Okagawa et al. Vet Res (2018) 49:50
https://doi.org/10.1186/s13567-018-0543-9

RESEARCH ARTICLE

Open Access

Cooperation of PD‑1 and LAG‑3
in the exhaustion of ­CD4+ and ­CD8+ T cells
during bovine leukemia virus infection
Tomohiro Okagawa1, Satoru Konnai1*, Asami Nishimori1, Naoya Maekawa1, Shinya Goto1, Ryoyo Ikebuchi1,6,
Junko Kohara2, Yasuhiko Suzuki3,4, Shinji Yamada5, Yukinari Kato5, Shiro Murata1 and Kazuhiko Ohashi1

Abstract
Bovine leukemia virus (BLV) is a retrovirus that infects B cells in cattle and causes bovine leukosis after a long latent
period. Progressive exhaustion of T cell functions is considered to facilitate disease progression of BLV infection. Programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are immunoinhibitory receptors that contributeto T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 andLAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- andLAG-3-expressing T cells were performed in BLV-infected cattle at different stages of the disease. The frequencies of
PD-1+LAG-3+ heavily exhausted T cells among ­CD4+ and ­CD8+ T cells was higher in the blood of cattle with B-cell
lymphoma over that of BLV-uninfected and BLV-infected cattle without lymphoma. In addition, blockade assays of
peripheral blood mononuclear cells were performed to examine whether inhibition of the interactions between PD-1
and LAG-3 and their ligands by blocking antibodies could restore T-cell function during BLV infection. Single or dual
blockade of the PD-1 and LAG-3 pathways reactivated the production of Th1 cytokines, interferon-γ and tumor necrosis factor-α, from BLV-specific T cells of the infected cattle. Taken together, these results indicate that PD-1 and LAG-3
cooperatively mediate the functional exhaustion of ­CD4+ and ­CD8+ T cells and are associated with the developmentof B-cell lymphoma in BLV-infected cattle.
Introduction
Bovine leukemia virus (BLV) is a member of the genus
Deltaretrovirus (subfamily Orthoretrovirinae, family Retroviridae) and is genetically related to human T-cell leukemia virus type 1 [1]. BLV infects B cells in cattle and is
integrated into the host genome as a provirus [2, 3]. The
majority (around 70%) of BLV infections in cattle remain
clinically asymptomatic, referred to as aleukemic (AL).
However, up to 30% of infected cattle develop persistent
lymphocytosis (PL), characterized by non-malignant
polyclonal expansion of ­IgM+CD5+ B cells, the majorityof which harbor BLV provirus. After a long latent period,
less than 5% of infected cattle develop malignant B-cell*Correspondence: konnai@vetmed.hokudai.ac.jp
1
Department of Disease Control, Faculty of Veterinary Medicine,
Hokkaido University, Sapporo 060‑0818, Japan
Full list of author information is available at the end of the article

lymphoma in various lymph nodes, so-called enzootic
bovine leukosis (EBL) [4]. During BLV infection in cattle,
T-cell response is known to be suppressed in PL and EBLanimals, but not in AL animals [5–12]. Suppression ofT-cell proliferation in response to BLV antigens and Th1
cytokine production are observed in PL and EBL cattle
and considered to be associated with disease progression[7–12].
To develop strategies to induce effective immune
responses to BLV infection, previous studies have investigated the mechanism responsible for T-cell exhaustion[13–16]. A previous report has shown that the immunoinhibitory receptor programmed death-1 (PD-1) is
upregulated in ­CD4+ and ­CD8+ T cells and is involvedin the exhaustion of T-cell functions in BLV-infected cattle bearing B-cell lymphoma [13]. Further studies haveconfirmed the correlation between upregulation of lymphocyte activation gene-3 (LAG-3) on ­CD4+ and ­CD8+© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License(http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/
publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Okagawa et al. Vet Res (2018) 49:50

T cells and disease progression in BLV infection [14, 15].
Respective ligands of PD-1 and LAG-3, programmed
death-ligand 1 (PD-L1) and major histocompatibility
complex class II (MHC II), were upregulated in B cells
including BLV-infected B cells in BLV-infected cattle [14–18]. We therefore hypothesized that PD-1 and LAG-3
cooperatively contribute to the development of T-cell
exhaustion during disease progression of BLV infection.
According to evidences from mouse models and humanpatients with chronic infections, PD-1+LAG-3+ T cells
is heavily exhausted functionally and strongly associatedwith progression of several chronic infections [19].
However, our previous studies on BLV infection wereanalyzed expression of PD-1 and LAG-3 separately
in independent experiments [13–15], thereby it was
unknown whether PD-1 and LAG-3 express in the same
T-cell populations and play immunosuppressive roles
cooperatively during BLV infection. In addition, LAG-3
expression has been investigated in AL and PL cattle, but
not in EBL animals yet [14, 15]. To unravel the immunosuppression mediated by LAG-3 during the developmentof bovine leukemia, expression level of the moleculeshould be investigated in EBL animals. Multi-color flow
cytometric analysis detecting PD-1+LAG-3+ T cells
requires sensitive monoclonal antibodies (mAbs) against
bovine immunoinhibitory molecules. Thus, we haveestablished anti-bovine PD-1 [13], anti-bovine PD-L1[20], and anti-bovine LAG-3 mAbs [21]. In this paper,
therefore, the dual expression of PD-1 and LAG-3 on T
cells were first analyzed among BLV-infected cattle at different stages of the disease.
Moreover, blockade of the PD-1/PD-L1 and LAG-3/
MHC II interactions using cross-reactive polyclonal antibodies increase Th1 cytokine responses and decrease
BLV proviral load ex vivo in our previous reports [15,16]. However, binding activity of the polyclonal antibodies was very weak and the blockade effect of the immunoinhibitory pathways seems to be underestimated in
the previous study [16]. To address this problem, in thisstudy, we tested the immunomodulatory activity of thenovel sensitive mAbs against bovine PD-L1 and LAG-3
established in recent studies [20, 21] by ex vivo T-cellblockade assays using peripheral blood mononuclear
cells (PBMCs) of BLV-infected cattle. This study will be
helpful to estimate the therapeutic potential of PD-1 andLAG-3 blockade for BLV infection.

