Developmental and Comparative Immunology 114 (2021) 103847

Contents lists available at ScienceDirect

Developmental and Comparative Immunology
journal homepage: www.elsevier.com/locate/devcompimm

Perspective

A TLR7 agonist activates bovine Th1 response and exerts antiviral activity
against bovine leukemia virus
Yamato Sajiki a, Satoru Konnai a, b, *, Tomohiro Okagawa b, Naoya Maekawa b,
Hayato Nakamura a, Yukinari Kato c, d, Yasuhiko Suzuki b, e, Shiro Murata a, b, Kazuhiko Ohashi a, b
a

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 Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japand
New Industry Creation Hatchery Center, Tohoku University, Sendai, 980-8575, Japane
Division of Bioresources, Research Center for Zoonosis Control, Hokkaido University, Sapporo, 001-0019, Japanbc

A R T I C L E I N F O

A B S T R A C T

Keywords:
Bovine leukemia virus
TLR7 agonist
CD11c+ cell
T cellAnti-PD-L1 antibody
CattleBovine leukemia virus (BLV) infection is a bovine chronic infection caused by BLV, a member of the genus
Deltaretrovirus. In this study, we examined the immunomodulatory effects of GS-9620, a toll-like receptor (TLR) 7
agonist, in cattle (Bos taurus) and its therapeutic potential for treating BLV infection. GS-9620 induced cytokine
production in peripheral blood mononuclear cells (PBMCs) as well as CD80 expression in CD11c+ cells andincreased CD69 and interferon (IFN)-γ expressions in T cells. Removing CD11c+ cells from PBMCs decreased
CD69 expression in T cells in the presence of GS-9620. These results suggest that TLR7 agonism promotes T-cell
activation via CD11c+ cells. Analyses using PBMCs from BLV-infected cattle revealed that TLR7 expression in
CD11c+ cells was upregulated during late-stage BLV infection. Furthermore, GS-9620 increased IFN-γ and TNF-α
production and inhibited syncytium formation in vitro, suggesting that GS-9620 may be used to treat BLV
infection.

1. Introduction

agonists have the potential to treat several chronic infections, such as
hepatitis B virus infection and human immunodeficiency virus infection(Lanford et al., 2013; Menne et al., 2015; Bam et al., 2016; Tsai et al.,2017). In bovine studies, several reports have demonstrated the asso­
ciation between TLR7 and the development of bovine viral infections(Zhang et al., 2006; Marin et al., 2014). Additionally, a previous study
has reported that TLR7 expression is upregulated in the peripheral bloodmononuclear cells (PBMCs) of bovine leukemia virus (BLV)-infected
cattle (Bos taurus), especially among those with high proviral loads(Farias et al., 2016). Therefore, TLR7 agonists have the potential to treat
bovine viral infections. However, in bovine research, the effects of TLR7
agonists on bovine immune cells have not been fully elucidated.
BLV is a member of the genus Deltaretrovirus, and is closely related tohuman T-cell leukemia virus type 1 (Sagata et al., 1985). BLV infects
bovine B cells and causes enzootic bovine leukosis (EBL). Most
BLV-infected cattle are asymptomatic carriers, called aleukemic (AL).
Meanwhile, around 30% of BLV-infected cattle show persistentToll-like receptors (TLRs) are pattern recognition receptors that
stimulate innate immune responses. TLRs enhance the adaptive immune
response by promoting cytokine secretion, dendritic cell (DC) matura­tion, and antigen presentation (Akira and Takeda, 2004). TLR7 recog­
nizes viral single-stranded RNA (Lund et al., 2004), and is
predominantly expressed in the endosomal compartment of DCs and B
cells in humans (Jarrossay et al., 2001; Kadowaki et al., 2001; Ito et al.,2005). Previous papers showed TLR7 agonists induce antiviral effects
via the activation of immune responses (Horscroft et al., 2011). TLR7
agonists promote DC maturation and type I interferon (IFN), such as
IFN-α, production (Gibson et al., 2002; Tsai et al., 2017). The secretionof type I IFNs promotes antiviral defense through interferon-stimulated
genes (Samuel, 2001). Additionally, type I IFNs act as a bridge between
innate and adaptive immunity, activating antiviral effects via the re­sponses of cytotoxic T cells (Schiavoni et al., 2013). Therefore, TLR7

* Corresponding author. Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo, 060-0818, Japan.
E-mail addresses: y.sajiki@vetmed.hokudai.ac.jp (Y. Sajiki), konnai@vetmed.hokudai.ac.jp (S. Konnai), okagawa@vetmed.hokudai.ac.jp (T. Okagawa),
maekawa@vetmed.hokudai.ac.jp (N. Maekawa), btbw-h314@eis.hokudai.ac.jp (H. Nakamura), yukinarikato@med.tohoku.ac.jp (Y. Kato), suzuki@czc.hokudai.
ac.jp (Y. Suzuki), murata@vetmed.hokudai.ac.jp (S. Murata), okazu@vetmed.hokudai.ac.jp (K. Ohashi).
https://doi.org/10.1016/j.dci.2020.103847
Received 5 June 2020; Received in revised form 27 August 2020; Accepted 27 August 2020
Available online 2 September 20200145-305X/© 2020 Elsevier Ltd. All rights reserved.

