PATHOGENESIS AND IMMUNITY

Combined Immune Checkpoint Blockade Enhances Antiviral
Immunity against Bovine Leukemia Virus
Hayato Nakamura,a Satoru Konnai,a,b,c Tomohiro Okagawa,b Naoya Maekawa,b Yamato Sajiki,a Kei Watari,a Kana Kamitani,dMaya Saito,d Yukinari Kato,e,f Yasuhiko Suzuki,b,c,g Shiro Murata,a,b Kazuhiko Ohashia,bDepartment of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japana

Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, JapanbcInstitute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan
Hokkaido Hayakita Meat Inspection Center, Hokkaido Government, Abira, Japand

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

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japanf

Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japang

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine
leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent
studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression
and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain andmucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection.
Therefore, this study was designed to further elucidate the immunoinhibitory role ofimmune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on
peripheral CD41 and CD81 T cells in BLV-infected cattle. Interestingly, in EBL cattle,
CD41 and CD81 T cells inﬁltrating lymphomas expressed TIM-3. TIM-3 and PD-1 wereupregulated and coexpressed in peripheral CD41 and CD81 T cells from BLV-infected
cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression
on T cells and gamma interferon (IFN-g ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated
the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition
of TIM-3 and PD-1 pathways signiﬁcantly enhanced IFN-g production and antiviral efﬁcacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3
and PD-1 pathways shows strong immune activation and antiviral effects and has
potential as a novel therapeutic method for BLV infection.
Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an
important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex.
Previously, it was found that immune checkpoint molecules, such as PD-1, suppress
BLV-speciﬁc Th1 responses as the disease progresses. To date, most studies havefocused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule,regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1
inhibition alone was insufﬁcient to exert anti-BLV effects in previous clinical studies.
In this study, the expression proﬁle of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected
cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in inﬂuencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral

IMPORTANCE

January 2023 Volume 97 Issue 1

Editor Frank Kirchhoff, Ulm University Medical
CenterCopyright © 2023 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Satoru Konnai,
konnai@vetmed.hokudai.ac.jp.
The authors declare no conﬂict of interest.
Received 11 October 2022
Accepted 8 December 2022
Published 4 January 2023

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ABSTRACTEnhanced Antiviral Immunity against BLV

Journal of Virology

cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically
restored antiviral immunity. This study could open new avenues for treating bovinechronic infections.
KEYWORDS bovine leukemia virus, TIM-3, PD-L1, lymphoma, cattle

