Combined Immune Checkpoint Blockade Enhances Antiviral Immunity against Bovine Leukemia Virus.

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 and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) 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-γ 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. IMPORTANCE 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 have focused 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 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 bovine chronic infections.

Chimeric provirus of bovine leukemia virus/SMAD family member 3 in cattle with enzootic bovine leukosis.

Bovine leukemia virus (BLV) is a member of the family Retroviridae that causes enzootic bovine leukemia (EBL). However, the association between BLV infection and EBL development remains unclear. In this study, we identified a BLV/SMAD3 chimeric provirus within CC2D2A intron 30 in monoclonal expanded malignant cells from a cow with EBL. The chimeric provirus harbored a spliced SMAD3 sequence composed of exons 3-9, encoding the short isoform protein, and the BLV-SMAD3 chimeric transcript was detectable in cattle with EBL. This is the first report of a BLV chimeric provirus that might be involved in EBL tumorigenesis.

Development of a high-affinity anti-bovine PD-1 rabbit-bovine chimeric antibody using an efficient selection and large production system.

Immune checkpoint molecules PD-1/PD-L1 cause T-cell exhaustion and contribute to disease progression in chronic infections of cattle. We established monoclonal antibodies (mAbs) that specifically inhibit the binding of bovine PD-1/PD-L1; however, conventional anti-PD-1 mAbs are not suitable as therapeutic agents because of their low binding affinity to antigen. In addition, their sensitivity for the detection of bovine PD-1 is low and their use for immunostaining PD-1 is limited. To address these issues, we established two anti-bovine PD-1 rabbit mAbs (1F10F1 and 4F5F2) and its chimeric form using bovine IgG1 (Boch1D10F1), which exhibit high binding affinity. One of the rabbit mAb 1D10F1 binds more strongly to bovine PD-1 compared with a conventional anti-PD-1 mAb (5D2) and exhibits marked inhibitory activity on the PD-1/PD-L1 interaction. In addition, PD-1 expression in bovine T cells could be detected with higher sensitivity by flow cytometry using 1D10F1. Furthermore, we established higher-producing cells of Boch1D10F1 and succeeded in the mass production of Boch1D10F1. Boch1D10F1 exhibited a similar binding affinity to bovine PD-1 and the inhibitory activity on PD-1/PD-L1 binding compared with 1D10F1. The immune activation by Boch1D10F1 was also confirmed by the enhancement of IFN-γ production. Finally, Boch1D10F1 was administered to bovine leukemia virus-infected cows to determine its antiviral effect. In conclusion, the high-affinity anti-PD-1 antibody developed in this study represents a powerful tool for detecting and inhibiting bovine PD-1 and is a candidate for PD-1-targeted immunotherapy in cattle.

Prostaglandin E2-Induced Immune Suppression via Cytotoxic T-Lymphocyte Antigen 4 in Paratuberculosis.

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

Characterization of a copper transporter 1 from Dermanyssus gallinae as a vaccine antigen.

Poultry red mites (Dermanyssus gallinae, PRM) are dangerous ectoparasites that infest chickens and threaten the poultry industry worldwide. PRMs usually develop resistance to chemical acaricides, necessitating the development of more effective preventive agents, and vaccination could be an alternative strategy for controlling PRMs. The suitability of plasma membrane proteins expressed in the midguts as vaccine antigens was evaluated because these molecules are exposed to antibodies in the ingested blood and the binding of antibodies could potentially induce direct damage to midgut tissue and indirect damage via inhibition of the functions of target molecules. Therefore, in the present study, a copper transporter 1-like molecule (Dg-Ctr1) was identified and its efficacy as a vaccine antigen was assessed in vitro. Dg-Ctr1 mRNA was expressed in the midguts and ovaries and in all the life stages, and flow cytometric analysis indicated that Dg-Ctr1 was expressed on the plasma membrane. Importantly, nymphs fed on plasma derived from chickens immunized with the recombinant protein of the extracellular region of Dg-Ctr1 showed a significant reduction in the survival rate. These data indicate that the application of Dg-Ctr1 as a vaccine antigen could reduce the number of nymphs in the farms, contributing to reduction in the economic losses caused by PRMs in the poultry industry. To establish an effective vaccination strategy, the acaricidal effects of the combined use of Dg-Ctr1 with chemical acaricides or other vaccine antigens must be examined.

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.

Genetic characterization of a Marek's disease virus strain isolated in Japan.