Y. Sajiki et al.

Developmental and Comparative Immunology 114 (2021) 103847

lymphocytosis (PL), which is characterized by the nonmalignant poly­clonal expansion of infected B cells in the peripheral blood. Between 1%
and 5% of BLV-infected cattle develop EBL, which is characterized byfatal lymphoma or lymphosarcoma after a long latent period (Schwartz
and Levy, 1994). The prevalence of BLV infection is high in many
countries including Japan. A previous epidemiology investigation has
shown that the seroprevalence of BLV infection among dairy cattle ismore than 40% in Japan (Murakami et al., 2013). The lack of aneffective treatment or vaccine is one of the reasons for the high preva­
lence of BLV in many countries. Thus, the establishment of a novel
control strategy against BLV infection is strongly required. To develop anovel strategy, our previous studies focused on immune checkpoint
molecules, such as programmed death 1 (PD-1) and PD-ligand 1 (PD-L1)(Konnai et al., 2017). PD-1 is an immunoinhibitory receptor expressedon T cells, and its upregulation plays a key role in T-cell exhaustion via
the interaction with its ligand PD-L1 (Okazaki and Honjo, 2007). In
bovine studies, previous reports have demonstrated that the PD-1/PD-L1
pathway regulates Th1 cytokine production and is associated with BLV
infection progression (Ikebuchi et al., 2010, 2011). The inhibition of thePD-1/PD-L1 pathway using specific antibodies (Abs) activates Th1 re­sponses, such as IFN-γ production and T-cell proliferation (Ikebuchi
et al., 2011; Nishimori et al., 2017). Additionally, the administration ofanti-PD-1/PD-L1 Abs to BLV-infected cattle, which were classified as AL,
significantly reduced BLV proviral loads (Nishimori et al., 2017; Oka­
gawa et al., 2017; Sajiki et al., 2019), suggesting a potential of theimmunotherapy targeting PD-1/PD-L1 as a novel control method
against BLV infection. However, a previous paper has shown that theadministration of anti-PD-L1 Abs does not reduce BLV proviral loads in
BLV-infected cattle with PL (Sajiki et al., 2019). Therefore, developing atherapeutic strategy that enhances the efficacy of anti-PD-1/PD-L1 Abs
is needed to overcome this situation. A previous paper has reported that
TLR7 agonists have the potential to be included in combination immu­
notherapies. Nishii et al. showed the potential of TLR7 agonists toovercome resistance to anti-PD-L1 Abs through the modification of DCs
in murine tumor models (Nishii et al., 2018). However, little informa­tion is available on the efficacy of combining anti-PD-L1 Abs with TLR7
agonists to treat viral infections.
In the present study, we found that a TLR7 agonist, GS-9620, acti­
vated Th1 responses via CD11c+ cells in cattle. Additionally, analyses
using samples from BLV-infected cattle showed that the expression
levels of TLR7 in CD11c+ cells were upregulated in BLV-infected cattle,
especially in PL cattle. GS-9620 treatment induced Th1 cytokine pro­duction and inhibited syncytium formation in vitro. Moreover, we
examined the therapeutic potential of combining the TLR7 agonist with
anti-PD-L1 Abs using PBMCs from BLV-infected cattle.

Percoll (GE Healthcare, Little Chalfont, UK). PBMCs were cultured in
RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing
10% heat-inactivated fetal calf serum (Thermo Fisher Scientific, Wal­
tham, MA, USA), 100 U/mL penicillin (Thermo Fisher Scientific), 100
μg/mL streptomycin (Thermo Fisher Scientific), and 2 mM L-glutamine(Thermo Fisher Scientific), and were grown in 96-well plates (Corning
Inc., Corning, NY, USA). CC81 cells were cultured for syncytium assay in
RPMI 1640 medium (Sigma-Aldrich) containing the same supplements
as shown above, and were grown in 24-well plates (Corning Inc.).
2.3. Functional analyses of GS-9620 using PBMCs
To examine whether TLR7 agonists activate the immune responses incattle, PBMCs from uninfected cattle were cultured with 1 μM of GS9620 (Chemscene, Monmouth Junction, NJ, USA) for three days.
Dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan) was used as avehicle control. After the incubation, culture supernatants werecollected, and the concentrations of IFN-α, IFN-γ, and TNF-α were
measured using Bovine IFN-α Do-It-Yourself ELISA (Kingfisher Biotech,
St. Paul, MN, USA), Bovine IFN-γ ELISA development kit (Mabtech,
Nacka Strand, Sweden), and Bovine TNF-α Do-It-Yourself ELISA (King­fisher Biotech), respectively. After the incubation, the cells were har­vested, and then, analyzed by flow cytometry and qPCR. For flow
cytometry, Fc blocking was performed by incubating PBMCs in phos­
phate buffered saline (PBS) containing 10% heat-inactivated goat serum(Thermo Fisher Scientific) for 15 min at 25 ◦ C to prevent nonspecificreactions. After Fc blocking, cells were stained with the following Abs:
Alexa Fluor 647-labeled anti-CD11c mAb (BAQ151A; Washington State
University Monoclonal Antibody Center, Pullman, WA, USA) and
PerCP/Cy 5.5-conjugated anti-CD80 mAb (IL-A159; Bio-Rad, Hercules,
CA, USA). BAQ151A was prelabeled using the Zenon Alexa Fluor 647
Mouse IgG1 Labeling Kit (Thermo Fisher Scientific). IL-A159 was con­jugated by using a Lightning-Link antibody labeling kit (Innova Bio­sciences, Cambridge, England, UK). After the staining for 20 min at
25 ◦ C, the cells were washed twice with PBS, and analyzed immediately
by using FACS Verse (BD Biosciences, San Jose, CA, USA). For qPCR,RNA extraction and cDNA synthesis were performed as described in aprevious paper (Sajiki et al., 2019). To measure the mRNA expression
levels of IFN-γ, TNF-α, IL-1β, TGF-β1, and Foxp3, qPCR was performed
using a thermal cycler (LightCycler 480 System II) with SYBR Premix
DimerEraser (Takara Bio), following the manufacturers’ instructions.
β-actin (ACTB) was used as a reference gene. Primers were 5′ -ATA ACC
AGG TCA TTC AAA GG-3′ and 5′ -ATT CTG ACT TCT CTT CCG CT-3′ for
IFN-γ, 5′ -TAA CAA GCC AGT AGC CCA CG-3′ and 5′ -GCA AGG GCT CTTGAT GGC AGA-3′ for TNF-α, 5′ -ACC TTC ATT GCC CAG GTT TCT-3′ and5′ -CTG TTT AGG GTC ATC AGC CTC AA-3′ for IL-1β, 5′ -CTG CTG AGG
CTC AAG TTA AAA GTG-3′ and 5′ -CAG CCG GTT GCT GAG GTA G-3′ for
TGF-β1, 5′ -CAC AAC CTG AGC CTG CAC AA-3′ and 5′ -TCT TGC GGAACT CAA ACT CAT C-3′ for Foxp3, and 5′ -TCT TCC AGC CTT CCT TCC
TG-3′ and 5′ -ACC GTG TTG GCG TAG AGG TC-3′ for ACTB. To examine
the effects of GS-9620 on CD69 expression in T cells, PBMCs from un­
infected cattle were cultivated with 1 μM of GS-9620 for two days. Afterthe incubation, the cells were collected, and the expression levels of
CD69 were measured by flow cytometry. After Fc blocking, the cells
were stained with the following Abs: PerCP/Cy 5.5-conjugated anti-CD3
mAb (MM1A; Washington State University Monoclonal Antibody Cen­ter), FITC-conjugated anti-CD4 mAb (CC8; Bio-Rad), PE-conjugatedanti-CD8 mAb (CC63; Bio-Rad), PE/Cy7-conjugated anti-IgM mAb(IL-A30; Bio-Rad), and Alexa Fluor 647-labeled anti-CD69 mAb(KTSN7A; Kingfisher Biotech). MM1A and IL-A30 were conjugated using
Lightning-Link antibody labeling kits. KTSN7A was prelabeled using the
Zenon Alexa Fluor 647 Mouse IgG1 Labeling Kit. After the staining for
20 min at 25 ◦ C, PBMCs were washed twice with PBS, and analyzed
immediately by FACS Verse. To examine the effect of GS-9620 on IFN-γ
production in T cells, PBMCs from uninfected cattle were cultivated with
1 μM of GS-9620 for three days. Cultures were stimulated by adding 1