Materials and methods
Samples from cattle and BLV diagnosis

Blood samples collected from cattle (Holstein–Friesianand Japanese black) in dairy and beef farms in Japanbetween 2014 and 2016. Informed consent was obtained
from all owners of the animals used in the study. BLV

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infection was diagnosed at Graduate School of Veterinary Medicine, Hokkaido University. Briefly, genomic
DNA was extracted from whole blood using the Wizard Genomic DNA Purification kit (Promega, Madison,
WI, USA). BLV was detected by nested PCR targeting
the BLV long terminal repeat region (LTR) using thefollowing primers: BLV-LTR1 5′-TGT ATG AAA GATCAT GCC GAC-3′ and BLV-LTR533 5′-AAT TGT TTG
CCG GTC TCT-3′ for the initial PCR, and BLV-LTR256
5′-GAG CTC TCT TGC TCC CGA GAC-3′ and BLVLTR453 5′-GAA ACA AAC GCG GGT GCA AGC CAG3′ for the second PCR [22]. The number of lymphocytes
in the blood was counted using an automatic hematology
analyzer Celltac α (Nihon Kohden, Tokyo, Japan). BLVpositive cattle were classified as AL or PL according to
the lymphocyte counts as follows: AL, < 10 000 cells/μLof blood; PL, ≥ 10 000 cells/μL of blood. Blood samples
from cattle clinically diagnosed as bovine leukosis wereconfirmed as B-cell lymphoma by phenotypic analysis oftumor cells using flow cytometry as described previously[13, 23].
Cell preparation

PBMCs were purified from the blood samples using density gradient centrifugation on Percoll (GE Healthcare,
Buckinghamshire, England, UK), washed three times
with phosphate-buffered saline (PBS), and suspended in
PBS.
Flow cytometric analysis of PD‑1 and LAG‑3 expression

Six-color analysis of PD-1- and LAG-3-expressing T cells
was performed using fresh PBMCs obtained from theanimals with various stages of the infection. PBMCs wereincubated in PBS containing 10% goat serum (Sigma–
Aldrich, St. Louis, MO, USA) at room temperature for
15 min to prevent nonspecific reactions. Cells were thenwashed and stained with anti-PD-1 monoclonal antibody (mAb) (5D2, rat I­gG2a) [13] or rat ­IgG2a isotype
control (R35-95, BD Biosciences, San Jose, CA, USA) for
20 min at room temperature, washed with PBS containing 1% bovine serum albumin (BSA; Sigma–Aldrich),and labeled with APC-conjugated anti-rat Ig antibody(Southern Biotech, Birmingham, AL, USA) for 20 min atroom temperature. Cells were then washed and stainedwith anti-LAG-3 mAb (71-2D8, rat ­IgG1) [21] or rat ­IgG1
isotype control (R3-34, BD Biosciences) for 20 min atroom temperature. Finally, cells were washed and incubated with anti-CD4-FITC mAb (CC8; Bio-Rad, Hercules, CA, USA) or anti-CD8-FITC mAb (CC63, Bio-Rad)in the presence of anti-TCR1-N24-APC/Cy7 mAb(anti-TCR δ chain; GB21A; Washington State University Monoclonal Antibody Center, Pullman, WA, USA),anti-CD3-PerCp/Cy5.5 mAb (MM1A; Washington State

Okagawa et al. Vet Res (2018) 49:50

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University Monoclonal Antibody Center), anti-IgM-PE/
Cy7 mAb (IL-A30; Bio-Rad), and PE-conjugated antirat ­IgG1 mAb (G17E7; Southern Biotech) for 20 min atroom temperature. mAbs GB21A, MM1A, and IL-A30
were conjugated with APC/Cy7, PerCp/Cy5.5, and PE/
Cy7, respectively, using Lightning-Link Conjugation Kits
(Innova Biosciences, Cambridge, England, UK). Cells
were then washed and immediately analyzed by FACS
Verse (BD Biosciences) and FCS Express 4 (De Novo
Software, Glendale, CA, USA). The primary antibodies
used in this experiment are shown in Table 1. More than
100 000 lymphocytes were analyzed among the samples.

in 96-well round-bottomed plates (Corning Inc., Corning, NY, USA) containing 1 × 106 PBMCs in 250 μL RPMI
1640 medium (Sigma–Aldrich) supplemented with 10%
heat-inactivated fetal bovine serum (Cansera International, Etobicoke, ON, Canada), 200 IU/mL penicillin,
200 μg/mL streptomycin, and 0.01% v/v l-glutamine(Thermo Fisher Scientific, Waltham, MA, USA). Culture
supernatants were then harvested and stored at −20 °C
for IFN-γ ELISA. IFN-γ concentrations in the supernatants were determined using a bovine IFN-γ ELISA(Mabtech, Nacka Strand, Sweden) performed in duplicate according to the manufacturer’s protocol.

PBMC blockade assay and IFN‑γ ELISAPBMC blockade assay and flow cytometric analysis
of IFN‑γ/TNF‑α‑producing T cells

To determine whether the blockade of the PD-1/PD-L1
and LAG-3/MHC II interactions alter BLV-specificIFN-γ responses, PBMCs prepared from BLV-infected
cattle were cultured with blocking mAbs anti-PD-L1(4G12, rat I­gG2a) [20], anti-LAG-3 (71-2D8, rat ­IgG1)[21], or rat IgG (Sigma–Aldrich) in the presence of heatinactivated supernatant (2%) of BLV-infected fetal lambkidney cells (FLK-BLV) at 37 °C with 5% C
­ O2 for 6 days.
FLK-BLV supernatant contained BLV antigens to stimulate BLV-specific T cells in PBMC cultures as described
previously [9]. The heat-inactivated supernatant (2%) of
BLV-uninfected fetal lamb kidney cells (FLK) was used asa negative control antigen. All cell cultures were grown

