis (1). Johne’s disease is common worldwide; indeed, no country
or region is reported to be free of M. avium subsp. paratuberculosis (2). During the earlystage of infection, the production of Th1 cytokines, such as gamma interferon (IFN-g),
is strongly induced by the pathogenic bacteria, which is killed by macrophages
activated by IFN-g (3–5). In contrast, M. avium subsp. paratuberculosis-speciﬁc Th1
responses are gradually suppressed during late subclinical and clinical stages (6–8).
Our previous studies revealed one mechanism of this suppression, which was observed
as the disease progresses (9–11). Programmed death 1 (PD-1) and PD-ligand 1 (PD-L1)
are immunoinhibitory molecules that contribute to T-cell dysfunction, i.e., exhaustion,
Month YYYY Volume XX Issue XX

Editor Sabine Ehrt, Weill Cornell Medical
College
Copyright © 2022 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Satoru Konnai,
konnai@vetmed.hokudai.ac.jp.
The authors declare no conﬂict of interest.
Received 19 May 2022
Returned for modiﬁcation 22 July 2022
Accepted 19 August 2022

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ABSTRACTInfection and Immunity

in humans and several animals, including cattle (12, 13). We showed previously thatthe expression levels of PD-1 and PD-L1 are upregulated in cattle infected with M.
avium subsp. paratuberculosis (9). In addition, we found that blockade of the PD-1/
PD-L1 pathway using anti-bovine PD-L1 antibody activates M. avium subsp. paratuberculosis-speciﬁc Th1 responses both in vitro and in vivo (10, 11). In addition, we demonstrated that prostaglandin E2 (PGE2) was associated with the suppression of Th1
responses in M. avium subsp. paratuberculosis-infected animals (10). In cattle, PGE2 induces PD-L1 expression in bovine immune cells (14) and suppresses Th1 responses via its
receptor, E prostanoid 4 (EP4) (10, 15). Serum PGE2 concentrations are upregulated in cattle with Johne’s disease, and treatment with a COX-2 inhibitor, which blocks PGE2 production, activates M. avium subsp. paratuberculosis-speciﬁc Th1 responses in vitro (10).
Collectively, these results suggest that PGE2 is involved in the progression of Johne’s disease. However, the mechanisms underlying the suppression of Th1 responses have yet
to be fully elucidated.
Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152, is an
immunoinhibitory molecule that is expressed in T cells and an essential negative regulator of T-cell responses (16). CTLA-4 binds to CD80 (B7-1) and CD86 (B7-2), which are
expressed in antigen-presenting cells, and suppresses host immunity (17, 18). In
humans, CTLA-4 has been associated with the progression of chronic diseases causedby human immunodeﬁciency virus (HIV) and hepatitis C virus (19, 20). In the veterinaryﬁeld, CTLA-4 has been associated with the progression of bovine leukemia virus (BLV)
infection (a bovine chronic infection); speciﬁcally, CTLA-4 expression in T cells isincreased in BLV-infected cattle, and the blockade of CTLA-4 using anti-bovine CTLA-4
monoclonal antibody (MAb) activates BLV-speciﬁc Th1 responses in vitro (21–23). In
addition, the involvement of CTLA-4 in cattle infected with M. avium subsp. paratuberculosis has been shown using anti-human CTLA-4 MAb (24). However, the involvementof CTLA-4 in M. avium subsp. paratuberculosis-infected cattle has yet to be investigatedusing anti-bovine CTLA-4 MAb.