2. Materials and methods
2.1. Blood samples and BLV diagnosis
The blood samples from BLV-infected and BLV-uninfected cattle
were obtained from several farmers and veterinarians in Japan. BLV
infection was confirmed via the detection of the anti-BLV antibodies
using a commercially available ELISA kit (JNC, Tokyo, Japan) and theprovirus using quantitative real-time polymerase chain reaction (qPCR)
using a LightCycler 480 System II (Roche Diagnostic, Mannheim, Ger­many) with BLV Detection Kit (Takara Bio). Lymphocytes in blood
samples from BLV-infected cattle were counted using a Celltac alphaMEK-6450 automatic hematology analyzer (Nihon Kohden, Tokyo,
Japan), and BLV-infected cattle were classified as AL or PL according toa previous paper (Ohira et al., 2016).
2.2. Cell preparation and culture
As described in our previous paper (Sajiki et al., 2018), PBMCs fromblood samples were purified using density-gradient centrifugation on
2

Y. Sajiki et al.

Developmental and Comparative Immunology 114 (2021) 103847

μg/mL of anti-CD3 mAb (MM1A) and 1 μg/mL of anti-CD28 mAb(CC220; Bio-Rad) to each well. Brefeldin A (10 μg/mL; Sigma-Aldrich)

BLV cells). As described in a previous paper, the FLK-BLV supernatantcontained BLV antigens to activate BLV-specific T cells in PBMC cultures(Mager et al., 1994). After the incubation, culture supernatants werecollected and the concentrations of IFN-γ and TNF-α were measured by
ELISA as described above. The cells were also collected and CD69
expression in CD4+ and CD8+ T cells was measured by flow cytometry as
described above.

was added for the final 6 h to enhance intracellular cytokine staining.
After the incubation, the cells were collected and Fc blocking was per­formed as described above. Then, surface staining was performed using
the following Abs: FITC-conjugated anti-CD4 mAb (CC8), PE-conjugatedanti-CD8 mAb (CC63), and PE/Cy7-conjugated anti-IgM mAb (IL-A30).
After the surface staining for 20 min at 25 ◦ C, intracellular cytokine
staining was performed using the following reagents: FOXP3 Fix/Permkit (BioLegend, San Diego, CA, USA), Biotinylated anti-bovine IFN-γ
mAb (MT307; Mabtech), and APC-conjugated streptavidin (BioLegend).
After final staining, the cells were analyzed immediately by FACS Verse.
PBMCs from BLV-infected cattle were cultured with 1 μM of GS-9620
for six days. Cultures were stimulated by adding a BLV antigen (fetallamb kidney (FLK)-BLV; 2% heat-inactivated culture supernatant of FLK-

2.4. Functional analysis of GS-9620 using CD11c+ cell-depleted PBMCs
To investigate whether GS-9620 induces the activation of T cells via
CD11c+ cells, CD11c+ cells were depleted from the PBMCs of uninfected
cattle. PBMCs were cultured with anti-bovine CD11c mAb (BAQ151A)
for 30 min at 4 ◦ C. Then, the cells were incubated with Anti-Mouse IgG1
MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min

Fig. 1. Functional analysis of GS-9620 using PBMCs. (a–i) PBMCs from uninfected cattle were incubated with GS-9620. (a–c) The concentrations of IFN-α (a, n = 8),
IFN-γ (b, n = 7), and TNF-α (c, n = 6) in culture supernatants were determined by ELISA. (d) The expression levels of CD80 in CD11c+ cells were measured by flow
cytometry (n = 10). (e–i) The gene expression levels of IFN-γ (e), TNF-α (f), IL-1β (g), TGF-β1 (h), and Foxp3 (i) were quantitated by qPCR (n = 6). (a–i) Statisticalsignificance was determined by the Wilcoxon signed-rank test.
3

Y. Sajiki et al.

Developmental and Comparative Immunology 114 (2021) 103847

Fig. 2. The effects of GS-9620 on T cell activation (aand b) PBMCs from uninfected cattle were incubatedwith GS-9620 (n = 10). CD69 expression in CD4+ (a)and CD8+ (b) T cells was assayed by flow cytometry. (cand d) PBMCs from BLV-uninfected cattle were incu­
bated with GS-9620 in the presence of anti-CD3 mAb
and anti-CD28 mAb (n = 8). The expression levels of
IFN-γ in CD4+ (c) and CD8+ (d) cells were quantified byflow cytometry. (a–d) Statistical significances were
determined by the Wilcoxon signed-rank test.

at 4 ◦ C. After the incubation, CD11c+ cell sorting was performed using
an autoMACS Pro (Miltenyi Biotec) according to the manufacturer’s
protocol. Negative fractions were used as CD11c+ cell-depleted PBMCs,
and positive fractions as CD11c+ cells. The purity of the fractions was
confirmed using FACS Verse. CD11c+ cell-depleted PBMCs contained
less than 3% of CD11c+ cells. The purity of CD11c+ cells was more than
95%. CD11c+ cell-depleted PBMCs were cultivated with 1 μM of GS9620 for two days, and then, the expression of CD69 in T cells wasmeasured by flow cytometry as described above.

concentration of IFN-γ was measured by ELISA as described above.
2.6. Identification of subpopulations in PBMCs
To analyze the percentage of T cells and B cells in uninfected and PL
cattle, after Fc blocking, the cells were stained with the following Abs:
PerCP/Cy 5.5-conjugated anti-CD3 mAb (MM1A), FITC-conjugated antiCD4 mAb (CC8), PE-conjugated anti-CD8 mAb (CC63), and PE/Cy7conjugated anti-IgM mAb (IL-A30). After staining for 20 min at 25 ◦ C,
PBMCs were washed twice with PBS, and analyzed immediately using
the FACS Verse. To analyze the percentage of monocytic cells in unin­
fected and PL cattle, PBMCs were stained with the following Abs after Fc
blocking: PerCP/Cy 5.5-conjugated anti-CD14 mAb (CAM36A; Wash­
ington State University Monoclonal Antibody Center) and Alexa Fluor
647-labeled anti-CD11c mAb (BAQ151A) for 20 min at 25 ◦ C. CAM36A
was conjugated using the Lightning-Link antibody labeling kit. After the
staining, PBMCs were washed twice with PBS, and analyzed immedi­
ately by FACS Verse.