To examine the effects of blocking mAbs on BLV-specificT-cell responses, in vitro blockade assay was performed.
PBMCs were cultivated with 10 μg/mL of blocking mAbs
anti-PD-L1 (4G12, rat I­gG2a) [20], anti-LAG-3 (71-2D8,
rat ­IgG1) [21], or rat IgG (Sigma–Aldrich) in the presence of 2% heat-inactivated FLK-BLV supernatant and1 μg/mL of agonist mAbs anti-CD3 (MM1A; Washington State University Monoclonal Antibody Center) andanti-CD28 (CC220; Bio-Rad) at 37 °C with 5% C
­ O2 for18 h. 2% heat-inactivated FLK supernatant was used as anegative control antigen. All cell cultures were grown in
96-well round-bottomed plates (Corning Inc.) containing

Table 1 Primary antibodies used in flow cytometric analysis of this paper
TargetIsotype

Clone

Source (reference)

Fluorochrome

Conjugation or labeling

Flow cytometric analysis of PD-1 and LAG-3
CD4

Mouse ­IgG2a

CC8

Bio-Rad

FITC(Conjugated primary antibody)

CD8

Mouse ­IgG2a

CC63

Bio-Rad

FITC(Conjugated primary antibody)

CD3

Mouse ­IgG1

MM1A

WSU monoclonal
Antibody Center

PerCp/Cy5.5

Lightning-Link PerCp/Cy5.5
Conjugation Kit (Innova Biosciences)

IgM

Mouse ­IgG1

IL-A30

Bio-Rad

PE/Cy7

Lightning-Link PE/Cy7
Conjugation Kit (Innova Biosciences)

TCR1-N24 (δ chain)

Mouse ­IgG2b

GB21A

WSU monoclonal
Antibody Center

APC/Cy7Lightning-Link APC/Cy7
Conjugation Kit (Innova Biosciences)

PD-1

Rat ­IgG2a

5D2

In house [13]APC

APC-conjugated anti-rat Ig antibody(Southern Biotech)

LAG-3

Rat ­IgG1

71-2D8In house [21]PE

PE-conjugated anti-rat ­IgG1 antibody(Southern Biotech)

Flow cytometric analysis of IFN-γ/TNF-α-producing T cells (intracellular staining)
CD4

Mouse ­IgG2a

CC8

Bio-Rad

FITC(Conjugated primary antibody)

CD8

Mouse ­IgG2a

CC63

Bio-Rad

PerCp/Cy5.5

Lightning-Link PerCp/Cy5.5
Conjugation Kit (Innova Biosciences)

TCR1-N24 (δ chain)

Mouse ­IgG2b

GB21A

WSU monoclonal
Antibody Center

Alexa Fluor 647

Zenon Alexa Fluor 647 Mouse ­IgG2bLabeling Kit (Thermo Fisher Scientific)

IFN-γ

Mouse ­IgG1

CC302

Bio-RadPE(Conjugated primary antibody)

TNF-α

Mouse ­IgG2b

CC328

Bio-RadAPC/eFluor780

Streptavidin APC/eFluor780(eBioscience)

FITC: fluorescein isothiocyanate, PerCp: peridinin-chlorophyll-protein complex, Cy: cyanin, PE: phycoerythrin, APC: allophycocyanin.

Okagawa et al. Vet Res (2018) 49:50

1 × 106 PBMCs in 250 μL RPMI 1640 medium (Sigma–
Aldrich) supplemented as described above. BFA (10 μg/
mL; Sigma–Aldrich) was added for the final 6 h toenhance intracellular cytokine staining signals by blocking transport processes. Cultured PBMCs were collected and incubated in PBS containing 10% goat serum
as described above. Cells were then washed and stainedwith anti-CD4-FITC mAb (CC8; Bio-Rad), anti-CD8PerCp/Cy5.5 mAb (CC63; Bio-Rad), and anti-TCR1N24-Alexa Fluor 647 mAb (anti-TCR δ chain; GB21A;
Washington State University Monoclonal Antibody
Center) for 30 min at 4 °C. CC63 was conjugated with
PerCp/Cy5.5 using Lightning-Link Antibody Labeling Kit(Innova Biosciences, Cambridge, England, UK). GB21A
was prelabeled with Zenon Alexa Fluor 647 Mouse­IgG2b Labeling Kit (Thermo Fisher Scientific). After surface staining, cells were fixed and permeabilized using
FOXP3 Fix/Perm kit (BioLegend, San Diego, CA, USA)according to the manufacturer’s protocol. Cells were thenstained with anti-IFN-γ-PE (CC302; Bio-Rad) and antiTNF-α-Biotin (CC328; Bio-Rad) for 30 min at 4 °C. Cells
were then washed and incubated with streptavidin APC/
eFluor780 (eBioscience, San Diego, CA) for 30 min at
4 °C. Finally, cells were washed and analyzed immediately
using FACS Verse (BD Biosciences) and FCS Express 4(De Novo Software). The primary antibodies used in thisexperiment are shown in Table 1. More than 10 000 lymphocytes were analyzed among the samples.
Statistics

Significant differences in the frequency of each T-cell
subset in expression analyses between groups wereidentified using Kruskal–Wallis test. A Friedman test
was used to compare the IFN-γ production and the frequency of each cytokine-producing T-cell subpopulation
between groups in blockade assays. All statistical tests
were performed using GraphPad Prism 6 (GraphPad
Software, San Diego, CA, USA). Differences were considered statistically significant when P < 0.05.

Results

Increased frequencies of heavily exhausted ­CD4+and ­CD8+ T cells in BLV‑infected cattle with B‑cell
lymphoma