BACKGROUND: Marek's disease virus (MDV) causes malignant lymphomas in chickens (Marek's disease, MD). MD is currently controlled by vaccination; however, MDV strains have a tendency to develop increased virulence. Distinct diversity and point mutations are present in the Meq proteins, the oncoproteins of MDV, suggesting that changes in protein function induced by amino acid substitutions might affect MDV virulence. We previously reported that recent MDV isolates in Japan display distinct mutations in Meq proteins from those observed in traditional MDV isolates in Japan, but similar to those in MDV strains isolated from other countries. METHODS: To further investigate the genetic characteristics in Japanese field strains, we sequenced the whole genome of an MDV strain that was successfully isolated from a chicken with MD in Japan. A phylogenetic analysis of the meq gene was also performed. RESULTS: Phylogenetic analysis revealed that the Meq proteins in most of the Japanese isolates were similar to those of Chinese and European strains, and the genomic sequence of the Japanese strain was classified into the Eurasian cluster. Comparison of coding region sequences among the Japanese strain and MDV strains from other countries revealed that the genetic characteristics of the Japanese strain were similar to those of Chinese and European strains. CONCLUSIONS: The MDV strains distributed in Asian and European countries including Japan seem to be genetically closer to each other than to MDV strains from North America. These findings indicate that the genetic diversities of MDV strains that emerged may have been dependent on the different vaccination-based control approaches.

Immune inhibitory function of bovine CTLA-4 and the effects of its blockade in IFN-γ production.

BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is known as an immune inhibitory receptor that is expressed on activated effector T cells and regulatory T cells. When CTLA-4 binds to CD80 or CD86, immunoinhibitory signals are transmitted to retain a homeostasis of the immune response. Recent studies have reported that CTLA-4 is upregulated in chronic infections and malignant neoplasms, contributing to host immune dysfunction. On the other hand, the blockade of CTLA-4 and CD80 or CD86 binding by antibody restores the immune response against these diseases. In a previous report, we indicated that the expression of CTLA-4 was closely associated with disease progression in cattle infected with the bovine leukemia virus (BLV). In this study, we established an anti-bovine CTLA-4 antibody to confirm its immune enhancing effect. RESULTS: Bovine CTLA-4-Ig binds to bovine CD80 and CD86 expressing cells. Additionally, CD80 and CD86 bind to CTLA-4 expressing cells in an expression-dependent manner. Bovine CTLA-4-Ig significantly inhibited interferon-gamma (IFN-γ) production from bovine peripheral blood mononuclear cells (PBMCs) activated by Staphylococcus enterotoxin B (SEB). An established specific monoclonal antibody (mAb) for bovine CTLA-4 specifically recognized only with bovine CTLA-4, not CD28, and the antibody blocked the binding of CTLA-4-Ig to both CD80 and CD86 in a dose-dependent manner. The bovine CTLA-4 mAb significantly restored the inhibited IFN-γ production from the CTLA-4-Ig treated PBMCs. In addition, the CTLA-4 mAb significantly enhanced IFN-γ production from CTLA-4 expressing PBMCs activated by SEB. Finally, we examined whether a CTLA-4 blockade by CTLA-4 mAb could restore the immune reaction during chronic infection; the blockade assay was performed using PBMCs from BLV-infected cattle. The CTLA-4 blockade enhanced IFN-γ production from the PBMCs in response to BLV-antigens. CONCLUSIONS: Collectively, these results suggest that anti-bovine CTLA-4 antibody can reactivate lymphocyte functions and could be applied for a new therapy against refractory chronic diseases. Further investigation is required for future clinical applications.

Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: potential for bovine inflammation therapy.

BACKGROUND: Refractory diseases, including bacterial infections, are causing huge economic losses in dairy farming. Despite efforts to prevent and treat those diseases in cattle, including the use of antimicrobials, it is not well controlled in the field. Several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), play important roles in disease progression; thus, blocking these cytokines can attenuate the acute and sever inflammation and may be a novel strategy for treatment. However, biological drugs targeting inflammatory cytokines have not been used in cattle. Therefore, in this study, bovine sTNFR1 and sTNFR2 IgG1 Fc-fusion proteins (TNFR1-Ig and TNFR2-Ig) were produced, and their anti-inflammatory functions were analyzed in vitro, to develop decoy receptors for bovine TNF-α. RESULTS: Both TNFR1-Ig and TNFR2-Ig were shown to bind with TNF-α, and TNFR2-Ig showed higher affinity toward TNF-α than TNFR1-Ig. We next stimulated murine fibroblast-derived cells (L929 cells) with TNF-α to induce cell death and analyzed cell viability in the presence of TNFR-Ig proteins. Both TNFR1-Ig and TNFR2-Ig suppressed TNF-α-induced cell death, significantly improving cell viability. In addition, cell death induced by TNF-α was suppressed, even at low TNFR2-Ig concentrations, suggesting TNFR2-Ig has higher activity to suppress TNF-α functions than TNFR1-Ig. Finally, to examine TNFR2-Ig's anti-inflammatory, we cultured peripheral blood mononuclear cells from cattle with TNF-α in the presence of TNFR2-Ig and analyzed the gene expression and protein production of the inflammatory cytokines IL-1ß and TNF-α. TNFR2-Ig significantly reduced the gene expression and protein production of these cytokines. Our results suggest that TNFR2-Ig inhibits inflammatory cytokine kinetics by blocking TNF-α to transmembrane TNFR, thereby attenuating excessive inflammation induced by TNF-α. CONCLUSIONS: Collectively, the findings of this study demonstrated the potential of TNFR2-Ig as a novel therapeutic for inflammatory diseases, such as bovine clinical mastitis. Further investigation is required for future clinical application.