2.5. Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining
CD11c+ cell-depleted PBMCs and CD11c+ cells were prepared from
PBMCs of uninfected cattle using the autoMACS Pro as described above.
CD11c+ cell-depleted PBMCs were labeled with CFSE (Sigma-Aldrich)and cultivated in culture medium for 24 h. Simultaneously, CD11c+ cells
were cultured with 1 μM of GS-9620 for 24 h. Then, CD11c+ cells were
washed with culture medium, and co-cultured with CD11c+ celldepleted PBMCs for 24 h. After the incubation, cells were harvested
and Fc blocking was performed as described above. The cells were
stained with the following Abs: Alexa Fluor 647-labeled anti-CD4 mAb(CC30; Bio-Rad), PerCP/Cy 5.5-conjugated anti-CD8 mAb (CC63), andPE/Cy7-conjugated anti-IgM mAb (IL-A30). CC30 was prelabeled using
the Zenon Alexa Fluor 647 Mouse IgG1 Labeling Kit. CC63 was conju­
gated by using a Lightning-Link antibody labeling kit. After staining for
20 min at 25 ◦ C, the cells were analyzed immediately by FACS Verse.
After the incubation, culture supernatants were also collected and the

2.7. Expression analysis of TLR7 gene in CD11c+ cells
To quantify the gene expression of TLR7, CD11c+ cells were isolated
from the PBMCs of BLV-infected and uninfected cattle as describedabove. TLR7 expression was measured by qPCR as described above.
Primers were 5′ -ACT CCT TGG GGC TAG ATG GT-3′ and 5′ -GCT GGA
GAG ATG CCT GCT AT-3′ for TLR7.
4

Y. Sajiki et al.

Developmental and Comparative Immunology 114 (2021) 103847

Fig. 3. T-cell activation by GS-9620 via CD11c+ cells.
(a and b) PBMCs from BLV-uninfected cattle wereincubated with GS-9620 (n = 10). The expression of
CD69 in CD4+ (a) and CD8+ (b) T cells was assayed byflow cytometry. (c and d) CD11c+ cell-depleted PBMCs
from BLV-uninfected cattle were cocultured with GS9620 treated-CD11c+ cells. (c) CD8+ cell proliferation
was assayed by flow cytometry (n = 9). (d) IFN-γ con­centrations in culture supernatants were determined by
ELISA (n = 8). (a–d) Statistical significance was deter­
mined by the Wilcoxon signed-rank test. CD11c (− ),CD11c+ cell-depleted PBMCs.

2.8. Functional analysis of the combined treatment of GS-9620 with antiPD-L1 Ab

from three independent culture wells.
2.10. Statistics

PBMCs from BLV-infected cattle were cultured with 1 μM of GS-9620
and/or 10 μg/mL of anti-PD-L1 Ab (Boch4G12; Nishimori et al., 2017)
for six days. DMSO and/or bovine IgG (Sigma-Aldrich) were used as
negative controls. Cultures were stimulated with FLK-BLV. After the
incubation, culture supernatants were collected and the concentrationsof IFN-γ and TNF-α were measured by ELISA as described above. The
cells were also collected and CD69 expression in CD4+ and CD8+ T cells
was measured by flow cytometry as described above.

In Fig. 4a–e, differences were identified using the Mann-Whitney U
test. In the other figures, statistical differences were identified using the
Wilcoxon signed-rank test for comparing two-group and the Steel-Dwass
test for comparing multiple groups. A p value of less than 0.05 wasconsidered statistically significant.
3. Results

2.9. Syncytium assay

3.1. The activation of immune responses by GS-9620

CC81 cells (0.6 × 105 cells/mL) were pre-cultured and used as in­dicator cells (Ferrer et al., 1981). After 24 h, PBMCs (5 × 105 cells/mL)from BLV-infected cattle were added to each well, and co-cultured with
CC81 cells in the presence of 1 μM of GS-9620 and/or 10 μg/mL ofanti-PD-L1 Ab (Boch4G12) for 70 h. DMSO and/or bovine IgG were usedas negative controls. After the incubation, the cells were fixed withmethanol (Sigma-Aldrich), and fixed cells were then stained with
Giemsa’s azur eosin methylene blue solution (Merck KGaA, Darmstadt,
Germany). Syncytium formation was observed using a ZOE Fluorescent
Cell Imager (Bio-Rad). The syncytium numbers represented the means

To examine whether TLR7 agonists activate immune the responses incattle, we analyzed the function of GS-9620 in cattle. PBMCs from un­
infected cattle were cultivated with the TLR7 agonist, and the levels ofcytokines in culture supernatants were measured using ELISA. As shownin Fig. 1a–c, GS-9620 significantly increased the production of IFN-α,
IFN-γ, and TNF-α (Fig. 1a–c). Additionally, GS-9620 upregulated theexpression of CD80, a maturation marker, in CD11c+ cells (Fig. 1d) andsignificantly increased the gene expression levels of IFN-γ, TNF-α, and IL1β in PBMCs (Fig. 1e–g). In contrast, GS-9620 decreased the expressionof TGF-β1 and Foxp3 in PBMCs (Fig. 1h and i). These results suggest that5

Y. Sajiki et al.

50
40
30
20
10

Uninfected

20

10

15

10

5

(e)

p < 0.01

500

Uninfected

CD11c+CD14+ cells in PBMCs (%)

CD8+ cells in CD3+IgM - cells (%)

30

PL

(d)
10

40

0

0

10

PL

Uninfected

(f)

n.s.

5

PL

PL

p < 0.05

6
5
4
3
2
1
0

0

Uninfectedp < 0.01

20

CD4+ cells in CD3+IgM - cells (%)

60

Fig. 4. The percentages of T cells, B
cells, and CD11c+ cells in PBMCs and
TLR7 expression in CD11c+ cells from
uninfected and BLV-infected cattle. The
percentages of B cells (a, CD3− IgM+), T
cells (b, CD3+ IgM− ), CD4+ T cells (c,CD3+ IgM− CD4+), CD8+ T cells (d,CD3+ IgM− CD8+), and CD11c+ cells (e)in PBMCs from uninfected and PL cattle
were analyzed by flow cytometry (n = 6each). (f) CD11c+ cells were isolated
from PBMCs of uninfected and BLVinfected cattle (uninfected, n = 8; AL,n = 7; PL, n = 6). TLR7 expression wasquantitated by qPCR. (a–f) Statisticalsignificance was determined by the
Mann-Whitney U test (a–e) and theSteel-Dwass test (f). Uninfected, cattle
not infected with BLV; PL, persistent
lymphocytosis; n. s., not significant; AL,
aleukemic.