Earlier studies have shown that immunoinhibitory
receptors such as PD-1 and LAG-3 were upregulated and play an immunomodulatory role in T-cell
exhaustion during BLV infection in cattle [13–15].
We therefore hypothesized that disease progression of BLV-infected cattle is associated with expansion of PD-1+LAG-3+ heavily exhausted T cells. To
test this hypothesis, the cell-surface expression of
PD-1 and LAG-3 on C
­ D4+, ­C D8+, and γδ T cells wasPage 4 of 12

investigated by multi-color flow cytometry of PBMCs
isolated from BLV-infected cattle in different diseasestages (AL, PL, and EBL). As shown in Figures 1A, 2A,
and 3A, ­C D4+γδTCR​−, ­C D8+γδTCR​−, or C
­ D8−γδTCR​++−
T cells were gated in ­C D3 IgM lymphocytes andthen analyzed for expression of PD-1 and LAG-3. The
percentage of PD-1+LAG-3+CD4+ T cells was significantly larger in the EBL animals than in the BLV-uninfected animals (Figure 1B). In the C­ D8+ T cell subset,++
the frequency of PD-1 LAG-3 CD8+ T cells was significantly higher in the EBL group than in the other
BLV-infected and BLV-uninfected groups (Figure 2B).
No remarkable changes were observed in the populations of PD-1+LAG-3−CD4+ and PD-1−LAG-3+CD8+
T cells of EBL animals (Figures 1B and 2C), but thepercentages of PD-1−LAG-3+CD4+ and PD-1+LAG3−CD8+ T cells were also significantly higher in the
EBL group (Figures 1C and 2B). In contrast, no significant differences were observed in any analyzed subsetsof ­C D8− γδ T cells among the tested groups (Figures 3B–D), although the number of samples available
for this analysis was small. To evaluate the status of all
the tested EBL animals and its contribution to the phenotype of T cells, the individual datasets were added as
Additional file 1. No tested parameters including ageof onset, lymphocyte counts, proviral load, tumor cell
type, and B-cell occupancy in blood were significantly
correlated with PD-1+LAG-3+CD4+ and PD-1+LAG3+CD8+ T-cell populations of the EBL animals. Taken
together, the frequency of heavily exhausted T cells(PD-1+LAG-3+) in the population of peripheral C­ D4++and ­C D8 T cells increases in BLV-infected cattle
bearing tumor.
Reactivation of IFN‑γ production by PD‑1 and LAG‑3
blockade

Several studies have shown that Th1 response including
IFN-γ production is impaired during disease progressionof BLV infection, especially in PL cattle [7–12]. In thisstudy, to determine whether BLV-specific IFN-γ response
was restored by the blockade using novel anti-PD-L1[20] and anti-LAG-3 mAbs [21], blockade assay were
performed using PBMCs of BLV-infected AL and PL
cattle. Single blockade using either anti-PD-L1 or antiLAG-3 mAbs showed significantly increased productionof IFN-γ from PBMCs of AL animals in response to BLV
antigen (7.5- and 4.6-fold over control IgG, respectively)(Figure 4A). In contrast, IFN-γ production in PBMCs of
PL animals were not significantly increased by PD-L1
or LAG-3 blockade, though the IFN-γ concentrations
tended to be increased by each blockade (3.3- and 4.6fold over control IgG, respectively) (Figure 4B). Notably, dual blockade by anti-PD-L1 and anti-LAG-3 mAbs

Okagawa et al. Vet Res (2018) 49:50

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Figure 1 Expression of PD-1 and LAG-3 on CD4+ T cells in BLV-infected cattle. A Gating strategy and representative dot plots for expression
analyses of PD-1 and LAG-3 on ­IgM−CD3+CD4+γδTCR​− T cells from peripheral blood of BLV-infected cattle (AL and EBL). Values in the quadrants
indicate percentages of cells. Percentages of PD-1+LAG-3+CD4+ T cells (B), PD-1+LAG-3−CD4+ T cells (C), and PD-1−LAG-3+CD4+ T cells (D) in­CD3+CD4+ T-cell population in peripheral blood from BLV-uninfected (BLV − ; n = 15), AL (n = 22), PL (n = 11), and EBL cattle (n = 7). Bars indicate
group median percentage. Significant differences between each group were determined using a Kruskal–Wallis test, where P < 0.05 and P < 0.001,
indicated by asterisks (* and ***, respectively).

significantly enhanced IFN-γ response to BLV antigen
in PBMCs of both AL and PL groups (16.4- and 6.7-foldover control IgG, respectively) (Figures 4A and B). There
results show that BLV-specific IFN-γ response can bereactivated by the in vitro blockade of PD-1/PD-L1 andLAG-3/MHC II interactions in BLV-infected cattle, evenwith progressed disease.

Okagawa et al. Vet Res (2018) 49:50

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Figure 2 Expression of PD-1 and LAG-3 on CD8+ T cells in BLV-infected cattle. A Gating strategy and representative dot plots for expression
analyses of PD-1 and LAG-3 on ­IgM−CD3+CD8+γδTCR​− T cells from peripheral blood of BLV-infected cattle (AL and EBL). Values in the quadrants
indicate percentages of cells. Percentages of PD-1+LAG-3+CD8+ T cells (B), PD-1+LAG-3−CD8+ T cells (C), and PD-1−LAG-3+CD8+ T cells (D) in­CD3+CD8+ T-cell population in peripheral blood from BLV − (n = 15), AL (n = 22), PL (n = 11), and EBL cattle (n = 7). Bars indicate group medianpercentage. Significant differences between each group were determined using a Kruskal–Wallis test, where P < 0.05, P < 0.01, and P < 0.001,
indicated by asterisks (*, **, and ***, respectively).

Reactivation of Th1 cytokine production from T cells
by PD‑1 and LAG‑3 blockade

Because we observed the enhancement of IFN-γ response
to BLV antigen by the blockade of the immunoinhibitory

pathways, it was important to determine which subsets
of T cells were induced to produce Th1 cytokines in
PBMC blockade. To address this issue, PBMC blockade
assay was performed in the presence of BLV antigen andOkagawa et al. Vet Res (2018) 49:50

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Figure 3 Expression of PD-1 and LAG-3 on γδ T cells in BLV-infected cattle. A Gating strategy and representative dot plots for expression
analyses of PD-1 and LAG-3 on ­IgM−CD3+CD8−γδTCR​+ T cells from peripheral blood of BLV-infected cattle (AL and EBL). Values in the quadrants
indicate percentages of cells. Percentages of PD-1+LAG-3+CD8− γδ T cells (B), PD-1+LAG-3−CD8− γδ T cells (C), and PD-1−LAG-3+CD8− γδ T cells(D) in ­CD3+CD8− γδ T-cell population in peripheral blood from BLV − (n = 6), AL (n = 13), PL (n = 5), and EBL cattle (n = 6). Bars indicate groupmedian percentage. Significant differences between each group were determined using a Kruskal–Wallis test.