n chronic infections and malignancies, T cells exposed to persistent antigenic stimuli
and inﬂammation become exhausted T cells that are unable to perform their naturalimmune functions even after antigen presentation due to decreased production of cytokines, such as interferon-g (IFN-g ), and decreased cellular proliferation (1). Exhausted T
cells exhibit increased expression of immunosuppressive factors, such as programmed
death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3(LAG-3), and T-cell immunoglobulin and mucin domain-3 (TIM-3). Exhausted T cells cannot respond to activation from T-cell receptors (TCRs) and costimulatory receptors via inhibition of activation signals, thereby suppressing T-cell effector functions (1). T-cell
exhaustion mediated by immunosuppressive factors is involved in the progression andmaintenance of pathological conditions in various chronic infections and malignancies.
Interestingly, T-cell exhaustion interferes with immune responses against infections andtumors, but inhibiting immunosuppressive factors with speciﬁc antibodies can reverse
this exhaustion and activate immune responses (2, 3). Thus, inhibitory antibodies against
immunosuppressive factors may be an effective treatment for chronic infections andmalignancies that have been difﬁcult to treat.
TIM-3 is a transmembrane protein on the surface of cell membranes belonging to
the TIM family and has been identiﬁed as a suppressor of CD41 Th1 cells (4). TIM-3 isexpressed on Th1 cells, CD81 cytotoxic T cells (5), dendritic cells and monocytes (6),and natural killer cells in humans and mice (7). The intracellular region of TIM-3 contains tyrosine kinase phosphorylation sites; TIM-3 functions as a tyrosine kinase-type
receptor (4, 8). Various factors have been reported as ligands for TIM-3, including galectin-9 (Gal-9) and carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1) (9–12). When TIM-3 binds to Gal-9 or Ceacam-1, it phosphorylates tyrosine residues
in the intracellular region and suppresses TCR signaling via FYN, another tyrosine kinase (12–14). As a result, production of cytokines (Th1 cytokines, such as IFN-g andinterleukin-2 [IL-2], and inﬂammatory cytokines, such as tumor necrosis factor alpha[TNF-a] and IL-6) and cell proliferation are inhibited, and T-cell death is induced (9, 15,16). Interestingly, the inhibitory effects of TIM-3 are ineffective when Ceacam-1 is deﬁcient (12, 17). Because TIM-3 is highly expressed, especially in IFN-g -secreting Th1 cells,
cellular immunity is suppressed by selectively inducing cell death in Th1 cells (9).
Studies using knockout mice have reported that Th1 cell-speciﬁc TIM-3 suppresses
autoimmune diseases and transplant immune responses (18). Thus, TIM-3 is considered
to be a suppressor of T-cell function.
In lymphocytic choriomeningitis virus (LCMV)-infected mice, a chronic infection
model, TIM-3 is highly expressed in LCMV-speciﬁc CD81 T cells (19). In HIV-infected
CD41 and HIV-speciﬁc CD81 T cells, TIM-3 expression, which increases with disease progression, promotes T-cell exhaustion (5). During hepatitis C virus (HCV) infection, suppression of CD41 and HCV-speciﬁc CD81 T-cell function by TIM-3 is associated withpathogenesis (20, 21). In contrast, the production of Gal-9, a ligand for TIM-3, is upregulated in liver Kupffer cells during HCV infection. Gal-9 induces the expansion of CD41
CD251 Foxp31 regulatory T cells (Tregs) and apoptosis of HCV-speciﬁc CD81 T cells (22).
Such disease progression involving elevated TIM-3 expression has been reported not
only in infectious diseases but also in tumor diseases, such as malignant melanoma (23).
Studies in mouse tumor models and human tumor patients have shown that increased
TIM-3 expression in tumor-inﬁltrating T cells (TILs) suppresses cellular immune responses,
including cytokine production (24, 25). These ﬁndings suggest that the TIM-3 pathway
may contribute to immune evasion mechanisms in chronic infectious and tumor
diseases.
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Bovine leukemia virus (BLV) infection is one of the most devastating bovine infectious diseases in Japan. BLV has a genetic structure most closely related tohuman T-cell leukemia virus (HTLV) (26) and persistently infects bovine lymphocytes, primarily B cells (27, 28). It causes enzootic bovine leukosis (EBL), leading tosystemic lymphosarcoma formation, poor prognosis, and death (29). Although viralfactors and the host immune response have been implicated in the development