(c)p < 0.01

50

TLR7 expression (relative)

CD3-IgM + cells in PBMCs (%)

70

(b)

p < 0.01

CD3+IgM - cells in PBMCs (%)(a)

Developmental and Comparative Immunology 114 (2021) 103847

Uninfected

UninfectedPL

GS-9620 treatment activates immune responses, in cattle, including in­flammatory responses and Th1 responses. In general, IFN-γ and TNF-α
are known as Th1-related cytokines. Therefore, we determined whether
GS-9620 activates Th1 responses in cattle. As shown in Fig. 2a and b, GS9620 induced CD69 expression, an activation marker, in CD4+ andCD8+ T cells (Fig. 2a and b). Additionally, GS-9620 significantly
increased the percentage of IFN-γ+ cells in CD4+ and CD8+ T cells(Fig. 2c and d). Taken together, GS-9620 treatment has the potential toactivate Th1 responses in vitro.

AL

PL

derived from PL cattle. As expected, the percentage of B cells(CD3− IgM+ cells) in PBMCs from PL cattle was significantly higher thanthe percentage of B cells in PBMCs from uninfected cattle (Fig. 4a). In
contrast, the percentages of T cells (CD3+IgM− cells), CD4+ T cells, andCD8+ T cells in PL cattle were dramatically lower than the percentagesin uninfected cattle (Fig. 4b–d). In addition, we analyzed the percentage
of monocytic cells in PBMCs from PL cattle. Interestingly, the percentage
of CD11c+CD14+ cells in PBMCs was not significantly different between
BLV-uninfected and PL cattle (Fig. 4e). Furthermore, TLR7 expression
was significantly upregulated in CD11c+ cells of PL cattle (Fig. 4f).

3.2. The induction of Th1 responses by the TLR7 agonist via CD11c+ cells
3.4. The effects of the combined treatment with the TLR7 agonist andanti-PD-L1 Abs on Th1 responses and syncytium formation

To examine whether GS-9620-induced upregulation of the Th1
response is caused by CD11c+ cell activation, CD11c+ cell-depleted
PBMCs were cultured with GS-9620. Flow cytometric analyses
revealed that CD69 expression in CD4+ and CD8+ T cells was lower in
the CD11c+ cell-depleted PBMCs compared to PBMCs containing
CD11c+ cells (Fig. 3a and b). Additionally, CD11c+ cells were precultured with GS-9620 or DMSO, and pretreated CD11c+ cells were
co-cultured with CD11c+ cell-depleted PBMCs. The pretreatment ofCD11c+ cells increased CD8+ cell proliferation (Fig. 3c) and IFN-γ pro­duction in culture supernatants (Fig. 3d). Collectively, these results
suggest that GS-9620 enhances the Th1 responses, at least in part, via
CD11c+ cells.

We then examined the effects of GS-9620 on the activation of Th1
responses using PBMCs from BLV-infected cattle in vitro. The treatment
with GS-9620 significantly increased IFN-γ and TNF-α production from
PBMCs of BLV-infected cattle in the presence of FLK-BLV, a BLV antigen(Fig. 5a and b). Additionally, CD69 expression in CD4+ and CD8+ T cells
was upregulated by the treatment with GS-9620 (Fig. 5c and d). To
examine whether the combined treatment with GS-9620 enhances theefficacy of anti-PD-L1 Abs, PBMCs from BLV-infected cattle were
cultured with anti-PD-L1 Abs and GS-9620 in the presence of FLK-BLV.
The combined treatment with GS-9620 and anti-PD-L1 Abs increased the
production of IFN-γ and TNF-α to a greater extent compared to treat­ment with anti-PD-L1 antibody or GS-9620 alone (Fig. 5e and f). In
addition, the combined treatment tended to upregulate CD69 expressionin CD4+ and CD8+ T cells (Fig. 5g and h). Finally, we examined theeffects of the combined treatment on syncytium formation. Represen­tative pictures of syncytium formation in each treatment were shown in
Fig. 6a. The treatment with GS-9620 significantly inhibited syncytium
formation by BLV compared to the control group (Fig. 6b, Table 1).
Moreover, the combined treatment tended to inhibit syncytium forma­tion compared to treatment with anti-PD-L1 antibody or GS-9620 alone

3.3. The percentages of T cells, B cells, and CD11c+ cells in PBMCs from
BLV-uninfected and PL cattle
Th1 response plays an important role for the control of bovinechronic infections including BLV infection (Orlik and Splitter, 1996;
Kabeya et al., 2001). During late-stage of BLV infection, the number ofcirculating B cells is dramatically increased, whereas the number ofcirculating T cells is dramatically decreased (Nieto Farias et al., 2018).
In this study, we confirmed the percentages of these cells in PBMCs
6

Y. Sajiki et al.

Developmental and Comparative Immunology 114 (2021) 103847

Fig. 5. Functional analysis of the combined treatmentof GS-9620 with anti-PD-L1 Abs. (a–d) PBMCs from
BLV-infected cattle were cultured with GS-9620 in thepresence of FLK-BLV. IFN-γ (a) and TNF-α (b) con­centrations in culture supernatants were measured by
ELISA (n = 9). CD69 expression in CD4+ (c) and CD8+(d) T cells was measured by flow cytometry (n = 8).
Statistical significance was determined by the Wil­coxon signed-rank test. (e–h) PBMCs from BLVinfected cattle were cultured with GS-9620 and/or
anti-PD-L1 Abs in the presence of FLK-BLV. IFN-γ (e)and TNF-α (f) concentrations in culture supernatants
were measured by ELISA (n = 20). CD69 expression in
CD4+ (g) and CD8+ (h) T cells was measured by flow
cytometry (n = 15). Statistical significance wasdetermined by the Steel-Dwass test.