Therefore, in the present study, we analyzed CTLA-4 expression in T cells in M.
avium subsp. paratuberculosis-infected cattle using anti-bovine CTLA-4 MAb, which
was established in our previous study (23). In addition, we investigated PGE2/EP4 signaling, ﬁnding that it was involved in CTLA-4 upregulation in T cells from cattle.
Furthermore, we established a bovine CTLA-4-immunogobulin fusion protein (CTLA-4Ig), using it and anti-bovine CTLA-4 MAb to examine whether CTLA-4 suppresses Th1
responses to antigens of M. avium subsp. paratuberculosis.
RESULTS
Expression analysis of CTLA-4 in M. avium subsp. paratuberculosis-infected cattle. In our previous studies, we demonstrated that CTLA-4 is associated with disease progression in BLV-infected cattle (21, 22). Here, we performed expression analysis of CTLA-4
in cattle infected with M. avium subsp. paratuberculosis using anti-bovine CTLA-4 MAb(4C2-D9). The gating strategy and representative plots of CTLA-4 expression analysis areshown in Fig. S1 in the supplemental material. Compared to cattle not infected with M.
avium subsp. paratuberculosis, CTLA-4 expression in CD81 T cells but not CD41 T cells was
signiﬁcantly increased in M. avium subsp. paratuberculosis-infected cattle (Fig. 1). Peripheral
blood mononuclear cells (PBMCs) from M. avium subsp. paratuberculosis-infected cattle
were cultivated with or without J-PPD, and ﬂow cytometric analysis revealed that stimulation with J-PPD signiﬁcantly upregulated CTLA-4 expression in both CD41 and CD81 T cells(Fig. 2). Thus, CTLA-4 expression is apparently upregulated by antigen stimulation in vitro
in M. avium subsp. paratuberculosis-infected cattle.
Effects of PGE2 on CTLA-4 upregulation. We previously showed that PGE2 production from the PBMCs of M. avium subsp. paratuberculosis-infected cattle was increasedby J-PPD stimulation (10). In the present study, we identiﬁed the major source of PGE2
production under antigen stimulation. PBMCs and CD141 cell-deleted PBMCs from M.
avium subsp. paratuberculosis-infected cattle were cultured with or without J-PPD, andPGE2 concentrations in culture supernatants were measured using enzyme-linked
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immunosorbent assay (ELISA). PGE2 production was signiﬁcantly induced from PBMCs
following J-PPD treatment in vitro, whereas PGE2 production was not induced from
CD141 cell-deleted PBMCs (see Fig. S2). Therefore, in M. avium subsp. paratuberculosisinfected cattle, CD141 cells could be a major source of PGE2 production under antigen
stimulation. To determine whether CTLA-4 expression is induced by PGE2 signaling,
PBMCs from uninfected cattle were cultivated with PGE2 or an EP4 agonist, and theexpression of CTLA-4 was measured using ﬂow cytometry. Treatment with PGE2 tended
to induce CTLA-4 expression in CD41 and CD81 T cells (Fig. 3a and b), and treatment
with the EP4 agonist signiﬁcantly promoted CTLA-4 expression in both CD41 andCD81 cells (Fig. 3a and b). In further tests, CD31 cells were sorted from uninfected cattle and cultured with PGE2 or the EP4 agonist. The gene expression of CTLA-4 in CD31
cells was signiﬁcantly increased by culture with PGE2 and the EP4 agonist (Fig. 3c and