and progression of EBL (30, 31), the exact pathogenesis of the disease has not yet
been elucidated. Comparative analysis of BLV-infected cattle by disease state
revealed that persistently lymphocytotic (PL) cattle with increased B-cell lymphocyte counts had decreased expression of Th1 cytokines such as IFN-g , IL-2, and IL12 compared to alymphocytotic (AL) cattle with normal lymphocyte counts (32–35). CD41 T cells from EBL and PL cattle exhibit a decreased T-cell proliferative
response to BLV antigens (33) and increased IL-10 mRNA expression, indicating ashift to humoral immunity (32). These ﬁndings suggest that immunosuppression,
primarily a decrease in cellular immunity, is a major factor in disease progressionin BLV-infected cattle.
Immunosuppression during BLV infection is associated with PD-1/PD-ligand 1 (PDL1) (36–42), LAG-3 (43–45), and CTLA-4 (46, 47) expression; inhibition of each of thesefactors can have an immunostimulatory effect. In addition, the proportion of PD-11
LAG-31 CD41 and PD-11 LAG-31 CD81 T cells is increased in EBL-affected cattle; simultaneous inhibition of PD-1/PD-L1 and LAG-3 pathways in peripheral blood mononuclear cells (PBMCs) from BLV-infected cattle has an immunostimulatory effect (45).
TIM-3 mRNA expression increases in CD41 and CD81 T cells as BLV infection progresses, and inhibition of the TIM-3 pathway increases IFN-g and IL-2 mRNA expression(48). Thus, these data suggest that TIM-3 is involved in immunosuppression during BLV
infection; the TIM-3 inhibition and simultaneous inhibition of TIM-3 and other immunosuppressive factors may have overall immunostimulatory effects. In this study, an antibovine TIM-3 monoclonal antibody (MAb) was generated to investigate whether TIM3-targeted therapies effectively improve immune function in BLV infection. First, TIM-3
expression on T cells from BLV-infected cattle was analyzed, and the association
between bovine TIM-3 and immunosuppression and the immunostimulatory effects of
TIM-3 pathway inhibition were examined using an anti-TIM-3 antibody. The immunostimulatory and antiviral effects of inhibiting both pathways were examined using antiTIM-3 and anti-PD-L1 antibodies.
RESULTS
Increased TIM-3 expression on T cells in BLV-infected cattle. First, TIM-3 expression on CD41 and CD81 T cells in PBMCs from uninfected and BLV-infected cattle wasanalyzed by ﬂow cytometry. Flow cytometric analyses showed that the percentage ofTIM-31 CD41 T cells was signiﬁcantly higher in BLV-infected cattle than in uninfected
cattle (Fig. 1A). The percentage of TIM-31 CD81 T cells was also signiﬁcantly higher in
BLV-infected cattle than in uninfected cattle (Fig. 1A), indicating that TIM-3 expression
was elevated in CD41 and CD81 T cells in peripheral blood of BLV-infected cattle. TIM3 expression on inﬁltrating CD41 and CD81 T cells in the lymph nodes of uninfected
cattle and cattle with EBL was analyzed. The percentages of TIM-31 CD41 and TIM-31
CD81 T cells were signiﬁcantly higher in tumorigenic, superﬁcial cervical lymph nodes(Fig. 1B) and mesenteric lymph nodes (Fig. 1C) derived from cattle with EBL than inthose from uninfected cattle.
Immune activation by TIM-3 blockade. To clarify the effects of TIM-3 blockade on
bovine T-cell responses, bioassays were ﬁrst conducted using PBMCs from healthy cattle. PBMCs cultured in vitro with anti-TIM-3 antibody increased the percentage of
CD69-expressing cells in CD41 and CD81 T cells (Fig. 2A). The percentage of CD41 andCD81 T cells expressing IFN-g (Fig. 2B) increased with in vitro culture with anti-TIM-3
antibody. Consistent with the increase in cells expressing intracellular IFN-g , blockadewith anti-TIM-3 antibody increased IFN-g production in the supernatant (Fig. 2C).
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FIG 1 Percentages of TIM-31 CD41 or TIM-31 CD81 T cells from BLV-infected and uninfected cattle.
(A) TIM-3 expression on CD41 and CD81 T cells in PBMCs from uninfected cattle [BLV (2); n = 11]
and BLV-infected cattle [BLV (1); n = 10] was measured by ﬂow cytometry. (B and C) TIM-3
expression was measured in inﬁltrating CD41 or CD81 T cells from tumorigenic shallow cervicallymph nodes (B) or tumorigenic mesenteric lymph nodes (C) in EBL cattle (n = 12). TIM-3 expression
on CD41 or CD81 T cells in superﬁcial cervical lymph nodes (B) or mesenteric lymph nodes (C) from
uninfected bovines is shown as controls [BLV (2); n = 7]. Statistical signiﬁcance was determined using
the Mann-Whitney U test. The median values for each group are indicated by red bars.