(Fig. 6b, Table 1). These data suggest that the combined treatment with
anti-PD-L1 Ab and the TLR7 agonist could be a novel control strategy fortreating BLV infection.

the control group. A previous study has shown the antiviral activity ofrecombinant bovine IFN-γ against BLV using syncytium assay (Sentsui
et al., 2001). Thus, the antiviral effect of GS-9620 is caused presumably
due to the induction of IFN-γ production. These data suggest the po­tential of TLR7 agonism as a novel control method for controlling BLV
infection. However, it is still unclear whether the TLR7 agonist activates
BLV-specific Th1 responses in vivo. Further clinical studies on the effi­cacy of the TLR7 agonist in BLV-infected cattle will be needed.
Our previous studies have shown that anti-PD-L1 Ab administration
significantly reduces BLV proviral loads in AL cattle (Nishimori et al.,2017; Sajiki et al., 2019). However, the anti-PD-L1 Ab did not reduce
BLV proviral loads in PL cattle (Sajiki et al., 2019). During late-stage of
BLV infection, Th1 responses are not only suppressed by the PD-1/PD-L1
pathway but also by other immunosuppressive factors such as PGE2 andregulatory T cells (Tregs) (Ohira et al., 2016; Konnai et al., 2017; Sajiki
et al., 2019). Thus, PL cattle might be resistant to the anti-PD-L1 Abs
alone due to Th1 suppression via several immunoinhibitory factors. To

4. Discussion
During BLV infection, the suppression of BLV-specific Th1 responses,
including T-cell proliferation and cytotoxic T cell responses against BLV
antigens, is associated with disease progression (Orlik and Splitter,
1996; Kabeya et al., 2001). Therefore, Th1 responses are important forcontrolling BLV infection. In this study, we found that the percentage ofCD11c+ cells in PBMCs was not changed between uninfected cattle andPL cattle, and TLR7 expression in CD11c+ cells was upregulated in PL
cattle. In addition, functional analyses of the TLR7 agonist revealed thatthe treatment of GS-9620 increased T-cell activation, including Th1
cytokine production, at least in part, through CD11c+ cells. Further­more, GS-9620 treatment reduced the number of syncytia compared to7

Developmental and Comparative Immunology 114 (2021) 103847

Y. Sajiki et al.

Fig. 6. The inhibition of syncytium formation by the combined treatment of GS-9620 with anti-PD-L1 Abs. (a and b) CC81 cells and PBMCs of BLV-infected cattle (n= 7) were co-cultured in the presence of GS-9620 and/or anti-PD-L1 Abs, and the number of syncytia was counted. (a) Representative pictures of syncytium for­mation in each treatment. (b) The results were shown as relative changes to no treatment group. Statistical significance was determined by the Steel-Dwass test.

enhances the antitumor immune response in murine tumor models(Nishii et al., 2018). In the present study, we demonstrated that the
combined treatment with anti-PD-L1 Ab and GS-9620 significantly
enhanced BLV-specific Th1 cytokine production in vitro. In addition, thecombined treatment tended to inhibit syncytium formation compared tothe single treatment with GS-9620 or anti-PD-L1 Abs. These data suggestthat this combined treatment could be an effective strategy for over­coming the resistance to PD-L1 blockade in BLV-infected cattle. To thebest of our knowledge, this is the first study showing that a TLR7 agonist
could be used in combination therapies to overcome the resistance to
PD-L1 blockade in bovine infections.
Tregs are immune cell populations which limit immune responses byproducing inhibitory cytokines, such as TGF-β and IL-10, and maintainhost immune homeostasis (Sakaguchi et al., 2008). Tregs dampen T-cell
immunity against cancers and infectious diseases, being an obstacle thattempers successful immunotherapy (Belkaid and Rouse, 2005; Zou,

Table 1
The number of syncytia in each treatment group.
Cattle
#1
#2
#3
#4
#5
#6
#7
MedianNumber of syncytia (/cm2)
No treatment

Control

GS-9620

Boch4G12

Combination

1118.61178.1
53.6
779.5
345.1
487.9
380.8
487.9

1313.01293.1
55.5
785.4
333.2
527.6
345.1
527.6

769.5
741.8
51.6
674.3
103.1
357.0
281.6
357.0

940.1
880.6
35.7
654.5
174.5
420.5
309.4
420.5

702.1
674.3
27.8
468.1
51.6
309.4
261.8
309.4

overcome this issue, combining anti-PD-L1 Abs with other drugs thatenhance the immune responses may be an effective strategy. A previous
study has reported that PD-L1 blockade combined with TLR7 agonists
8Developmental and Comparative Immunology 114 (2021) 103847

Y. Sajiki et al.

2006). It is reported that inducing TGF-β secretion from Tregs in
BLV-infected cattle reduces Th1 cytokine production (Ohira et al.,2016). Thus, the inhibition of Tregs would be important for the control
of BLV infection. Interestingly, previous reports have demonstrated that
TLR7 stimulation suppresses the function of Tregs via DC activation(Hackl et al., 2011; Wang et al., 2015). Also, the anti-PD-L1 Abs with
TLR7 agonists overcome the resistance to anti-PD-L1 immunotherapy byattenuating Tregs via DC modification (Nishii et al., 2018). Our results
showed that the treatment with GS-9620 decreased the gene expressionof TGF-β1 and Foxp3 in bovine PBMCs. Foxp3 is a transcription factor
that is required for the development of Tregs (Hori et al., 2003).
Therefore, these results suggest that GS-9620 activates the Th1 immune
response via the inhibition of Tregs. Additional experiments are needed
to reveal the effect of TLR7 agonists on Treg function in cattle.