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.

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.

Cooperation of PD-1 and LAG-3 Contributes to T-Cell Exhaustion in Anaplasma marginale-Infected Cattle.

The CD4(+) T-cell response is central for the control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhaustion of antigen-specific CD4(+) T cells in cattle immunized with A. marginale outer membrane proteins or purified outer membranes (OMs), which presumably facilitates the persistence of this rickettsia. In the present study, we hypothesize that T-cell exhaustion following infection is induced by the upregulation of immunoinhibitory receptors on T cells, such as programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3). OM-specific T-cell responses and the kinetics of PD-1-positive (PD-1(+)) LAG-3(+) exhausted T cells were monitored in A. marginale-challenged cattle previously immunized with OMs. Consistent with data from previous studies, OM-specific proliferation of peripheral blood mononuclear cells (PBMCs) and interferon gamma (IFN-γ) production were significantly suppressed in challenged animals by 5 weeks postinfection (wpi). In addition, bacteremia and anemia also peaked in these animals at 5 wpi. Flow cytometric analysis revealed that the percentage of PD-1(+) LAG-3(+) T cells in the CD4(+), CD8(+), and γδ T-cell populations gradually increased and also peaked at 5 wpi. A large increase in the percentage of LAG-3(+) γδ T cells was also observed. Importantly, in vitro, the combined blockade of the PD-1 and LAG-3 pathways partially restored OM-specific PBMC proliferation and IFN-γ production at 5 wpi. Taken together, these results indicate that coexpression of PD-1 and LAG-3 on T cells contributes to the rapid exhaustion of A. marginale-specific T cells following infection and that these immunoinhibitory receptors regulate T-cell responses during bovine anaplasmosis.

Bovine Immunoinhibitory Receptors Contribute to Suppression of Mycobacterium avium subsp. paratuberculosis-Specific T-Cell Responses.

Johne's disease (paratuberculosis) is a chronic enteritis in cattle that is caused by intracellular infection with Mycobacterium avium subsp. paratuberculosis. This infection is characterized by the functional exhaustion of T-cell responses to M. avium subsp. paratuberculosis antigens during late subclinical and clinical stages, presumably facilitating the persistence of this bacterium and the formation of clinical lesions. However, the mechanisms underlying T-cell exhaustion in Johne's disease are poorly understood. Thus, we performed expression and functional analyses of the immunoinhibitory molecules programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC-II) in M. avium subsp. paratuberculosis-infected cattle during the late subclinical stage. Flow cytometric analyses revealed the upregulation of PD-1 and LAG-3 in T cells in infected animals, which suffered progressive suppression of interferon gamma (IFN-γ) responses to the M. avium subsp. paratuberculosis antigen. In addition, PD-L1 and MHC-II were expressed on macrophages from infected animals, consistent with PD-1 and LAG-3 pathways contributing to the suppression of IFN-γ responses during the subclinical stages of M. avium subsp. paratuberculosis infection. Furthermore, dual blockade of PD-L1 and LAG-3 enhanced M. avium subsp. paratuberculosis-specific IFN-γ responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-γ production from M. avium subsp. paratuberculosis-specific CD4(+) and CD8(+) T cells. Taken together, the present data indicate that M. avium subsp. paratuberculosis-specific T-cell exhaustion is in part mediated by PD-1/PD-L1 and LAG-3/MHC-II interactions and that LAG-3 is a molecular target for the control of M. avium subsp. paratuberculosis-specific T-cell responses.

Detection of bovine leukemia virus and identification of its genotype in Mongolian cattle.

Epidemiological studies have indicated that bovine leukemia virus (BLV) infection is globally distributed. However, no information regarding the disease and genetic diversity of the virus in the cattle of Mongolia is currently available. In this study, the prevalence of BLV was assessed using PCR, and the genetic diversity was analyzed through DNA sequencing. Of the 517 samples tested, 20 positives were identified. Phylogenetic analysis showed that six, one, and four isolates were classified into genotype 4, 7, and 1, respectively. Most isolates were clustered with isolates from Eastern Europe and Russia. This study is the first to investigate the BLV genotype in Mongolia.