FIG 2 Upregulation of CTLA-4 expression through stimulation with J-PPD. PBMCs from cattle experimentally infected with
M. avium subsp. paratuberculosis (n = 6) were cultured with J-PPD, and the expression of CTLA-4 in CD41 T cells (a) andCD81 T cells (b) was assayed using ﬂow cytometry. N.S., no stimulation; J-PPD, Johnin-puriﬁed protein derivative. P values
were obtained using Mann-Whitney U tests.
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FIG 1 Expression analysis of CTLA-4 in T cells from M. avium subsp. paratuberculosis-infected cattle. The
expression of CTLA-4 in CD41 T cells (CD31 IgM2 CD41) (a) and CD81 T cells (CD31 IgM2 CD81) (b) was analyzed
using ﬂow cytometry (uninfected [cattle not infected with M. avium subsp. paratuberculosis], n = 10; infected [M.
avium subsp. paratuberculosis-infected cattle], n = 10; n.s., not signiﬁcant [the P value was obtained using MannWhitney U test]).

Infection and Immunity

FIG 3 Upregulation of CTLA-4 expression via treatment with PGE2 or an EP4 agonist. (a and b) First, PBMCs of cattle not infected with M. avium subsp.
paratuberculosis were cultured with PGE2 or the EP4 agonist, and dimethyl sulfoxide (DMSO) was used as a vehicle control. Subsequently, CTLA-4
expression in CD41 T cells (a) and CD81 T cells (b) was measured by using ﬂow cytometry (n = 10; P values were obtained using Mann-Whitney U tests). (cand d) CD31 cells were sorted from the PBMCs of cattle not infected with M. avium subsp. paratuberculosis cattle and cultured with PGE2 (c) or the EP4
agonist (d), and DMSO was used as a vehicle control. The gene expression of CTLA-4 was quantitated using qPCR (n = 8; P values were obtained using
Steel-Dwass tests).

d). Collectively, these data suggest that J-PPD-induced PGE2 is involved in the upregulation of CTLA-4 expression, at least in part, via its receptor EP4.
The PGE2/EP4 pathway is known to stimulate the production of intracellular cyclic
AMP (cAMP) (25). In the present study, we examined whether the upregulation of intracellular cAMP is involved in upregulation of CTLA-4 expression in cattle. Treatment withdibutyryl cAMP (dbcAMP), a cell-permeable analog of cAMP that activates cAMP-dependent protein kinases, signiﬁcantly induced the gene expression of CTLA-4 in T cells (Fig.
4a). In addition, ﬂow cytometric analysis revealed that treatment with dbcAMP upregulated CTLA-4 expression in both CD41 and CD81 T cells (Fig. 4b and c), suggesting that
CTLA-4 expression is induced by the PGE2/EP4/cAMP pathway in cattle.
Establishment and functional analysis of CTLA-4-Ig. To investigate whether CTLA4 is involved in the suppression of M. avium subsp. paratuberculosis-speciﬁc immune
responses, we generated an Fc-fusion protein of bovine CTLA-4, namely, CTLA-4-Ig,
which was expressed using a pDC62c5-U533 expression vector and CHO-DG44 cell
expression system in vitro. After puriﬁcation, SDS-PAGE was performed under nonreducing (Fig. 5a) and reducing (Fig. 5b) conditions. Flow cytometric analysis revealed
that CTLA-4-Ig bound to both bovine CD80 and CD86 (Fig. 5c and d). In addition, ﬂow
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cytometric analysis showed that CTLA-4-Ig inhibited the binding of CD80-Ig or CD86-Ig