Belkaid, Y., Rouse, B., 2005. Natural regulatory T cells in infectious disease. Nat.
Immunol. 6, 353–360.
Farias, M.V., Lendes, P.A., Marin, M., Quintana, S., Martínez Cuesta, L., Ceriani, C.,
Dolcini, G.L., 2016. Toll-like receptors, IFN-γ and IL-12 expression in bovine
leukemia virus-infected animals with low or high proviral load. Res. Vet. Sci. 107,
190–195.
Ferrer, J.F., Cabradilla, C., Gupta, P., 1981. Use of a feline cell line in the syncytia
infectivity assay for the detection of bovine leukemia virus infection in cattle. Am. J.
Vet. Res. 42, 9–14.
Gibson, S.J., Lindh, J.M., Riter, T.R., Gleason, R.M., Rogers, L.M., Fuller, A.E.,
Oesterich, J.L., Gorden, K.B., Qiu, X., McKane, S.W., Noelle, R.J., Miller, R.L.,
Kedl, R.M., Fitzgerald-Bocarsly, P., Tomai, M.A., Vasilakos, J.P., 2002. Plasmacytoid
dendritic cells produce cytokines and mature in response to the TLR7 agonists,
imiquimod and resiquimod. Cell. Immunol. 218, 74–86.
Hackl, D., Loschko, J., Sparwasser, T., Reindl, W., Krug, A.B., 2011. Activation ofdendritic cells via TLR7 reduces Foxp3 expression and suppressive function in
induced Tregs. Eur. J. Immunol. 41, 1334–1343.
Hori, S., Nomura, T., Sakaguchi, S., 2003. Control of regulatory T cell development by
the transcription factor Foxp3. Science 299, 1057–1061.
Horscroft, N.J., Pryde, D.C., Bright, H., 2011. Antiviral applications of Toll-like receptor
agonists. J. Antimicrob. Chemother. 67, 789–801.
Ikebuchi, R., Konnai, S., Sunden, Y., Onuma, M., Ohashi, K., 2010. Molecular cloning andexpression analysis of bovine programmed death-1. Microbiol. Immunol. 54,
291–298.
Ikebuchi, R., Konnai, S., Shirai, T., Sunden, Y., Murata, S., Onuma, M., Ohashi, K., 2011.
Increase of cells expressing PD-L1 in bovine leukemia virus infection andenhancement of anti-viral immune responses in vitro via PD-L1 blockade. Vet. Res.
42, 103.
Ito, T., Wang, Y.H., Liu, Y.J., 2005. Plasmacytoid dendritic cell precursors/type I
interferon-producing cells sense viral infection by Toll-like receptor (TLR) 7 andTLR9. Springer Semin. Immunopathol. 26, 221–229.
Jarrossay, D., Napolitani, G., Colonna, M., Sallusto, F., Lanzavecchia, A., 2001.
Specialization and complementarity in microbial molecule recognition by human
myeloid and plasmacytoid dendritic cells. Eur. J. Immunol. 31, 3388–3393.
Kabeya, H., Ohashi, K., Onuma, M., 2001. Host immune responses in the course of bovine
leukemia virus infection. J. Vet. Med. Sci. 63, 703–708.
Kadowaki, N., Ho, S., Antonenko, S., Malefyt, R.W., Kastelein, R.A., Bazan, F., Liu, Y.J.,
2001. Subsets of human dendritic cell precursors express different Toll-like receptors
and respond to different microbial antigens. J. Exp. Med. 194, 863–869.
Konnai, S., Murata, S., Ohashi, K., 2017. Immune exhaustion during chronic infections incattle. J. Vet. Med. Sci. 79, 1–5.
Lanford, R.E., Guerra, B., Chavez, D., Giavedoni, L., Hodara, V.L., Brasky, K.M.,
Fosdick, A., Frey, C.R., Zheng, J., Wolfgang, G., Halcomb, R.L., Tumas, D.B., 2013.
GS-9620, an oral agonist of Toll-like receptor-7, induces prolonged suppression ofhepatitis B virus in chronically infected chimpanzees. Gastroenterology 144,
1508–1517.
Lund, J.M., Alexopoulou, L., Sato, A., Karow, M., Adams, N.C., Gale, N.W., Iwasaki, A.,
Flavell, R.A., 2004. Recognition of single-stranded RNA viruses by Toll-like receptor
7. Proc. Natl. Acad. Sci. U.S.A. 101, 5598–5603.
Mager, A., Masengo, R., Mammerickx, M., Letesson, J.J., 1994. T cell proliferative
response to bovine leukaemia virus (BLV): identification of T cell epitopes on the
major core protein (p24) in BLV-infected cattle with normal haematological values.
J. Gen. Virol. 75, 2223–2231.
Marin, M.S., Quintana, S., Faverin, C., Leunda, M.R., Odeon, A.C., Perez, S.E., 2014. Tolllike receptor activation and expression in bovine alpha-herpesvirus infections. Res.
Vet. Sci. 96, 196–203.
Menne, S., Tumas, D.B., Liu, K.H., Thampi, L., AlDeghaither, D., Baldwin, B.H.,
Bellezza, C.A., Cote, P.J., Zheng, J., Halcomb, R., Fosdick, A., Fletcher, S.P.,
Daffis, S., Li, L., Yue, P., Wolfgang, G.H., Tennant, B.C., 2015. Sustained efficacy andseroconversion with the Toll-like receptor 7 agonist GS-9620 in the Woodchuckmodel of chronic hepatitis B. J. Hepatol. 62, 1237–1245.
Murakami, K., Kobayashi, S., Konishi, M., Kameyama, K., Tsutsui, T., 2013. Nationwide
survey of bovine leukemia virus infection among dairy and beef breeding cattle inJapan from 2009-2011. J. Vet. Med. Sci. 75, 1123–1126.
Nieto Farias, M.V., Souza, F.N., Lendez, P.A., Martínez-Cuesta, L., Santos, K.R., Della
Libera, A.M.M.P., Ceriani, M.C., Dolcini, G.L., 2018. Lymphocyte proliferation andapoptosis of lymphocyte subpopulations in bovine leukemia virus-infected dairy
cows with high and low proviral load. Vet. Immunol. Immunopathol. 206, 41–48.
Nishii, N., Tachinami, H., Kondo, Y., Xia, Y., Kashima, Y., Ohno, T., Nagai, S., Li, L.,
Lau, W., Harada, H., Azuma, M., 2018. Systemic administration of a TLR7 agonist
attenuates regulatory T cells by dendritic cell modification and overcomes resistanceto PD-L1 blockade therapy. Oncotarget 9, 13301–13312.
Nishimori, A., Konnai, S., Okagawa, T., Maekawa, N., Ikebuchi, R., Goto, S., Sajiki, Y.,
Suzuki, Y., Kohara, J., Ogasawara, S., Kato, Y., Murata, S., Ohashi, K., 2017. In vitro
and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) ratbovine chimeric antibody against bovine leukemia virus infection. PloS One 12,
e0174916.
Ohira, K., Nakahara, A., Konnai, S., Okagawa, T., Nishimori, A., Maekawa, N.,
Ikebuchi, R., Kohara, J., Murata, S., Ohashi, K., 2016. Bovine leukemia virus reduces
anti-viral cytokine activities and NK cytotoxicity by inducing TGF-β secretion from
regulatory T cells. Immun. Inflamm. Dis. 4, 52–63.
Okagawa, T., Konnai, S., Nishimori, A., Maekawa, N., Ikebuchi, R., Goto, S.,
Nakajima, C., Kohara, J., Ogasawara, S., Kato, Y., Suzuki, Y., Murata, S., Ohashi, K.,
2017. Anti-bovine programmed death-1 rat-bovine chimeric antibody for
immunotherapy of bovine leukemia virus infection in cattle. Front. Immunol. 8, 650.

