The bovine leukemia virus-derived long non-coding RNA AS1-S binds to bovine hnRNPM and alters the interaction between hnRNPM and host mRNAs.

Viruses utilize several strategies to cause latent infection and evade host immune responses. Long non-coding RNA (lncRNA), a class of non-protein-encoding RNA that regulates various cellular functions by interacting with RNA-binding proteins, plays important roles for viral latency in several viruses, such as herpesviruses and retroviruses, due to its lack of antigenicity. Bovine leukemia virus (BLV), which belongs to the family Retroviridae, encodes the BLV-derived lncRNA AS1-S, which is a major transcript expressed in latently infected cells. We herein identified bovine heterogeneous nuclear ribonucleoprotein M (hnRNPM), an RNA-binding protein located in the nucleus, as the binding partner of AS1-S using an RNA-protein pull-down assay. The pull-down assay using recombinant hnRNPM mutants showed that RNA recognition motifs (RRMs) 1 and 2, located in the N-terminal region of bovine hnRNPM, were responsible for the binding to AS1-S. Furthermore, RNA immunoprecipitation (RIP) assay results showed that the expression of AS1-S increased the number of mRNAs that co-immunoprecipitated with bovine hnRNPM in MDBK cells. These results suggested that AS1-S could alter the interaction between hnRNPM and host mRNAs, potentially interfering with cellular functions during the initial phase of mRNA maturation in the nucleus. Since most of the identified mRNAs that exhibited increased binding to hnRNPM were correlated with the KEGG term "Pathways in cancer," AS1-S might affect the proliferation and expansion of BLV-infected cells and contribute to tumor progression. IMPORTANCE BLV infects bovine B cells and causes malignant lymphoma, a disease that greatly affects the livestock industry. Due to its low incidence and long latent period, the molecular mechanisms underlying the progression of lymphoma remain enigmatic. Several non-coding RNAs (ncRNAs), such as miRNA and lncRNA, have recently been discovered in the BLV genome, and the relationship between BLV pathogenesis and these ncRNAs is attracting attention. However, most of the molecular functions of these transcripts remain unidentified. To the best of our knowledge, this is the first report describing a molecular function for the BLV-derived lncRNA AS1-S. The findings reported herein reveal a novel mechanism underlying BLV pathogenesis that could provide important insights for not only BLV research but also comparative studies of retroviruses.

Upregulation of host genes during disease progression in bovine leukemia virus infection is independent of overexpression of viral transcriptional regulators in vitro.

Bovine leukemia virus (BLV) is a member of the genus Deltaretrovirus within the family Retroviridae that infects bovine B cells, causing persistent lymphocytosis and enzootic bovine leukosis (EBL) in a small fraction of infected cattle. As changes in the transcriptome of infected cells are important for BLV disease progression, comprehensive analysis of gene expression in different disease states is required. In this study, we performed an RNA-seq analysis using samples from non-EBL cattle with and without BLV infection. Subsequently, a transcriptome analysis was conducted in combination with previously obtained RNA-seq data from EBL cattle. We found several differentially expressed genes (DEGs) between the three groups. After screening and confirmation of target DEGs using real-time reverse transcription polymerase chain reaction, we found that 12 target genes were significantly upregulated in EBL cattle compared to BLV-infected cattle without lymphoma. In addition, the expression levels of B4GALT6, ZBTB32, EPB4L1, RUNX1T1, HLTF, MKI67, and TOP2A were significantly and positively correlated with the proviral load in BLV-infected cattle. Overexpression experiments revealed that these changes were independent of BLV tax or BLV AS1-S expression in vitro. Our study provides additional information on host gene expression during BLV infection and EBL development, which may be helpful for understanding the complexity of transcriptome profiles during disease progression.

Bovine leukemia virus-associated B cell lymphoma with severe pleomorphism in a steer.