agonist mAbs to CD3 and CD28, and then cells producing IFN-γ and TNF-α were analyzed in each T-cell subset by flow cytometry (AL n = 6, PL n = 2). As shownin Figure 5A, ­CD4+, ­CD8+γδTCR​−, or ­CD8−γδTCR​+
T cells were gated in lymphocytes and then analyzed

for expression of IFN-γ and TNF-α. Consistent with thedata of Figure 4, the frequency of IFN-γ producing cells
in total lymphocytes significantly increased by the dual
blockade using anti-PD-L1 and anti-LAG-3 mAbs (Figure 5B, left). Additionally, LAG-3 blockade significantly

Okagawa et al. Vet Res (2018) 49:50

Page 8 of 12anti-LAG-3 mAb with or without anti-PD-L1 mAb (Figure 5E, left). Overall, these results indicate that BLV-specific Th1 response can be reactivated by the blockade of
the PD-1 and LAG-3 pathways in BLV-infected cattle.

Figure 4 Effect of PD-1/PD-L1 and LAG-3/MHC II blockade
on BLV-specific IFN-γ response. PBMCs of BLV-infected AL (A;n = 12) and PL cattle (B; n = 6) were cultured with blocking mAbs
(anti-PD-L1 and anti-LAG-3 mAbs; 10 μg/mL) or rat IgG control in
the presence of FLK-BLV antigen (2%) for 6 days. IFN-γ production
for each animal was measured by ELISA in duplicate. Bars indicate
group median response. Significant differences between each groupwere determined using Friedman test, where P < 0.05 and P < 0.01,
indicated by asterisks (* and **, respectively).

enhanced TNF-α production in lymphocytes (Figure 5A,
center). Remarkably, IFN-γ/TNF-α double producers
are activated by anti-LAG-3 mAb alone and in combination with anti-PD-L1 mAb (Figure 5B, right). Phenotypic analyses of cytokine-producing T cells revealed that
­CD4+ and C­ D8+ T cells, but not C­ D8− γδ T cells, were
induced to produce the larger amounts of Th1 cytokines
by the blockade of the PD-1 and LAG-3 pathways (Figures 5C–E). Single or dual treatment of anti-PD-L1 andanti-LAG-3 mAbs induced significant increases in thepercentages of IFN-γ+TNF-α+CD4+ T cells (Figure 5C,
right). TNF-α production in ­CD4+ and ­CD8+ T cells
was significantly enhanced by single blockade using antiPD-L1 or anti-LAG-3 mAb (Figures 5C and D, center).
Unexpectedly, the frequencies of IFN-γ-producing ­CD8−γδ T cells were significantly higher in cultures using

Discussion
Cases of BLV infection and BLV-induced EBL in cattle have been increasing in Japan [24, 25]. Nevertheless,there is neither effective treatment nor vaccination tocontrol BLV infection, which is due in part to the lack ofunderstanding of the immunological mechanisms leading
to immune evasion. During BLV infection, especially in
the PL and EBL stages, T-cell exhaustion characterized by
impaired proliferation of BLV-specific T cells and downregulation of Th1 cytokines accelerates disease progression [7–12]. Previous studies have clarified the molecularmechanism responsible for this T-cell exhaustion, implicating immunoinhibitory receptors such as PD-1 andLAG-3 in this process [13–15]. PD-1 upregulation has
been observed in C
­ D4+ and ­CD8+ T cells in peripheral
blood and lymph nodes of EBL cattle [13]. Expression
level of LAG-3 upregulated on peripheral ­CD4+ and+­CD8 T cells in PL cattle and correlated positively withlymphocytosis and negatively with IFN-γ expression in­CD4+ T cells [15]. In the present study, we conducted
immunophenotypic and functional analyses of PD-1- andLAG-3-expressing exhausted T cells in BLV-infected cattle with different stages of disease.
The immunophenotypic analyses have revealed that
PD-1+LAG-3+ T cells are expanded in peripheral among­CD4+ and ­CD8+ T cells during the EBL stage but not
the PL stage. The tested EBL animals developed monoclonal or oligoclonal expansion of B cells in peripheral
blood (data not shown); therefore, these observations
indicate that PD-1+LAG-3+ T cells can be functionally exhausted and are associated with the tumor development. However, the contribution of these exhausted
T-cell subsets to tumor phenotype and pathogenesis is
not clear. In this study, no correlation was found between
the frequency of exhausted T-cell subsets in EBL animals
and age of the onset, disease parameters, or clinical signs(data not shown). Further immunophenotypic analysis of
PD-1+LAG-3+ T cells in tumor lesions of EBL animals isdesired to be conducted to address this issue.
In healthy cattle, PD-1 and LAG-3 are expressed mainlyon ­CD4+ and ­CD8+ T cells, respectively [13, 21]. Indeed,
PD-1+LAG-3− and PD-1−LAG-3+ T cells were mainly
observed in C
­ D4+ and C­ D8+ T-cell subsets, respectively, of BLV-uninfected, AL and PL animals. In the EBLgroup, not only the median percentages of PD-1+LAG3+CD4+ and PD-1+LAG-3+CD8+ T cells but also thatof PD-1−LAG-3+CD4+ and PD-1+LAG-3−CD8+ T cells
were larger than the other groups. These results imply

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Figure 5 Effect of PD-1/PD-L1 and LAG-3/MHC II blockade on Th1 cytokine production in BLV-specific T cells. PBMCs (n = 8; AL n = 6, PL n = 2)
were cultivated with 10 µg/mL each of blocking mAbs (anti-PD-L1 and anti-LAG-3 mAbs; 10 μg/mL) or rat IgG control in the presence of FLK-BLV antigen(2%) and anti-CD3 and anti-CD28 agonist mAbs (1 µg/mL each) for 18 h. A Gating strategy and representative dot plots for expression analyses of IFN-γ­ D8+γδTCR​− T cells, and C­ D8−γδTCR​+ T cells from peripheral blood of an AL animal. Values in the quadrants
and TNF-α on total lymphocytes, C­ D4+ T cells, C
indicate percentages of cells. Percentages of IFN-γ+ (left), TNF-α+ (center), IFN-γ+TNF-α+ cells (right) among total lymphocytes (B), ­CD4+ T cells (C),­CD8+γδTCR​− T cells (D), and C­ D8−γδTCR​+ T cells (E) in PBMCs treated with blocking mAbs. Bars indicate group median response. Significant differences
between each group were determined using Friedman test, where P < 0.05, P < 0.01, and P < 0.001, indicated by asterisks (*, **, and ***, respectively).