These results indicated that inhibition of the TIM-3 pathway activates bovine T cells
and increases IFN-g production.
Next, PBMCs from BLV-infected cattle were used to examine the effects of the TIM-3 inhibition on the activation of T-cell responses to BLV antigen. PBMCs derived from BLVinfected cattle were cultured under BLV antigen stimulation along with anti-TIM-3 antibody.
CD69 expression and IFN-g production were analyzed. In vitro culture of PBMCs from BLVinfected cattle with anti-TIM-3 antibody increased the percentage of CD69-expressing
CD41 and CD81 T cells (Fig. 3A) and IFN-g production in the supernatant (Fig. 3B). These
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FIG 2 Effects of T-cell activation by inhibiting the TIM-3 pathway using PBMCs derived from healthy
cattle. Bovine PBMCs stimulated with anti-bovine CD3 and CD28 MAbs were cultured with anti-TIM-3
antibody for 24 h. CD69 expression (A; n = 9) and IFN-g intracellular expression (B; n = 18) were
measured by ﬂow cytometry. The number of IFN-g 1 CD41 T cells was reported relative to T-cellstimulation alone. After 3 days of cultivation, IFN-g in the culture supernatant was measured by ELISA(C; n = 9). Mouse IgG1 antibody was used as a negative control (Cont.). Each antibody was added toa ﬁnal concentration of 10 m g/mL. Statistical signiﬁcance was determined by the Wilcoxon signedrank test.

results indicated that inhibition of the TIM-3 pathway could reactivate T-cell responses to
BLV antigens in exhausted T cells from BLV-infected cattle.
Enhancement of antiviral immunity against BLV by combined immune checkpoint blockade. In previous studies, PD-1 expression was upregulated in BLV infection. In
this study, TIM-3 and PD-1 expression levels in CD41 and CD81 T cells in PBMCs from BLVinfected cattle were simultaneously analyzed by ﬂow cytometry (Fig. 4A). The percentage
of TIM-31 PD-11 CD41 T cells was signiﬁcantly higher in BLV-infected cattle than in uninfected cattle (Fig. 4B). The percentage of TIM-31 PD-11 CD81 T cells was also signiﬁcantly
higher in PBMCs from BLV-infected cattle than in uninfected cattle (Fig. 4B).
The percentage of T cells coexpressing TIM-3 and PD-1 increased in BLV-infected
cattle. Thus, it was expected that the simultaneous inhibition of TIM-3 and PD-1/PD-L1
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FIG 3 Effects of T-cell activation by inhibiting the TIM-3 pathway using PBMCs derived from BLVinfected cattle. PBMCs derived from BLV-infected cattle were incubated with anti-TIM-3 antibody inthe presence of BLV antigen (Ag) for 24 h. CD69 expression on CD41 and CD81 T cells was measured
by ﬂow cytometry (A; n = 8). IFN-g production in the culture supernatant was measured by ELISA (B;n = 18). Mouse IgG1 antibody was used as a negative control. Each antibody was added to a ﬁnal
concentration of 10 m g/mL.