to CD28-expressing cells on the cell surface (Fig. 5e and f). We also used CTLA-4-Ig toassess the involvement of CTLA-4 in the suppression of M. avium subsp. paratuberculosis-speciﬁc immune responses. PBMCs from M. avium subsp. paratuberculosis-infected
cattle were cultured with CTLA-4-Ig in the presence of J-PPD, and CTLA-4-Ig treatment
signiﬁcantly downregulated the production of IFN-g from PBMCs (Fig. 6). Therefore,
CTLA-4 may be associated with the immune suppression in M. avium subsp. paratuberculosis-infected cattle.
Activation of J-PPD-stimulated immunity via CTLA-4 blockade. As described previously, the blockade of CTLA-4 activates immune responses in BLV-infected cattle invitro (22, 23). To examine the effects of CTLA-4 blockade on immune responses in M.
avium subsp. paratuberculosis-infected cattle, PBMCs derived from the infected animals
were cultured with anti-bovine CTLA-4 MAb (4C2-D9) (23) in the presence of J-PPD.
The production of Th1 cytokines, i.e., IFN-g and tumor necrosis factor alpha (TNF-a),
was signiﬁcantly increased in the group treated with anti-bovine CTLA-4 MAb (Fig. 7).
DISCUSSION
In the present study, as well as a previous study (10), we have shown that antigen
stimulation activates PGE2 production from the PBMCs of M. avium subsp. paratuberculosis-infected cattle. Furthermore, we showed here that PGE2 upregulated CTLA-4
expression in T cells via the EP4/cAMP pathway (see Fig. S3). cAMP is known to play a
critical role in upregulating CTLA-4 expression in T cells (26) and has been associatedwith the progression of chronic infections, such as HIV (27). However, information waspreviously lacking on the relationship between antigen stimulation and cAMP-induced
CTLA-4; thus, the present study provides new insights in this regard. CTLA-4 expression
in T cells is upregulated in many other chronic infections, including HIV (19), hepatitis
C virus (20), and BLV (21, 22) infections. Therefore, further studies will be needed toinvestigate whether antigen stimulation is involved in CTLA-4 expression via the PGE2/
EP4/cAMP pathway in bovine chronic infections. In the present study, M. avium subsp.
paratuberculosis-experimentally infected cattle showed high expression of CTLA-4 but
did not exhibit clear clinical signs because they might be in the latent period. The
detailed involvement of CTLA-4 in the process leading to the development of granulomas in Johne’s disease should also continue to be investigated.
CTLA-4 is expressed in regulatory T cells (Tregs) and is known as a Treg-associated molecule (28, 29). In M. avium subsp. paratuberculosis-infected cattle, Tregs have been associatedwith immune responses to antigen stimulation (30). However, the mechanism underlying
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FIG 4 Upregulation of CTLA-4 expression via treatment with dbcAMP. (a) CD31 cells were sorted from the PBMCs of cattle not infected with M. avium
subsp. paratuberculosis (n = 6) and cultured with dbcAMP. Double-distilled water (DDW) was used as a vehicle control. Gene expression of CTLA-4 wasquantitated using qPCR. (b and c) PBMCs of cattle not infected with M. avium subsp. paratuberculosis (n = 7) were cultured with dbcAMP, DDW was used
as a vehicle control, and CTLA-4 expression in CD41 T cells (b) and CD81 T cells (c) was assayed by using ﬂow cytometry. All P values were obtained using
Mann-Whitney U tests.