5. Conclusion
Our data in the present study showed that TLR7 is a potential ther­apeutic target for enhancing antiviral immunity through CD11c+ cell
activation in cattle. In addition, the combined treatment of the TLR7
agonist with anti-PD-L1 Abs enhanced Th1 cytokine production from
PBMCs of BLV-infected cattle in vitro. Future studies, which further
analyze the efficacy of the treatment of the TLR7 agonist with or withoutanti-PD-L1 Abs in BLV-infected cattle, will open up new avenues for thetreatment of BLV infection.
Funding
This work was supported by JSPS KAKENHI grant number 19KK0172[to S.K.], grants from the Project of the NARO, Bio-oriented Technology
Research Advancement Institution (Research Program on Developmentof Innovative Technology 26058 BC [to S.K.] and Special Scheme Project
on Regional Developing Strategy, Grant 16817557 [to S.K.]), regulatory
research projects for food safety, animal health and plant protection(JPJ008617.17935709 [to S.K.]) funded by the Ministry of Agriculture,
Forestry and Fisheries of Japan and AMED under grant number
JP20am0101078 [to Y.K.]. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of themanuscript.
Author contributions
YSa, SK, SM, and KO: designed the work; YSa, TO, NM, and HN:
performed the experiments; YSa, SK, TO, NM, and HN: acquired,analyzed, and interpreted the data; YK and YSu: provided laboratory
materials and reagents; YSa and SK: wrote the manuscript; TO, NM, YK,
YSu, SM, and KO: revised the manuscript. All authors reviewed andapproved the final manuscript.
Declaration of competing interest
The authors declare that they have no competing interests.
Acknowledgments
We are grateful to Dr. Hideyuki Takahashi, Dr. Yasuyuki Mori, andDr. Tomio Ibayashi for valuable advice and discussions. We thank Enago(http://www.enago.jp) for the English language review.
References
Akira, S., Takeda, K., 2004. Toll-like receptor signaling. Nat. Rev. Immunol. 4, 499–511.
Bam, R.A., Hansen, D., Irrinki, A., Mulato, A., Jones, G.S., Hesselgesser, J., Frey, C.R.,
Cihlar, T., Yant, S.R., 2016. TLR7 agonist GS-9620 is a potent inhibitor of acute HIV1 infection in human peripheral blood mononuclear cells. Antimicrob. Agents
Chemother. 61 e01369-16.

9Y. Sajiki et al.

Developmental and Comparative Immunology 114 (2021) 103847

Okazaki, T., Honjo, T., 2007. PD-1 and PD-1 ligands: from discovery to clinicalapplication. Int. Immunol. 19, 813–824.
Orlik, O., Splitter, G.A., 1996. Progression to persistent lymphocytosis and tumor
development in bovine leukemia virus (BLV)-infected cattle correlates with impaired
proliferation of CD4+ T cells in response to gag- and env-encoded BLV proteins.
J. Virol. 70, 7584–7593.
Sagata, N., Yasunaga, T., Tsuzuku-Kawamura, J., Ohishi, K., Ogawa, Y., Ikawa, Y., 1985.
Complete nucleotide sequence of the genome of bovine leukemia virus: its
evolutionary relationship to other retroviruses. Proc. Natl. Acad. Sci. U.S.A. 82,
677–681.
Sajiki, Y., Konnai, S., Okagawa, T., Nishimori, A., Maekawa, N., Goto, S., Ikebuchi, R.,
Nagata, R., Kawaji, S., Kagawa, Y., Yamada, S., Kato, Y., Nakajima, C., Suzuki, Y.,
Murata, S., Mori, Y., Ohashi, K., 2018. Prostaglandin E2 induction suppresses the Th1
immune responses in cattle with johne’s disease. Infect. Immun. 86, e00910–e00917.
Sajiki, Y., Konnai, S., Okagawa, T., Nishimori, A., Maekawa, N., Goto, S., Watari, K.,
Minato, E., Kobayashi, A., Kohara, J., Yamada, S., Kaneko, M.K., Kato, Y.,
Takahashi, H., Terasaki, N., Takeda, A., Yamamoto, K., Toda, M., Suzuki, Y.,
Murata, S., Ohashi, K., 2019. Prostaglandin E2-induced immune exhaustion andenhancement of antiviral effects by anti-PD-L1 antibody combined with COX-2
inhibitor in bovine leukemia virus infection. J. Immunol. 203, 1313–1324.
Sakaguchi, S., Yamaguchi, T., Nomura, T., Ono, M., 2008. Regulatory T cells and immune
tolerance. Cell 133, 775–787.

Samuel, C.E., 2001. Antiviral actions of interferons. Clin. Microbiol. Rev. 14, 778–809.
Schiavoni, G., Mattei, F., Gabriele, L., 2013. Type I interferons as stimulators of DCmediated cross-priming: impact on anti-tumor response. Front. Immunol. 4, 483.
Schwartz, I., Lévy, D., 1994. Pathobiology of bovine leukemia virus. Vet. Res. 25,
521–536.
Sentsui, H., Murakami, K., Inoshima, Y., Yokoyama, T., Inumaru, S., 2001. Anti-viraleffect of recombinant bovine interferon gamma on bovine leukaemia virus. Cytokine
16, 227–231.
Tsai, A., Irrinki, A., Kaur, J., Cihlar, T., Kukolj, G., Sloan, D.D., Murry, J.P., 2017. Tolllike receptor 7 agonist GS-9620 induces HIV expression and HIV-specific immunity
in cells from HIV-infected individuals on suppressive antiretroviral therapy. J. Virol.
91 e02166–16.
Wang, C., Zhou, Q., Wang, X., Wu, X., Chen, X., Li, J., Zhu, Z., Liu, B., Su, L., 2015. The
TLR7 agonist induces tumor regression both by promoting CD4⁺ T cells proliferation
and by reversing T regulatory cell-mediated suppression via dendritic cells.
Oncotarget 6, 1779–1789.
Zhang, Z., Bashiruddin, J.B., Doel, C., Horsington, J., Durand, S., Alexandersen, S., 2006.
Cytokine and toll-like receptor mRNAs in the nasal-associated lymphoid tissues ofcattle during foot-and-mouth disease virus infection. J. Comp. Pathol. 134, 56–62.
Zou, W., 2006. Regulatory T cells, tumour immunity and immunotherapy. Nat. Rev.
Immunol. 6, 295–307.

10