pathways would enhance the immunostimulatory effects. Therefore, PBMCs derived
from BLV-infected cattle were cultured with anti-TIM-3 and anti-PD-L1 antibodies
under BLV antigen stimulation. The production of Th1 cytokines, IFN-g and TNF-a, wasanalyzed by enzyme-linked immunosorbent assay (ELISA). These results indicated thatthe addition of anti-TIM-3 and anti-PD-L1 antibodies as single agents signiﬁcantly
increased IFN-g production compared to control antibodies (Fig. 5A). The combined
treatment with anti-TIM-3 and anti-PD-L1 antibodies resulted in a further signiﬁcant
increase in IFN-g production compared to treatment with each alone (Fig. 5A). In addition, TNF-a production increased after combined treatment with anti-TIM-3 and antiPD-L1 antibodies, although there was no signiﬁcant difference from that after thetreatment with each alone (Fig. 5B).
The simultaneous inhibition of TIM-3 and PD-1/PD-L1 pathways in BLV-infected cattle can reactivate exhausted T cells and strongly enhance T-cell responses to BLV.
Therefore, the antiviral effects of TIM-3 and PD-1/PD-L1 pathway inhibition were evaluated by syncytium formation assay (Fig. 6A). Treatment with anti-TIM-3 or anti-PD-L1
antibodies signiﬁcantly inhibited BLV-induced syncytium formation compared to controls; the combination of antibodies resulted in a stronger antiviral effect than singleagent treatment (Fig. 6B). These results indicated that inhibition of TIM-3 and PD-1/PDL1 pathways has antiviral effects against BLV.
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FIG 4 TIM-3/PD-1 coexpression in CD41 and CD81 T cells in PBMCs derived from BLV-infected cattle. (A) Gating strategy used for
TIM-3 and PD-1 staining. Total lymphocytes were ﬁrst gated in a forward scatter (FSC)/side scatter (SSC) plot and then in a single
population. Cells were subsequently gated for the CD31 population and further gated for CD4 or CD8 expression. Gated CD41 andCD81 T cells were analyzed for TIM-3 and PD-1 coexpression, respectively. (B) Percentage of TIM-31 PD-11 CD41 or TIM-31 PD-11
CD81 T cells in uninfected cattle [BLV (2); n = 10] and BLV-infected cattle [BLV (1); n = 13]. The Steel-Dwass test was used forstatistical analysis.

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FIG 5 Effects of the dual inhibition of TIM-3 and PD-1 pathways on antiviral cytokine production
using PBMCs derived from BLV-infected cattle. PBMCs from BLV-infected cattle (n = 20) were cultured
with anti-TIM-3 and/or anti-PD-L1 antibodies in the presence of BLV antigen. IFN-g (A) and TNF-a (B)concentrations in culture supernatants were measured by ELISA. Statistical signiﬁcance wasdetermined by the Steel-Dwass test.

DISCUSSION
Previous studies have shown that immunosuppressive receptors, such as PD-1,
LAG-3, and CTLA-4, suppress cellular immunity during BLV infection (36–47). Anotherimmunosuppressive receptor, TIM-3, has also been identiﬁed in cattle. A preliminary
study showed that TIM-3 mRNA expression increases in CD41 and CD81 T cells in the
blood as BLV infection progresses (49). However, the details of TIM-3-mediated immunosuppressive mechanisms in BLV-infected cattle were unknown. This study analyzed
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FIG 6 Antiviral effects of the dual inhibition of TIM-3 and PD-1 pathways using PBMCs derived from BLVinfected cattle. Indicator cells (CC811; 0.6  105) were cultured at 37°C for 24 h in the presence of 5% CO2.
Anti-TIM-3 and anti-PD-L1 antibodies were added simultaneously with PBMCs derived from BLV-infected cattle(n = 12; 5.0  105 cells) and cultured for 3 days. Mouse and rat IgG antibodies were used as negative controls.
Each antibody was added at a ﬁnal concentration of 10 m g/mL. After incubation, Giemsa staining was
performed, and the number of syncytia containing >10 nuclei was measured. (A) Representative pictures ofsyncytium formation in each treatment. (B) Number of syncytia in each treatment. Three wells were cultured
with PBMCs from each specimen bovine, and results were expressed as the mean from three wells. The SteelDwass test was used for statistical analysis. Median values for each group are indicated by red bars.