Infection and Immunity

FIG 5 Establishment and functional analyses of CTLA-4-Ig. (a and b) Puriﬁed CTLA-4-Ig was conﬁrmed via SDS-PAGE under
nonreducing (a) and reducing (b) conditions. (c and d) Binding of CTLA-4-Ig to bovine CD80 (c) and bovine CD86 (d) was determine
using ﬂow cytometry. (e and f) CTLA-4-Ig inhibited the binding of bovine CD80 (e) or bovine CD86 (f) to bovine CD28. 2ME, 2mercaptoethanol; isotype: puriﬁed bovine IgG1; control-Ig, puriﬁed rabbit IgG.

Treg proliferation in M. avium subsp. paratuberculosis-infected cattle is still unclear. Our previous studies showed that PGE2 concentrations in sera are increased in infected cattle andthat PGE2 induces the expression of the Treg-associated molecules Foxp3 and TGF-b 1 in cattle (10, 31). Thus, in M. avium subsp. paratuberculosis-infected cattle, antigen stimulationinduced PGE2 might contribute to increasing the Treg population via its receptor EP4.
The cell-mediated immunity plays an essential role in controlling the progression ofJohne’s disease. However, M. avium subsp. paratuberculosis-speciﬁc immune responses,
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especially IFN-g production, are gradually suppressed in M. avium subsp. paratuberculosis-infected cattle, contributing to progression to the clinical stage (6–8, 32). In thepresent study, we found that CTLA-4-Ig treatment reduced IFN-g production from the
PBMCs of M. avium subsp. paratuberculosis-infected cattle. In addition, anti-bovine
CTLA-4 MAb increased anti-bacterial cytokine productions in vitro. These data suggestthat CTLA-4 is involved in the suppression of anti-bacterial responses in M. avium
subsp. paratuberculosis-infected cattle.
On farms, calves become infected with M. avium subsp. paratuberculosis mainly via
the fecal-oral route (1). Therefore, M. avium subsp. paratuberculosis-infected cattle thatexhibit bacterial fecal shedding can be considered a major infection source. Our previous study demonstrated that treatment with anti-bovine PD-L1 antibody signiﬁcantly
activated Th1 responses in M. avium subsp. paratuberculosis-infected cattle in vivo,
although the inhibitory effect of anti-PD-L1 antibody alone on bacterial shedding was
limited (11). Thus, to reduce bacterial load, the development of a novel control methodthat induces potent antibacterial responses is desirable. The upregulation of PGE2 is

FIG 7 Activation of Th1 responses via treatment with anti-CTLA-4 MAb. PBMCs from M. avium subsp.
paratuberculosis-infected cattle (n = 8) were cultured with anti-CTLA-4 MAb in the presence of J-PPD, and theconcentrations of IFN-g (a) and TNF-a (b) in culture supernatants were measured using ELISA. P values wereobtained using Mann-Whitney U tests. Control-Ig, mouse IgG1 isotype control.
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FIG 6 Suppression of IFN-g production via CTLA-4-Ig. PBMCs from M. avium subsp. paratuberculosisinfected cattle (n = 6) were cultured with CTLA-4-Ig or Control-Ig (39) in the presence of J-PPD. IFN-g
production was measured using ELISA. P values were obtained using Steel-Dwass tests.