Okagawa et al. Vet Res (2018) 49:50

that upregulations of LAG-3 on ­CD4+ T cells and PD-1
on ­CD8+ T cells and subsequent co-expressions withother inhibitory receptors are remarkable change during the development of B-cell lymphoma in BLV-infected
cattle.
A large number of γδ T cells are found in T-cell population of cattle, comprising up to 80% of T cells inperipheral blood of young calves, and 10–20% of T cells
in adult cattle [26, 27]. Bovine γδ T cells have cytolytic
and cytokine secretory properties similar to those of αβT cells, thereby playing key roles in adaptive immunityof cattle [26, 27]. PD-1 expression is known to impair
IFN-γ production and cytolytic function in human γδ
T cells [28]. In addition, a previous study demonstrated
increase of LAG-3+ γδ T cells in peripheral blood of cattle during acute anaplasmosis [29]. In contrast, this study
found that the expression levels of PD-1 and LAG-3 were
not changed on ­CD8− γδ T cells during BLV infection.
Further experimentation is required to confirm immunomodulatory role of PD-1 and LAG-3 on bovine T
cells, but it seems that these immunoinhibitory receptors
might play the major role in C
­ D4+ and ­CD8+ αβ T-cell
subsets, not in γδ T cells, in cattle bearing lymphoma.
The mechanism underlying the upregulation of PD-1
and LAG-3 is unclear in BLV infection, but evidencesin humans and mice suggest that expression of PD-1
and LAG-3 is dependent on massive antigen presentation and the cytokine environment for a long period
during chronic diseases [19, 30, 31]. Therefore, period
post-infection could be associated with viral load andtumor development as well as the upregulation of PD-1
and LAG-3. The period post-infection of each tested
animal is unknown because these animals are naturally infected with BLV. The experimental infection is
helpful to investigate kinetics of the immunoinhibitory molecules throughout development of the disease.
Co-upregulation of PD-1 and LAG-3 was observed in­C D8+ T cells specific for viral or tumor antigens during chronic infection with lymphocytic choriomeningitis virus and human ovarian cancer [32, 33]. In
BLV-infected cattle, PD-1 and LAG-3 are expected tobe co-expressed on BLV-specific T cells. Confirmationof the relationship between PD-1+LAG-3+ T cells andantigen-specific immunosuppression in BLV infection
requires MHC-peptide tetramers for the detection of
BLV-specific T cells by flow cytometry.
A previous study has shown that proliferation of C
­ D4+
T cells in response to gag- and env-encoded BLV proteins is impaired in PL animals and is almost lost in EBLanimals, which is a critical evidence of the exhaustionof BLV-specific ­CD4+ T cells [8]. In this study, blockadeof the immunoinhibitory receptor-ligand interactions
using our novel blocking mAbs, anti-PD-L1 [20] andPage 10 of 12anti-LAG-3 mAbs [21], resulted in successful reactivationof IFN-γ production in response to whole BLV antigen
even in PL animals. According to flow cytometric analysis of IFN-γ- and TNF-α-producing T cells, blockade ofeither PD-1/PD-L1 or LAG-3 was sufficient to activate
double cytokine-producing multifunctional C­ D4+ T cells.
These results suggest that the dual blockade has a potential to prevent disease progression by restoring anti-viral
immunity during BLV infection. However, no significant
correlation between the blockade effects and clinicalparameters (proviral load, lymphocyte counts, and B cell
occupancy in blood) was identified in the tested animals(data not shown). Additionally, most of the expression
analyses and blockade assays were performed separately.
The data of PD-1 and LAG-3 expression is available only
for four animals which PBMC samples were also tested
for the blockade assay at the same time. Therefore, association between the frequency of PD-1+LAG-3+ T-cell subsets and the blockade outcomes is unclear. In this study,
the blockade assays were also performed using PBMCs of
EBL animals, but the number of T cells was so small thatthe level of IFN-γ production could not analyzed by IFN-γELISA and flow cytometry (data not shown). To evaluate
the blockade effect in EBL animals, more sensitive methods to detect IFN-γ production from BLV-specific T cells
are required, such as Enzyme-Linked ImmunoSpot assay
with epitope peptides or immunogenic proteins, notwhole viral protein [34, 35].
We have conducted immunophenotypic and functional analyses of exhausted T cells in various chronic
infections in cattle, such as BLV infection, Johne’s disease, bovine anaplasmosis, and bovine mycoplasmosis[21, 29, 36, 37]. Findings in these studies reveal that
T-cell exhaustion is caused by the upregulation of PD-1
and LAG-3 and presumably facilitates persistent infection and disease progression in cattle with chronic
infections. BLV infection is one of the good models of
infectious diseases causing T-cell exhaustion in cattle
as well as the disease to be addressed.
Numbers of researchers and pharmaceutical companies have investigated antibody treatments that blockthese “immune checkpoint” in human, and anti-PD-1,
anti-PD-L1, and anti-LAG-3 antibodies have been
approved for various human cancers or evaluated onclinical trials with cancer patients [38, 39]. We recently
established anti-bovine PD-1 and anti-bovine PD-L1
rat–bovine chimeric antibodies as candidate agentsfor blocking the PD-1/PD-L1 pathway in vivo [40, 41].
Administration of these chimeric antibodies to BLVinfected calves enhanced BLV-specific ­C D4+ T-cell proliferation and reduced BLV proviral load in vivo. Thus,targeting immunoinhibitory pathway is a significant
strategy for the regulation of T-cell response to BLV

Okagawa et al. Vet Res (2018) 49:50

infection in cattle. In order to establish novel immunotherapies against BLV infection, additional studies are
required to confirm the mechanisms underlying immunomodulatory and anti-viral effects of blocking antibody treatment in BLV-infected cattle.