We examined a 26-month-old steer with neoplastic lesions in the spleen, lymph nodes, heart and kidneys, characterized by pleomorphic lymphoid cells that were immunohistochemically positive for CD20. The presence of bovine leukemia virus (BLV) at >200,000 copies per 100,000 cells by quantitative RT-PCR was considered to be due to random integration of the provirus into the neoplastic cells´ genomes. Inverse PCR identified the presence of one, two, two and three different malignant clones in the heart, spleen, mesenteric node and blood, respectively. Because BLV can rapidly induce lymphoma and a high proviral load facilitates B-cell carcinogenesis, multiclonal tumor development was suspected in the present case.

Endemic infections of bovine viral diarrhea virus genotypes 1b and 2a isolated from cattle in Japan between 2014 and 2020.

Bovine viral diarrhea virus (BVDV) is a causative agent of bovine viral diarrhea. In Japan, a previous study reported that subgenotype 1b viruses were predominant until 2014. Because there is little information regarding the recent epidemiological status of BVDV circulating in Japan, we performed genetic characterization of 909 BVDV isolates obtained between 2014 and 2020. We found that 657 and 252 isolates were classified as BVDV-1 and BVDV-2, respectively, and that they were further subdivided into 1a (35 isolates, 3.9%), 1b (588, 64.7%), 1c (34, 3.7%), and 2a (252, 27.7%). Phylogenetic analysis using entire E2 coding sequence revealed that a major domestic cluster in Japan among BVDV-1b and 2a viruses were unchanged from a previous study conducted from 2006 to 2014. These results provide updated information concerning the epidemic strain of BVDV in Japan, which would be helpful for appropriate vaccine selection.

Novel single nucleotide polymorphisms in the bovine leukemia virus genome are associated with proviral load and affect the expression profile of viral non-coding transcripts.

Bovine leukemia virus (BLV) infects bovine B-cells and causes malignant lymphoma, resulting in severe economic losses in the livestock industry. To control the spread of BLV, several studies have attempted to clarify the molecular mechanisms of BLV pathogenesis, but the details of the mechanism are still enigmatic. Currently, viral non-coding RNAs are attracting attention as a novel player for BLV pathogenesis because these transcripts can evade the host immune response and are persistently expressed in latent infection. One of the viral non-coding RNA, AS1, is encoded in the antisense strand of the BLV genome and consists of two isoforms, AS1-L and AS1-S. Although the function of the AS1 is still unknown, the AS1 RNA might also have some roles because it keeps expressing in tumor tissues. In the present study, we identified novel single nucleotide polymorphisms (SNPs) located in the AS1 coding region and indicated that individuals infected with BLV with minor SNPs showed low proviral load. To evaluate the effect of identified SNPs, we constructed infectious clones with these SNPs and found that their introduction affected the expression profile of AS1 RNA; the amount of AS1-L isoform increased compared with the wild type, although the total amount of AS1 RNA remained unchanged. Prediction analysis also suggested that the introduction of SNPs changed the secondary structure of AS1 RNA. These results explain part of the relationship between BLV expansion in vivo and the expression profile of AS1, although further analysis is required.

Establishment of a simplified inverse polymerase chain reaction method for diagnosis of enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma of cattle caused by infection with bovine leukemia virus (BLV). It is defined by clonal and neoplastic expansion of BLV-infected B cells. Currently, multiple examinations are able to comprehensively diagnose this condition. Inverse polymerase chain reaction (PCR) is a useful method to determine retrovirus integration sites. Here, we established a simplified inverse PCR method, involving the evaluation of clonality and similarity of BLV integration sites, to clinically diagnose EBL, and we also assessed its reliability. We found that the novel BLV inverse PCR could detect clonal expansion of infected cells even if they constituted only 5% of the total number of cells, while not amplifying any fragments from BLV-uninfected cells, thus confirming its sufficient sensitivity and specificity for use in EBL diagnosis. Furthermore, 50 clinical cases of bovine leukemia were analyzed using BLV inverse PCR and other PCR-based methods, wherein our method most efficiently determined virus-dependent bovine leukemia, including unidentified clinical cases observed in a previous report. Following further clinical investigations to enhance its reliability, the proposed BLV inverse PCR method has the potential to be applied to EBL diagnosis.

Development of an in situ hybridization assay using an AS1 probe for detection of bovine leukemia virus in BLV-induced lymphoma tissues.