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the expression and function of TIM-3 protein in BLV-infected cattle and its association
with immunosuppression during the pathological progression of BLV infection.
TIM-3 protein expression in CD41 and CD81 T cells in peripheral blood of BLVinfected cattle was higher than in uninfected cattle, consistent with previous TIM-3
mRNA kinetic analysis (49). A study that analyzed TIM-3 expression in patients infectedwith HIV, a member of the same family, retroviridae, as BLV, also reported that CD41
and CD81 T cells in peripheral blood had expressed higher levels of TIM-3 protein (5).
This suggested that TIM-3 may also be involved in suppressing cellular immunity in
BLV infection. In contrast, TIM-3 expression decreased in patients infected with HTLV, avirus closely related to BLV (49, 50). All BLV-infected cattle analyzed in this study were
naturally infected; the timing of BLV infection and the stage of infection at the time ofthis analysis were unknown. Therefore, the detailed role of TIM-3 in BLV pathogenesis
from infection to the onset of EBL requires further investigation. Interestingly, analysis
of TIM-3 expression in BLV-infected cattle with EBL revealed increased TIM-3 protein
expression in CD41 and CD81 T cells inﬁltrating tumorigenic lymph nodes. Similar tothis ﬁnding, increased PD-1 expression was also observed in CD41 and CD81 T cells inperipheral blood and lymph nodes of EBL cattle (38). Studies in mouse models of colon
adenocarcinoma and human renal cell carcinoma reported increased TIM-31 tumorinﬁltrating lymphocytes in tumors and decreased production of Th1 cytokines, such as
IFN-g , in TIM-31 TILs (24, 25). These ﬁndings suggested that T cells with high expression of TIM-3 are exhausted, and Th1 responses are reduced in tumorigenic lymph
nodes in EBL and other cancers in mice and humans. In this study, the number of collected TILs was limited, and functional analysis was not possible. It remains to be elucidated whether TIM-31 TILs, like TIM-31 T cells in peripheral blood, decrease the production of Th1 cytokines, such as IFN-g , and inhibit tumor clearance.
Although the mechanism of elevated TIM-3 expression in BLV infection is unknown
in this study, prostaglandin E2 (PGE2) may trigger the induction of TIM-3 expression.
Previous studies showed that the PD-1/PD-L1 pathway suppresses cellular immunity inbovine chronic infectious diseases, such as BLV infection (36–42), Johne’s disease (51–53), bovine anaplasmosis (54), and bovine mycoplasmosis (55, 56). In addition to thefunction of the PD-1/PD-L1 pathway in cattle, PGE2 is involved in suppressing Th1
responses in bovine chronic infections. Speciﬁcally, this study focused on the functionof PGE2 because serum PGE2 levels are elevated in bovine chronic infectious diseases,
including BLV infection. PGE2 induces PD-L1 expression in bovine immune cells via its
receptor, E-prostanoid 4, thereby suppressing Th1 responses. More recently, PGE2
induced the expression of not only PD-1 and PD-L1 but also LAG-3 and CTLA-4 (42, 57).
Based on these data thus far, it is possible that PGE2 induced by BLV also induces TIM-3
expression, which will be veriﬁed in future investigations.
Previously, several clinical studies were performed using an anti-bovine PD-L1 or
PD-1 rat-bovine chimeric antibody (chAb) administered to BLV-infected cattle (40–42).
The administration of these chAbs decreased the BLV provirus load in cattle. However,certain BLV-infected cattle did not respond to this treatment, suggesting the presenceof immunosuppressive mechanisms using molecules other than those in the PD-1/PDL1 pathway. In this study, increased TIM-3 expression was observed in T cells in peripheral blood and tumorigenic lymph nodes of BLV-infected cattle. Interestingly, the percentage of PD-11 TIM-31 CD41 and PD-11 TIM-31 CD81 T cells increased in peripheral
blood of BLV-infected cattle. These results suggested that PD-11 TIM-31 T cells may be
involved in pathological progression in BLV infection by inducing stronger immune
exhaustion. In addition, Tregs that suppress immunity may be involved. Previous studies have suggested that Tregs increase with the progression of BLV infection, causing
immunosuppression (35, 46). In mouse models of colorectal cancer, PD-1 expression
increased on TIM-31 Tregs, and expression of inhibitory factors, such as IL-10 and
CTLA-4, is elevated compared to that in TIM-3-negative Tregs (58, 59). In this study, asigniﬁcant increase in the percentage of PD-11 TIM-31 CD41 T cells was observed in
BLV-infected cattle. This suggested that BLV infection may increase PD-1 and TIM-3
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expression on Tregs and suppress the immune function of effector T cells. Further analysis of PD-1 and TIM-3 expression in Tregs is needed to clarify the immunomodulatory
function of Tregs in BLV infection.
In a previous report, IFN-g and IL-2 mRNA expression was upregulated when PBMCs
from BLV-infected cattle were cultured with Cos-7 cells expressing TIM-3 to inhibit the
TIM-3/Gal-9 pathway (48). Moreover, the combined blockade of TIM-3/Gal-9 and PD-1/
PD-L1 pathways signiﬁcantly promoted IFN-g mRNA expression compared to theblockade of the PD-1/PD-L1 pathway alone (48). Although preliminary results suggested that TIM-3 is involved in suppressing T-cell function during BLV infection, theyalso suggested that TIM-3 may be a therapeutic target for restoring antiviral efﬁcacy.
Indeed, TIM-3 blockade by anti-bovine TIM-3 MAb restored T-cell activation and IFN-g
production in PBMCs from BLV-infected cattle in this study. Dual inhibition of bovine
TIM-3 and PD-1/PD-L1 pathways by their respective antibodies enhanced IFN-g and
TNF-a production compared to that after inhibition of either pathway alone, resulting
in potent antiviral activity against BLV. Presumably, dual inhibition of TIM-3 and PD-1/
PD-L1 pathways effectively activated severely exhausted CD41 and CD81 T cells,
enhancing their antiviral activity. However, in human cancers, inhibition of the PD-1/
PD-L1 pathway produces dramatic antitumor effects in some patients but is partial orineffective in most patients, requiring new strategies to overcome resistance to PD-1/
PD-L1 therapy (60, 61). Similarly, when chAbs to PD-L1 were administered to BLVinfected cattle, certain BLV-infected cattle were found not to respond to the treatment(42). Interestingly, PD-1/PD-L1-targeted therapy increases TIM-3 expression in peripheral T cells and TILs in mouse models of lung adenocarcinoma and squamous cell carcinoma (62, 63). TNF-a, an antiviral cytokine, increases TIM-3 expression (64). In thisstudy, enhanced TNF-a production was observed when both bovine TIM-3 and PD-1/
PD-L1 pathways were inhibited either alone or simultaneously. Recently, anti-PD-L1 Ab
treatment induced TNF-a production, and TNF-a blockade by TNF-RII-Ig reduces PGE2
production in the presence of anti-PD-L1 Ab, suggesting that anti-PD-L1 antibodyinduced TNF-a impairs T-cell activation by PGE2 upregulation (65). Thus, TNF-a,although important as an antiviral cytokine, may be responsible for the immunosuppression associated with TIM-3. It is possible that cytokines (including TNF-a) increased
via PD-1/PD-L1 pathway inhibition induce increased TIM-3 expression, leading to resistance to PD-1/PD-L1-targeted therapy through compensatory immunosuppression of
the TIM-3 pathway. Therefore, combination therapy with anti-TIM-3 and anti-PD-L1
antibodies is also expected to be an effective strategy to overcome resistance to PDL1-targeted therapies. It is possible that the reason for the limited antiviral efﬁcacy ofanti-bovine PD-L1 or PD-1 rat-bovine chAb in previous clinical trials against BLV wasthe compensatory induction of TIM-3. This point should be investigated in the future.
These results suggested that bovine TIM-3 is upregulated in T cells during BLV
infection and plays a role in immunosuppressive mechanisms in pathogenesis. In
addition, targeting the TIM-3 pathway in cattle, as well as in humans and mice, has
immunostimulatory effects, as there are many immunosuppressive factors associatedwith T-cell exhaustion, such as TIM-3 and PD-1 (1, 66). Inhibition of these immunosuppressive pathways restores and enhances the function of exhausted T cells andthus has potential therapeutic applications in chronic infectious diseases and tumor
diseases. This study demonstrated the potential of targeting TIM-3 as a new control
strategy against bovine chronic infections. Future studies need to determine the relationship between TIM-3-mediated immunosuppressive mechanisms and pathologicalprogression in various bovine chronic diseases and apply this new immunoregulation
method to bovine diseases.
MATERIALS AND METHODSBovine blood and tumor samples. This study was conducted at the Faculty of Veterinary Medicine,
Hokkaido University, which is approved by the Association for Assessment and Accreditation ofLaboratory Animal Care International. All experimental procedures were conducted with the approval of
the Hokkaido University Laboratory Animal Committee (approval no. 17-0024). Blood samples wereJanuary 2023 Volume 97 Issue 1