Page 11 of 12

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Received: 23 December 2017 Accepted: 3 May 2018

Additional file
Additional file 1. Disease status and T-cell phenotype of individual
EBL animals tested in this study. To evaluate the status of all the tested
EBL cattle and its contribution to the phenotype of T cells, the individual
data are shown as Additional file 1. The dataset includes age of onset,
lymphocyte count in peripheral blood, BLV proviral load, tumor cell type,the percentage of B cells in peripheral blood, and the percentages of
PD-1+LAG-3+ cells, PD-1+LAG-3− cells, and PD-1−LAG-3+ cells in C
­ D4+,­CD8+, and γδTCR​+ T-cell subset of seven EBL animals tested in this study.
Materials and methods related to only this dataset are also shown in
additional file.

Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SK, SM, and KO were responsible for the conception and design of the study.
TO, SK, AN, NM, SG, and, RI performed the experiments. TO, SK, JK, YS, YK, SM,and KO analyzed the data. JK, YS, SY, and YK provided intellectual input, laboratory materials, reagents and/or analytic tools. TO wrote the manuscript. SK, JK,
YS, YK, SM, and KO contributed to the revision of the manuscript. All authors
read and approved the final manuscript.
Acknowledgements
We are grateful to Dr Hideyuki Takahashi, Dr Yasuyuki Mori, and Dr Tomio
Ibayashi for their valuable advice and discussions. We would like to thankEnago for the English language review.
Ethics approval and consent to participate
Not applicable.
Funding
This work was supported by grants-in-aid for Scientific Research from Japan
Society for the Promotion of Science (JSPS), by the Research Project for
Improving Animal Disease Prevention Technologies to Combat Antimicrobial
Resistance 2017-2021 FY, and by grants from the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, andFood Industry, Japan (number 26058B; to SK) and the NARO, Bio-oriented
Technology Research Advancement Institution (the special scheme project
on regional developing strategy; grant 16817557 to SK). This research waspartially supported by the Platform Project for Supporting Drug Discovery andLife Science Research (Basis for Supporting Innovative Drug Discovery and Life
Science Research, BINDS) and a grant from the Ministry of Education, Culture,
Sports, Science, and Technology (MEXT), Japan for the Joint Research Programof the Research Center for Zoonosis Control, Hokkaido University to YS. The
funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Author details
1
Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido
University, Sapporo 060‑0818, Japan. 2 Animal Research Center, Agriculture
Research Department, Hokkaido Research Organization, Shintoku 081‑0038,
Japan. 3 Division of Bioresources, Research Center for Zoonosis Control,
Hokkaido University, Sapporo 001‑0020, Japan. 4 Global Station for Zoonosis
Control, Global Institution for Collaborative Research and Education (GI‑CoRE),
Hokkaido University, Sapporo 001‑0020, Japan. 5 Department of Antibody
Drug Development, Graduate School of Medicine, Tohoku University, Sendai 980‑8575, Japan. 6 Present Address: Laboratory of Immunology, Facultyof Pharmacy, Osaka Ohtani University, Tondabayashi 584‑8540, Japan.

References
1. 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
2. Schwartz I, Bensaid A, Polack B, Perrin B, Berthelemy M, Levy D (1994)
In vivo leukocyte tropism of bovine leukemia virus in sheep and cattle. J
Virol 68:4589–4596
3. Mirsky ML, Olmstead CA, Da Y, Lewin HA (1996) The prevalence of proviral
bovine leukemia virus in peripheral blood mononuclear cells at two
subclinical stages of infection. J Virol 70:2178–2183
4. Gillet N, Florins A, Boxus M, Burteau C, Nigro A, Vandermeers F, Balon H,
Bouzar A-B, Defoiche J, Burny A, Reichert M, Kettmann R, Willems L (2007)
Mechanisms of leukemogenesis induced by bovine leukemia virus:
prospects for novel anti-retroviral therapies in human. Retrovirology 4:18
5. Kabeya H, Ohashi K, Onuma M (2001) Host immune responses in thecourse of bovine leukemia virus infection. J Vet Med Sci 63:703–708
6. Frie MC, Coussens PM (2015) Bovine leukemia virus: a major silent threat
to proper immune responses in cattle. Vet Immunol Immunopathol
163:103–114
7. 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
8. Orlik O, Splitter GA (1996) Progression to persistent lymphocytosis andtumor development in bovine leukemia virus (BLV)-infected cattle correlates with impaired proliferation of ­CD4+ T cells in response to gag- andenv-encoded BLV proteins. J Virol 70:7584–7593
9. Mager A, Masengo R, Mammerickx M, Letesson JJ (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 withnormal haematological values. J Gen Virol 75:2223–2231
10. Amills M, Ramiya V, Norimine J, Olmstead CA, Lewin HA (2002) Reduced
IL-2 and IL-4 mRNA expression in ­CD4+ T cells from bovine leukemia
virus-infected cows with persistent lymphocytosis. Virology 304:1–9
11. Pyeon D, O’Reilly KL, Splitter GA (1996) Increased interleukin-10 mRNA
expression in tumor-bearing or persistently lymphocytotic animals
infected with bovine leukemia virus. J Virol 70:5706–5710
12. Pyeon D, Splitter GA (1998) Interleukin-12 p40 mRNA expression inbovine leukemia virus-infected animals: increase in alymphocytosis but
decrease in persistent lymphocytosis. J Virol 72:6917–6921
13. Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y,
Murata S, Ohashi K (2013) Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro. Vet
Res 44:59
14. Shirai T, Konnai S, Ikebuchi R, Okagawa T, Suzuki S, Sunden Y, Onuma
M, Murata S, Ohashi K (2011) Molecular cloning of bovine lymphocyte
activation gene-3 and its expression characteristics in bovine leukemia
virus-infected cattle. Vet Immunol Immunopathol 144:462–467
15. Konnai S, Suzuki S, Shirai T, Ikebuchi R, Okagawa T, Sunden Y, Mingala
CN, Onuma M, Murata S, Ohashi K (2013) Enhanced expression of LAG-3
on lymphocyte subpopulations from persistently lymphocytotic cattle
infected with bovine leukemia virus. Comp Immunol Microbiol Infect Dis
36:63–69
16. 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 and enhancement of anti-viral immune responses in vitro via PD-L1
blockade. Vet Res 42:103
17. Stone DM, Hof AJ, Davis WC (1995) Up-regulation of IL-2 receptor alphaand MHC class II expression on lymphocyte subpopulations from bovine

Okagawa et al. Vet Res (2018) 49:50

18.