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one possible mechanism underlying the inhibition of anti-bacterial responses during
PD-1 blockade (15). Our previous study revealed that PGE2 concentrations in sera wereincreased in BLV-infected cattle after treatment with anti-bovine PD-L1 antibody (15).
In the present study, we revealed the role played by PGE2 in the upregulation of CTLA4 expression in cattle. Therefore, CTLA-4 levels might increase after PD-1 blockade, andCTLA-4 could be a target molecule for inducing potent immune responses. Indeed, incancer research, the dual blockade of PD-1/PD-L1 and CTLA-4 using speciﬁc antibodies
enhances therapeutic effects compared to the effects achieved using single blockadeof PD-1/PD-L1 or CTLA-4 (33). Moreover, combined treatment with anti-PD-1 antibody
and anti-CTLA-4 antibody leads to potent antitumor effects in patients with advanced
melanoma (34, 35). In our previous study, we found that the dual blockade of PD-L1
and CTLA-4 enhances antiviral cytokine production from the PBMCs of BLV-infected
cattle in vitro (23). Therefore, combination therapy involving anti-PD-1 and CTLA-4 antibodies could be a novel control strategy for Johne’s disease. Future studies should
investigate the synergistic effects of dual blockade in cattle infected with M. avium
subsp. paratuberculosis.

Animals and blood samples. Blood samples of cattle (adults of the Holstein breed) not infectedwith M. avium subsp. paratuberculosis, which were tested for speciﬁc antibodies against M. avium subsp.
paratuberculosis using a commercial ELISA test kit (Kyoritsu Seiyaku Corporation, Tokyo, Japan), wereobtained at the Field Science Center for Northern Biosphere, Hokkaido University and from dairy farmers
in Hokkaido. Blood samples of cattle experimentally infected with M. avium subsp. paratuberculosis wereobtained from the National Institute of Animal Health, Tsukuba, Japan. For the experimental infection,
Holstein calves 1 to 4 weeks of age were inoculated orally with intestinal tissue homogenate (6.8  106
CFU) from M. avium subsp. paratuberculosis-infected cattle once daily for 20 days (10) or once with ahigh concentration of homogenate (1.36 or 2.50  108 CFU), as described previously (11). All infected
cattle were kept in a biosafety level II animal facility at this institute and did not show any clinical symptoms. All experiments using bovine blood samples were approved by the National Institute of Animal
Health Ethics Committee (approval numbers 15-001, 18-004, and 18-077) and/or the local committee foranimal studies at Hokkaido University (approval number 17-0024).
Cell preparation and culture. PBMCs from blood samples were puriﬁed using density gradient centrifugation on Percoll (GE Healthcare, Little Chalfont, UK) as described previously (36). CD31 and CD141
cells were isolated from PBMCs using an autoMACS Pro (Miltenyi Biotec, Bergisch Gladbach, Germany) as
described previously (15, 31). All cell cultures were grown in 96-well plates (Corning, Inc., Corning, NY)
using RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) including 10% heat-inactivated fetal calf serum(Thermo Fisher Scientiﬁc, Waltham, MA), 100 U/mL of penicillin (Thermo Fisher Scientiﬁc), 100 m g/mL of
streptomycin (Thermo Fisher Scientiﬁc), and 2 mM L-glutamine (Thermo Fisher Scientiﬁc).
Flow cytometry. To analyze CTLA-4 expression, ﬂow cytometric analysis was performed using
PBMCs from cattle infected or not infected with M. avium subsp. paratuberculosis. To prevent nonspeciﬁcreactions, Fc blocking was performed by incubating PBMCs with phosphate-buffered saline (PBS) including 10% goat serum (Thermo Fisher Scientiﬁc) for 15 min at 25°C. The cells were then stained with antibovine CTLA-4 MAb (4C2-D9) (23) or mouse IgG1 isotype control (15H6; Southern Biotech, Birmingham,AL) for 20 min at 37°C. After being washed twice with PBS containing 1% bovine serum albumin (BSA;
Sigma-Aldrich), the cells were stained with Alexa Fluor 647-conjugated anti-mouse IgG(H1L) F(ab9)2(Invitrogen, Carlsbad, CA) for 20 min at 25°C. They were then washed twice with PBS containing 1% BSA
and stained with the following antibodies for 20 min at 25°C: PerCP/Cy 5.5-conjugated anti-bovine CD3
MAb (MM1A; Washington State University Monoclonal Antibody Center, Pullman, WA), FITC-conjugatedanti-bovine CD4 MAb (CC8; Bio-Rad, Hercules, CA), PE-conjugated anti-bovine CD8 MAb (CC63; Bio-Rad),and PE/Cy7-conjugated anti-bovine IgM MAb (IL-A30; Bio-Rad). MM1A and IL-A30 were conjugatedusing Lightning-Link antibody labeling kits (Innova Biosciences, Cambridge, England). After the ﬁnal
staining, the cells were washed twice with PBS containing 1% BSA and analyzed immediately using
FACSVerse (BD Biosciences, San Jose, CA, USA).
Enzyme-linked immunosorbent assay. To measure the concentrations of IFN-g, TNF-a, and PGE2 inculture supernatants, ELISAs were performed using a bovine IFN-g ELISA development kit (Mabtech,
Nacka Strand, Sweden), a bovine TNF-a Do-It-Yourself ELISA (Kingﬁsher Biotech), and a PGE2 Express
ELISA kit (Cayman Chemical, Ann Arbor, MI), respectively.
Quantitative PCR. RNA extraction and cDNA synthesis were performed in accordance with methods
reported in a previous study (31). The gene expression of CTLA-4 was quantitated using a thermal cycler(LightCycler 480 System 2; Roche Diagnostic, Mannheim, Germany) and SYBR Premix DimerEraser(TaKaRa Bio, Otsu, Japan) according to the manufacturer’s instructions. b -actin (ACTB) was used as a reference gene. Relative expression levels were calculated using the DDCt method, and the results wererepresented as changes relative to expression levels in the untreated group. The primers used were as
follows: 59-CCA GAG TCA TGG GAC TTG GT-39 and 59-TCA CAT GAG AAG CTG GCA AC-39 for CTLA-4 and
59-TCT TCC AGC CTT CCT TCC TG-39 and 59-ACC GTG TTG GCG TAG AGG TC-39 for ACTB.
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MATERIALS AND METHODSInfection and Immunity