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collected from cattle (Holstein breed, adult cattle) at the Field Science Center for the Northern
Biosphere, Hokkaido University, and dairy farmers in Hokkaido. The samples were submitted for analysis
within 2 days. PBMCs were isolated from the collected blood by density gradient centrifugation using
Percoll (GE Healthcare, Chicago, IL, USA) for ﬂow cytometry and cell bioassays.
Shallow cervical and mesenteric lymph nodes from cattle (Holstein breed, Japanese Black breed, andcrossbreed) with or without lymphoma were donated by the Hokkaido Hayakita Meat Inspection Center(Abira, Hokkaido). Those lymph nodes were randomly collected for this study. The lymph nodes wereshredded with scissors, separated into a single-cell suspension using a 40-m m cell strainer (BDBiosciences, Franklin Lakes, NJ, USA), and washed twice with phosphate-buffered saline (PBS; pH 7.2)
supplemented with 0.5 mg/mL disodium EDTA (Dojindo Molecular Technologies, Kumamoto, Japan).
The isolated single cells were suspended in deactivated fetal bovine serum (Thermo Fisher Scientiﬁc,Waltham, MA, USA) supplemented with 10% dimethyl sulfoxide (Nacalai Tesque, Inc., Kyoto, Japan) andstored at 280°C until analysis.
BLV infection was conﬁrmed by detection of anti-BLV antibodies in serum using a commercial ELISA
kit (JNC, Tokyo, Japan) and quantitative real-time PCR using a LightCycler 480 System II (Roche
Diagnostics, Mannheim, Germany) with a BLV detection kit (TaKaRa Bio, Shiga, Japan). BLV-associatedlymphomas were further diagnosed via immunophenotyping using ﬂow cytometry (40) to determine if
EBL was due to BLV infection.
Analysis of TIM-3 and PD-1 expression by ﬂow cytometry. TIM-3 and PD-1 expression on CD41
and CD81 T cells in PBMCs or EBL-derived cells was measured by ﬂow cytometry using the antibodies
listed in Table 1. To inhibit the nonspeciﬁc binding of antibodies to Fc receptors, PBMCs (1  106) or cells
derived from lymph nodes (5  106) from cattle were pretreated with PBS supplemented with 10% inactivated goat serum (Thermo Fisher Scientiﬁc) at 25°C for 15 min. Pretreated cells were reacted with antibovine TIM-3 mouse MAb or mouse IgG1 isotype control antibody at 37°C for 20 min. After the reaction,
cells were reacted with Alexa Fluor 647-conjugated anti-mouse IgG(H1L) goat polyclonal F(ab9)2(Thermo Fisher Scientiﬁc) at 25°C for 20 min. Cells were washed twice and reacted with ﬂuorescein isothiocyanate-conjugated anti-bovine CD4 mouse MAb, phycoerythrin (PE)-conjugated anti-bovine CD8mouse MAb, peridinin chlorophyll protein-cyanin 5.5 (PerCp/Cy5.5)-labeled anti-bovine CD3 mouse
MAb, and PE/Cy7-conjugated anti-bovine IgM MAb for 20 min at 25°C. Anti-bovine CD3 mouse MAb andanti-bovine IgM mouse MAb were labeled with PerCp/Cy5.5 or PE/Cy7 using Lighting-Link antibody
labeling kits (Novus Biological, Centennial, CO, USA). After the reactions, cells were washed twice andimmediately analyzed by ﬂow cytometry (FACS Verse; BD Biosciences). PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) was used for antibody dilution and cell washing.
Dual TIM-3 and PD-1 expression on CD41 and CD81 T cells was also measured by ﬂow cytometry.
PBMCs were pretreated with inactivated goat serum as described above and reacted with anti-bovine
TIM-3 mouse MAb and anti-bovine PD-1 rabbit MAb at 37°C for 20 min. Mouse IgG1 and rabbit IgG isotype control antibodies were used as negative-control antibodies. After the reaction, cells were washedtwice and reacted with Alexa Fluor 488-conjugated anti-mouse IgG(H1L) goat polyclonal F(ab9)2(Thermo Fisher Scientiﬁc) and Alexa Fluor 647-conjugated anti-rabbit IgG(H1L) goat polyclonal F(ab9)2(Thermo Fisher Scientiﬁc) at 25°C for 20 min. Cells were reacted with MAbs against CD4, CD8, and CD3,
as listed in Table 1, and analyzed by ﬂow cytometry, as described above.
Blockade assay by TIM-3 antibody. PBMCs (1  106) from BLV-infected or uninfected cattle were
cultivated with 10 m g/mL anti-bovine TIM-3 mouse MAb (1H2-E2) or mouse IgG1 isotype control antibody (15H6; Southern Biotech) at 37°C for 1 to 6 days in the presence of 5% CO2. PBMCs from uninfected
cattle were incubated with 1 m g/mL anti-bovine CD3 mouse MAb (MM1A; Washington State University
Monoclonal Antibody Center, Pullman, WA, USA) and 1 m g/mL anti-bovine CD28 mouse MAb (CC220;
Bio-Rad, Hercules, CA, USA). PBMCs derived from BLV-infected cattle were cultivated in the presence of
BLV antigen, 2% heat-inactivated culture supernatant of BLV-infected FLK cell line, as described previously (42).
To evaluate lymphocyte activation, 24-h-cultivated PBMCs were analyzed for CD69 expression by
ﬂow cytometry, as described previously (42). Cell staining was performed as described above, exceptthat Alexa Fluor 647-conjugated anti-bovine CD69 mouse MAb (Table 1) was also used.
IFN-g production was analyzed by ﬂow cytometry and ELISA, as described previously (42). Whenmeasuring IFN-g intracellular expression of 24-h-cultivated cells, 10 m g/mL brefeldin A (Sigma-Aldrich)
was added 5 h before cell collection. Cell staining for CD4, CD8, and CD3 was performed as describedabove, and stained cells were permeabilized using Foxp3 Perm buffer (BioLegend, San Diego, CA, USA)at 25°C for 15 min. After treatment, cells were washed twice and reacted with biotin-conjugated antibovine IFN-g mouse MAb at 25°C for 20 min (Table 1). After the reaction, cells were treated with allophycocyanin-labeled streptavidin (BioLegend) for 20 min and immediately analyzed by ﬂow cytometry (FACS
Verse; BD Biosciences) after washing. Cells were cultured under the culture conditions for 6 days, as
described above, and IFN-g production in the culture supernatant was measured using the bovine IFN-g
ELISA development kit (Mabtech, Nacka Strand, Sweden). Absorbance was measured at 450 nm using a
microplate reader (MTP-900; Corona Electric, Hitachinaka, Japan).
Dual blockade assay by TIM-3 and PD-L1 antibodies. PBMCs (1  106) from BLV-infected cattle
were treated with anti-bovine TIM-3 mouse MAb (1H2-E2) and anti-bovine PD-L1 rat MAb (4G12-C1) inthe presence of BLV antigen, as described above. The blockade antibody was added at 10 m g/mL toeach well and incubated at 37°C for 7 days in the presence of 5% CO2. Mouse serum-derived IgG (SigmaAldrich) or rat serum-derived IgG (Sigma-Aldrich) was used as a negative-control antibody. IFN-g and
TNF-a production in the culture was measured using the bovine IFN-g ELISA development kit and thebovine TNF-a Do-It-Yourself ELISA (Kingﬁsher Biotech, Saint Paul, MN, USA).
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Journal of Virology