Enzootic bovine leukosis (EBL) is a malignant B cell lymphoma caused by infection with bovine leukemia virus (BLV). Histopathological examination is commonly used for diagnosis of the disease, but observation of lymphoma alone does not confirm EBL because cattle may be affected by sporadic forms of lymphoma that are not associated with BLV. Detection of BLV in tumor cells can be definitive evidence of EBL, but currently, there is no technique available for such a purpose. In this study, we focused on a viral non-coding RNA, AS1, and developed a novel in situ hybridization assay for the detection of BLV from formalin-fixed paraffin-embedded (FFPE) tissues. RNA-seq analysis revealed that all examined B lymphocytes derived from clinical EBL abundantly expressed AS1 RNA, indicating a possible target for detection. The in situ hybridization assay using an AS1 probe clearly detected AS1 RNA in fetal lamb kidney cells persistently infected with BLV. The utility of this assay in clinical samples was assessed using three EBL-derived lymph node specimens and one BLV-negative specimen, and AS1 RNA was detected specifically in the EBL-derived tissues. These results suggest that AS1 RNA is a useful target for the detection of BLV from FFPE specimens of tumor tissues. This technique is expected to become a powerful tool for EBL diagnosis.

The chemokines CCL2 and CXCL10 produced by bovine endometrial epithelial cells induce migration of bovine B lymphocytes, contributing to transuterine transmission of BLV infection.

Bovine leukemia virus (BLV) causes a lymphoproliferative disease in cattle and is transmitted horizontally and vertically via infected lymphocytes. Although transplacental infection is considered the predominant route of vertical transmission of BLV, the molecular mechanisms of this process remain to be elucidated. Notably, how BLV passes through the blood-placental barrier remains unclear, given that BLV is transmitted primarily by cell-to-cell contact. One hypothesis is that B cell migration to the placenta may be induced by certain endometrium-expressed chemokines. To test this hypothesis, we performed an in vitro cell migration assay using bovine B cell lines and endometrial epithelial cells. Cell migration assays showed that two bovine B cell lines, BL2M3 and BL3.1 cells, were attracted to the supernatant of bovine endometrial epithelial cells (BEnEpCs). Quantitative real-time RT-PCR showed that expression levels of mRNAs encoding the chemokines CCL2 and CXCL10 were higher in BEnEpCs than in MDBK cells. Additionally, an inhibition assay using immune serum against CCL2 and CXCL10 showed suppression of migration of bovine B cell lines. A syncytium assay showed that cells expressing BLV envelope (Env) protein fused with BEnEpCs. Here we found that bovine B cells are attracted by chemokines produced in the endometrium and that cells expressing BLV Env protein fused with endometrium epithelial cells. These results explain part of the molecular mechanism of transplacental transmission during BLV infection, although further analysis will be required. Advances in these areas are expected to contribute to controlling the spread of BLV.

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

Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.

Cooperation of PD-1 and LAG-3 in the exhaustion of CD4+ and CD8+ T cells during bovine leukemia virus infection.

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 contribute to T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 and LAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- and LAG-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 development of B-cell lymphoma in BLV-infected cattle.

Prostaglandin E2 Induction Suppresses the Th1 Immune Responses in Cattle with Johne's Disease.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8+ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8+ T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8+ T-cell proliferation in vitro, suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease.

Intrauterine infection with bovine leukemia virus in pregnant dam with high viral load.

Enzootic bovine leukemia is caused by the bovine leukemia virus (BLV). BLV is transmitted vertically or horizontally through the transfer of infected cells via direct contact, through milk, insect bites and contaminated iatrogenic procedures. However, we lacked direct evidence of intrauterine infection. The purpose of this study was to confirm intrauterine BLV infection in two pregnant dams with high viral load by cesarean delivery. BLV was detected in cord and placental blood, and the BLV in the newborns showed 100% nucleotide identity with the BLV-env sequence from the dams. Notably, a newborn was seropositive for BLV but had no colostral antibodies. In this study, we presented a direct evidence of intrauterine BLV transmission in pregnant dam with a high proviral load. These results could aid the development of BLV control measures targeting viral load.

A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma.

Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells. A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.