Establishment of CTLA-4-Ig. To examine the effects of CTLA-4 on M. avium subsp. paratuberculosisspeciﬁc Th1 responses, we generated bovine CTLA-4-Ig using Chinese hamster ovary (CHO)-DG44 cells.
The amino acid sequence of CTLA-4-Ig consisted of an extracellular domain fragment of bovine CTLA-4
and bovine IgG1, triggering reduced Fc-mediated effector functions (ADCC2) (37). A plasmid vector
encoding CTLA-4-Ig (pUCIDT-Kan-CTLA-4-Ig) was digested using AscI and AsiSI (both New England
Biolabs, Ipswich, MA), after which the fragment was inserted into pDC62c5-U533 (38) using a DNA ligation kit (Mighty Mix; TaKaRa Bio). The vector (pDC62c5-U533-CTLA-4-Ig) was then transfected into CHODG44 cells using Lipofectamine LTX (Life Technologies, Carlsbad, CA). Transfectants (5  105 cells/mL)
were shaken for 14 days on a rotary shaker at 125 rpm and 37°C under 8% CO2 using CD OptiCHO
Medium (Thermo Fisher Scientiﬁc). Subsequently, the supernatants were collected and ﬁltered using
Centricon Plus-70 (Merck Millipore, Billerica, MA), and CTLA-4-Ig was puriﬁed using Ab-Capture ExTra(ProteNova, Tokushima, Japan) according to the manufacturer’s protocol. After puriﬁcation, SDS-PAGE
was performed using a 12% SDS-polyacrylamide gel under reducing and nonreducing conditions. The
gel was stained using a Quick-CBB kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), andPrecision Plus Protein All Blue prestained protein standards (Bio-Rad) were used as a marker.
Functional analysis of CTLA-4 Ig. To conﬁrm the binding of CTLA-4-Ig to bovine CD80 and CD86,
Cos-7 cells expressing bovine CD80 or CD86 (22) were incubated with 10 m g/mL of CTLA-4-Ig for 30 min
at 25°C. Puriﬁed bovine IgG1 (10 m g/mL; Bethyl Laboratories, Montgomery, TX) was used as a negative
control. After being washed, the cells were stained with Alexa Fluor 647-conjugated AfﬁniPure goat antibovine IgG (H1L) (Jackson ImmunoResearch Laboratories, West Grove, PA) for 20 min at 25°C and analyzed immediately using FACSVerse. Competitive and blocking assays were also performed to conﬁrm
whether CTLA-4-Ig inhibits interactions with CD28 and CD80 or CD86. CTLA-4-Ig was incubated with
CD80-Ig or CD86-Ig (22) for 30 min at 25°C. Puriﬁed rabbit IgG (Southern Biotech) was used as a negative
control of CTLA-4-Ig. Cos-7 cells expressing bovine CD28 were then incubated with a reaction mixture of
Fc-fusion proteins for 30 min at 25°C. After incubation, the cells were stained with Alexa Fluor 647-conjugated anti-rabbit IgG(H1L) (Thermo Fisher Scientiﬁc) and analyzed immediately using FACSVerse.
Cell cultures. PBMCs or CD141 cell-depleted PBMCs from cattle experimentally infected with M. avium
subsp. paratuberculosis were incubated with 1 m g/mL of Johnin-puriﬁed protein derivative (J-PPD). After 24 h,
CTLA-4 expression in T cells was analyzed using ﬂow cytometry as described above. After 72 h, PGE2 concentrations in culture supernatants were measured using ELISA.
To determine whether the PGE2/EP4/cAMP pathway upregulates CTLA-4 expression, PBMCs from cattle
not infected with M. avium subsp. paratuberculosis were cultured with 2.5 m M PGE2 (Cayman Chemical),1 m g/mL of Rivenprost (an EP4 agonist; Cayman Chemical), or 400 m M dbcAMP (an analog of cAMP; SigmaAldrich). After 24 h, the cells were collected, and CTLA-4 expression was analyzed as described above.
Puriﬁed CD31 cells of cattle not infected with M. avium subsp. paratuberculosis were cultured with 250
nM PGE2, 1 m g/mL of Rivenprost, or 400 m M dbcAMP. After 24 h, the gene expression of CTLA-4 was measured using quantitative PCR (qPCR).
To examine the effects of CTLA-4-Ig on Th1 responses, PBMCs of M. avium subsp. paratuberculosisinfected cattle were cultured with 10 or 1 m M CTLA-4-Ig or with Control-Ig (39) in the presence of 1 m g/mLof J-PPD. After 24 h, IFN-g concentrations in culture supernatants were measured using ELISA.
To determine whether anti-bovine CTLA-4 MAb activates M. avium subsp. paratuberculosis-speciﬁc
Th1 responses, PBMCs from M. avium subsp. paratuberculosis-infected cattle were cultivated with 10 m g/
mL of anti-bovine CTLA-4 MAb (4C2-D9) in the presence of 1 m g/mL of J-PPD. After 120 h, the concentrations of IFN-g and TNF-a in culture supernatants were measured using ELISA.
Statistical analysis. Statistical signiﬁcance was determined using Mann-Whitney U tests and SteelDwass tests. MEPHAS (http://www.gen-info.osaka-u.ac.jp/MEPHAS/) was used to conduct statistical analyses. Statistical signiﬁcance was set at P , 0.05.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF ﬁle, 0.6 MB.
ACKNOWLEDGMENTS
We are grateful to Hideyuki Takahashi and Tomio Ibayashi for valuable advice anddiscussions. We thank Enago (http://www.enago.jp) for the English language review.
This study was supported by JSPS KAKENHI grant 22H02503 (to S.K.); grants from theProject of the NARO, Bio-oriented Technology Research Advancement Institution (Research
Program on Development of Innovative Technology grant 26058 BC [to S.K.] and Special
Scheme Project on Regional Developing Strategy grant 16817557 [to S.K.]); regulatory
research projects for food safety, animal health, and plant protection (JPJ008617.17935709)
funded by the Ministry of Agriculture, Forestry, and Fisheries of Japan; and AMED under
grants JP20am0101078 and JP22ama121008 (to Y.K.). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
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