TABLE 1 Primary antibodies used in ﬂow cytometric analyses of this studya

Isotype

Clone

SourceFluorochrome

Conjugation or labeling

Mouse IgG1

MM1A

WSU

PerCp/Cy5.5

CD4
CD8
IgM
TIM-3

Mouse IgG2a
Mouse IgG2a
Mouse IgG1
Mouse IgG1

CC8
CC63
IL-A30
1H2-E2

Bio-Rad
Bio-Rad
Bio-RadThis study

FITCPE
PE/Cy7

Mouse IgG1 isotype
control

Mouse IgG1

15H6

Southern Biotech

Lightning-Link PerCp/Cy5.5 conjugation kit (Novus
Biosciences)(Conjugated primary antibody)(Conjugated primary antibody)
Lightning-Link PE/Cy7 conjugation kit (Novus Biosciences)
2nd Ab: Alexa Fluor 647-conjugated anti-mouse IgG(H1L)
goat polyclonal F(ab9)2 (Thermo Fisher Scientiﬁc)
2nd Ab: Alexa Fluor 647-conjugated anti-mouse IgG(H1L)
goat polyclonal F(ab9)2 (Thermo Fisher Scientiﬁc)

Mouse IgG1

MM1A

WSU

PerCp/Cy5.5

CD4
CD8
TIM-3

Mouse IgG2a
Mouse IgG2a
Mouse IgG1

CC8
CC63
1H2-E2

Bio-Rad
Bio-RadThis study

FITC
PE

PD-1

Rabbit IgG

1D10F1

In house

Mouse IgG1 isotype
control
Rabbit IgG isotype
control

Mouse IgG1

15H6

Southern Biotech

Rabbit IgG-

Southern Biotech

CD691 CD41 T cells andCD691 CD81 T cells
CD4
CD8
IgM
CD69

Mouse IgG2a
Mouse IgG2a
Mouse IgG1
Mouse IgG1

CC8
CC63
IL-A30KTSN7A

Bio-Rad
Bio-Rad
Bio-Rad
WSU

FITC
PE
PE/Cy7Alexa Fluor 647(Conjugated primary antibody)(Conjugated primary antibody)
Lightning-Link PE/Cy7 conjugation kit (Novus Biosciences)Zenon Alexa Fluor 647 mouse IgG1 labeling kit (Thermo
Fisher Scientiﬁc)

IFN-g 1 CD41 T cells and
IFN-g 1 CD81 T cells
CD3

Mouse IgG1

MM1A

WSU

PerCp/Cy5.5

Mouse IgG2a
Mouse IgG2a
Mouse IgG21

CC8
CC63
MT307

Bio-Rad
Bio-RadMabtech

FITC
PE
APC

Lightning-Link PerCp/Cy5.5 conjugation kit (Novus
Biosciences)(Conjugated primary antibody)(Conjugated primary antibody)
APC-conjugated streptavidin (BioLegend)

TIM-31 PD-11 CD41
T cells and TIM-31
PD-11 CD81 T cells
CD3

CD4
CD8
IFN-g

Lightning-Link PerCp/Cy5.5 conjugation kit (Novus
Biosciences)(Conjugated primary antibody)(Conjugated primary antibody)
2nd Ab: Alexa Fluor 488-conjugated anti-mouse IgG(H1 L)
goat polyclonal F(ab9)2 (Thermo Fisher Scientiﬁc)
2nd Ab: Alexa Fluor 647-conjugated anti-rabbit IgG(H1L)
goat polyclonal F(ab9)2 (Thermo Fisher Scientiﬁc)
2nd Ab: Alexa Fluor 488-conjugated anti-mouse IgG(H1 L)
goat polyclonal F(ab9)2 (Thermo Fisher Scientiﬁc)
2nd Ab: Alexa Fluor 647-conjugated anti-rabbit IgG(H1L)
goat polyclonal F(ab9)2 (Thermo Fisher Scientiﬁc)aWSU, WSU Monoclonal Antibody Center; FITC, ﬂuorescein isothiocyanate; APC, allophycocyanin.

The antiviral efﬁcacy of the dual blockade by anti-TIM-3 and anti-PD-L1 antibodies was evaluated by
the syncytium formation assay using the number of syncytia formed by BLV-infected cells as an indicator, as described previously (67). Brieﬂy, CC81 cells (0.6  105) were seeded onto 24-well plates (Corning,Inc., Corning, NY, USA) and cultivated for 24 h at 37°C in the presence of 5% CO2. Next, PBMCs (5  105
cells) from BLV-infected cattle, anti-bovine TIM-3 mouse MAb (1H2-E2; 10 m g/mL), and anti-bovine PDL1 rat antibody (4G12-C1; 10 m g/mL) were added and incubated for an additional 72 h. Mouse serumderived IgG (Sigma-Aldrich) and rat serum-derived IgG (Sigma-Aldrich) were used as negative-control
antibodies. After incubation, cells were ﬁxed in methanol (Sigma-Aldrich) and stained with Giemsa’s azure-eosin-methylene blue solution (Merck, Kenilworth, NJ, USA). The number of syncytia containing $10
cell nuclei was measured in 10 ﬁelds of view in each well using the ZOE ﬂuorescent cell imager (Bio-Rad)
for stained cells. Three wells were cultured per specimen cow. The results were expressed as the average
from three wells.
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Type of cells analyzed
by ﬂow cytometry andtargetTIM-31 CD41 T cells andTIM-31 CD81 T cells
CD3Enhanced Antiviral Immunity against BLV

Journal of Virology

Statistical analysis. Statistical analysis was performed using the statistical analysis software JMP(SAS), using the Mann-Whitney U test for two matched groups, the Wilcoxon signed-rank sum test for
two independent groups, and the Steel-Dwass test for more than three groups. A difference was considered statistically signiﬁcant if P was ,0.05.
Data availability. The data sets used and analyzed in this study are available from the corresponding author on reasonable request.

ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI grant number 19KK0172, 22H02503, and22K19232 (to S.K.), grants from the Project of the NARO, Bio-oriented Technology
Research Advancement Institution (Research Program on Development of Innovative
Technology 26058 BC [to S.K.] and Special Scheme Project on Regional Developing
Strategy, grant 16817557 [to S.K.]), and grants from the Ito Memorial Foundation (toS.K.) and regulatory research projects for food safety, animal health and plant
protection (JPJ008617.17935709 [to S.K.]) and was also supported by the Japan Agency
for Medical Research and Development (AMED) under grants JP223fa627005 (to S.K.),
JP21am0101078 (to Y.K.), and JP22ama121008 (to Y.K.). The funders had no role instudy design, data collection and analysis, decision to publish, or preparation of themanuscript.
We thank Enago (http://www.enago.jp) for the English language review.

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