Anti-Bovine Programmed Death-1 Rat-Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle.

Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1)/PD-ligand 1 (PD-L1), is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat-bovine chimeric monoclonal antibody 5D2 (Boch5D2) was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV). Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4+ T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.

Identification of an Atypical Enzootic Bovine Leukosis in Japan by Using a Novel Classification of Bovine Leukemia Based on Immunophenotypic Analysis.

Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLV-associated lymphoma occurs predominantly in adult cattle of >3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection.

Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis.

INTRODUCTION: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. METHODS: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. RESULTS: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+ CD4+ and PD-L1+ CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+ CD4+ and PD-1+ CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. CONCLUSIONS: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.

In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection.

Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM+ B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4+ T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.

Immunohistochemical Analysis of PD-L1 Expression in Canine Malignant Cancers and PD-1 Expression on Lymphocytes in Canine Oral Melanoma.

Spontaneous cancers are common diseases in dogs. Among these, some malignant cancers such as oral melanoma, osteosarcoma, hemangiosarcoma, and mast cell tumor are often recognized as clinical problems because, despite their high frequencies, current treatments for these cancers may not always achieve satisfying outcomes. The absence of effective systemic therapies against these cancers leads researchers to investigate novel therapeutic modalities, including immunotherapy. Programmed death 1 (PD-1) is a costimulatory receptor with immunosuppressive function. When it binds its ligands, PD-ligand 1 (PD-L1) or PD-L2, PD-1 on T cells negatively regulates activating signals from the T cell receptor, resulting in the inhibition of the effector function of cytotoxic T lymphocytes. Aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancers. In clinical trials, anti-PD-1 or anti-PD-L1 antibodies induced tumor regression for several malignancies, including advanced melanoma, non-small cell lung carcinoma, and renal cell carcinoma. In this study, to assess the potential of the PD-1/PD-L1 axis as a novel therapeutic target for canine cancer immunotherapy, immunohistochemical analysis of PD-L1 expression in various malignant cancers of dogs was performed. Here, we show that dog oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumor, mammary adenocarcinoma, and prostate adenocarcinoma expressed PD-L1, whereas some other types of cancer did not. In addition, PD-1 was highly expressed on tumor-infiltrating lymphocytes obtained from oral melanoma, showing that lymphocytes in this cancer type might have been functionally exhausted. These results strongly encourage the clinical application of PD-1/PD-L1 inhibitors as novel therapeutic agents against these cancers in dogs.

Bovine leukemia virus reduces anti-viral cytokine activities and NK cytotoxicity by inducing TGF-ß secretion from regulatory T cells.

CD4(+)CD25(high)Foxp3(+) T cells suppress excess immune responses that lead to autoimmune and/or inflammatory diseases, and maintain host immune homeostasis. However, CD4(+)CD25(high)Foxp3(+) T cells reportedly contribute to disease progression by over suppressing immune responses in some chronic infections. In this study, kinetic and functional analyses of CD4(+)CD25(high)Foxp3(+) T cells were performed in cattle with bovine leukemia virus (BLV) infections, which have reported immunosuppressive characteristics. In initial experiments, production of the Th1 cytokines IFN-γ and TNF-α was reduced in BLV-infected cattle compared with uninfected cattle, and numbers of IFN-γ or TNF-α producing CD4(+) T cells decreased with disease progression. In contrast, IFN-γ production by NK cells was inversely correlated with BLV proviral loads in infected cattle. Additionally, during persistent lymphocytosis disease stages, NK cytotoxicity was depressed as indicated by low expression of the cytolytic protein perforin. Concomitantly, total CD4(+)CD25(high)Foxp3(+) T cell numbers and percentages of TGF-ß(+) cells were increased, suggesting that TGF-ß plays a role in the functional declines of CD4(+) T cells and NK cells. In further experiments, recombinant bovine TGF-ß suppressed IFN-γ and TNF-α production by CD4(+) T cells and NK cytotoxicity in cultured cells. These data suggest that TGF-ß from CD4(+)CD25(high)Foxp3(+) T cells is immunosuppressive and contributes to disease progression and the development of opportunistic infections during BLV infection.