19.
20.

21.

22.

23.

24.
25.

26.
27.
28.
29.

30.

leukemia virus infected lymphocytotic cows. Vet Immunol Immunopathol 48:65–76
Isaacson JA, Flaming KP, Roth JA (1998) Increased MHC class II and CD25
expression on lymphocytes in the absence of persistent lymphocytosis incattle experimentally infected with bovine leukemia virus. Vet Immunol
Immunopathol 64:235–248
Wherry EJ (2011) T cell exhaustion. Nat Immunol 12:492–499
Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y,
Murata S, Ohashi K (2014) Influence of PD-L1 cross-linking on cell deathin PD-L1-expressing cell lines and bovine lymphocytes. Immunology
142:551–561
Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R,
Kawaji S, Tanaka S, Kagawa Y, Murata S, Mori Y, Ohashi K (2016) Bovine
immunoinhibitory receptors contribute to the suppression of Mycobacterium avium subsp. paratuberculosis-specific T-cell responses. InfectImmun 84:77–89
Tajima S, Takahashi M, Takeshima S, Konnai S, Yin SA, Watarai S, Tanaka
Y, Onuma M, Okada K, Aida Y (2003) A mutant form of the tax proteinof bovine leukemia virus (BLV), with enhanced transactivation activity,
increases expression and propagation of BLV in vitro but not in vivo. J
Virol 77:1894–1903
Nishimori A, Konnai S, Okagawa T, Maekawa N, Goto S, Ikebuchi R,
Nakahara A, Chiba Y, Ikeda M, Murata S, Ohashi K (2017) Identification of
an atypical enzootic bovine leukosis in Japan by using a novel classification of bovine leukemia based on immunophenotypic analysis. Clin Vacc
Immunol 24:e00067–e00117
Murakami K, Kobayashi S, Konishi M, Kameyama K, Yamamoto T, Tsutsui
T (2011) The recent prevalence of bovine leukemia virus (BLV) infection
among Japanese cattle. Vet Microbiol 148:84–88
Murakami K, Kobayashi S, Konishi M, Kameyama K, Tsutsui T (2013)
Nationwide survey of bovine leukemia virus infection among dairy
and beef breeding cattle in Japan from 2009–2011. J Vet Med Sci
75:1123–1126
Guzman E, Price S, Poulsom H, Hope J (2012) Bovine γδ T cells: cells withmultiple functions and important roles in immunity. Vet Immunol Immunopathol 148:161–167
Baldwin CL, Telfer JC (2015) The bovine model for elucidating the role ofγδ T cells in controlling infectious diseases of importance to cattle andhumans. Mol Immunol 66:34–47
Iwasaki M, Tanaka Y, Kobayashi H, Murata-Hirai K, Miyabe H, Sugie T, Toi M,
Minato N (2011) Expression and function of PD-1 in human γδ T cells thatrecognize phosphoantigens. Eur J Immunol 41:345–355
Okagawa T, Konnai S, Deringer JR, Ueti MW, Scoles GA, Murata S, Ohashi
K, Brown WC (2016) Cooperation of PD-1 and LAG-3 contributes to
T-cell exhaustion in Anaplasma marginale-infected cattle. Infect Immun
84:2779–2790
Kamphorst AO, Ahmed R (2013) Manipulating the PD-1 pathway to
improve immunity. Curr Opin Immunol 25:381–388

Page 12 of 12

31. Sierro S, Romero P, Speiser DE (2011) The CD4-like molecule LAG-3, biology and therapeutic applications. Expert Opin Ther Targets 15:91–101
32. Blackburn SD, Shin H, Haining WN, Zou T, Workman CJ, Polley A, Betts MR,
Freeman GJ, Vignali DAA, Wherry EJ (2009) Coregulation of ­CD8+ T cell
exhaustion by multiple inhibitory receptors during chronic viral infection.
Nat Immunol 10:29–37
33. Matsuzaki J, Gnjatic S, Mhawech-Fauceglia P, Beck A, Miller A, Tsuji T,
Eppolito C, Qian F, Lele S, Shrikant P, Old LJ, Odunsi K (2010) Tumorinfiltrating NY-ESO-1–specific C­ D8+ T cells are negatively regulated
by LAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci U S A
107:7875–7880
34. Han S, Norimine J, Palmer GH, Mwangi W, Lahmers KK, Brown WC (2008)
Rapid deletion of antigen-specific ­CD4+ T cells following infection
represents a strategy of immune evasion and persistence for Anaplasma
marginale. J Immunol 181:7759–7769
35. Maggioli MF, Palmer MV, Thacker TC, Vordermeier HM, Waters WR (2015)
Characterization of effector and memory T cell subsets in the immune
response to bovine tuberculosis in cattle. PLoS One 10:e0122571
36. Goto S, Konnai S, Okagawa T, Nishimori A, Maekawa N, Gondaira S, Higuchi H, Koiwa M, Tajima M, Kohara J, Ogasawara S, Kato Y, Suzuki Y, Murata
S, Ohashi K (2017) Increase of cells expressing PD-1 and PD-L1 andenhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine
mycoplasmosis. Immun Inflamm Dis 5:355–363
37. Konnai S, Murata S, Ohashi K (2017) Immune exhaustion during chronic
infections in cattle. J Vet Med Sci 79:1–5
38. Lipson EJ, Forde PM, Hammers HJ, Emens LA, Taube JM, Topalian SL(2015) Antagonists of PD-1 and PD-L1 in cancer treatment. Semin Oncol
42:587–600
39. Sheridan C (2015) Immuno-oncology moves beyond PD-1. Nat Biotechnol 33:673–675
40. 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
41. 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 (PDL1) rat-bovine chimeric antibody against bovine leukemia virus infection.
PLoS One 12:e0174916