Influence of PD-L1 cross-linking on cell death in PD-L1-expressing cell lines and bovine lymphocytes.

Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-γ (IFN-γ) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-γ production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1.

Differences in cellular function and viral protein expression between IgMhigh and IgMlow B-cells in bovine leukemia virus-infected cattle.

Bovine leukemia virus (BLV) induces abnormal B-cell proliferation and B-cell lymphoma in cattle, where the BLV provirus is integrated into the host genome. BLV-infected B-cells rarely express viral proteins in vivo, but short-term cultivation augments BLV expression in some, but not all, BLV-infected B-cells. This observation suggests that two subsets, i.e. BLV-silencing cells and BLV-expressing cells, are present among BLV-infected B-cells, although the mechanisms of viral expression have not been determined. In this study, we examined B-cell markers and viral antigen expression in B-cells from BLV-infected cattle to identify markers that may discriminate BLV-expressing cells from BLV-silencing cells. The proportions of IgM(high) B-cells were increased in blood lymphocytes from BLV-infected cattle. IgM(high) B-cells mainly expressed BLV antigens, whereas IgM(low) B-cells did not, although the provirus load was equivalent in both subsets. Several parameters were investigated in these two subsets to characterize their cellular behaviour. Real-time PCR and microarray analyses detected higher expression levels of some proto-oncogenes (e.g. Maf, Jun and Fos) in IgM(low) B-cells than those in IgM(high) B-cells. Moreover, lymphoma cells obtained from the lymph nodes of 14 BLV-infected cattle contained IgM(low) or IgM(-) B-cells but no IgM(high) B-cells. To our knowledge, this is the first study to demonstrate that IgM(high) B-cells mainly comprise BLV-expressing cells, whereas IgM(low) B-cells comprise a high proportion of BLV-silencing B-cells in BLV-infected cattle.

Identification and characterization of bovine programmed death-ligand 2.

Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)-1 receptor and its ligand, PD-L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD-L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD-L2 was analyzed in vitro. Recombinant PD-L2 (PD-L2-Ig), which comprises an extracellular domain of bovine PD-L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD-L2. Bovine PD-L2-Ig bound to bovine PD-1-expressing cells and addition of soluble bovine PD-1-Ig clearly inhibited the binding of PD-L2-Ig to membrane PD-1 in a dose-dependent manner. Cell proliferation and IFN-γ production were significantly enhanced in the presence of PD-L2-Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD-L2-Ig significantly enhanced IFN-γ production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-γ production was further enhanced by treatment with anti-bovine PD-1 antibody. These data suggest potential applications of bovine PD-L2-Ig as a therapy for bovine diseases.

Characterization of a Very Short Meq Protein Isoform in a Marek's Disease Virus Strain in Japan.

Marek's disease virus (MDV) causes malignant lymphoma (Marek's disease; MD) in chickens. The Meq protein is essential for tumorigenesis since it regulates the expression of host and viral genes. Previously, we reported that the deletion of the short isoform of Meq (S-Meq) decreases the pathogenicity of MDV. Recently, we identified a further short isoform of Meq (very short isoform of Meq, VS-Meq) in chickens with MD in Japan. A 64-amino-acid deletion was confirmed at the C-terminus of VS-Meq. We measured the transcriptional regulation by VS-Meq in three gene promoters to investigate the effect of VS-Meq on protein function. Wild-type VS-Meq decreased the transrepression of the pp38 promoter but did not alter the transactivation activity of the Meq and Bcl-2 promoters. The deletion in VS-Meq did not affect the activity of the pp38 promoter but enhanced the transactivation activities of the Meq and Bcl-2 promoters. Collectively, the deletion of VS-Meq potentially enhanced the activity of the Meq promoter, while other amino acid sequences in wild-type VS-Meq seemed to affect the weak transrepression of the pp38 promoter. Further investigation is required to clarify the effects of these changes on pathogenicity.

In Vivo Characterization of the Anti-Glutathione S-Transferase Antibody Using an In Vitro Mite Feeding Model.

Poultry red mites (Dermanyssus gallinae, PRMs), tropical fowl mites (Ornithonyssus bursa, TFMs), and northern fowl mites (O. sylviarum, NFMs) are blood-feeding pests that debilitate poultry worldwide. Glutathione S-transferase (GST) plays an important role in the detoxification and drug metabolism of mites. However, research on avian mite GSTs as vaccine antigens is still lacking. Therefore, we aimed to evaluate the potential of avian mite GSTs for vaccine development. We identified GST genes from TFMs and NFMs. We prepared recombinant GST (rGST) from TFMs, NFMs, and PRMs, and assessed their protein functions. Moreover, we evaluated the cross-reactivity and acaricidal effect of immune plasma against each rGST on TFMs, NFMs, and PRMs. The deduced amino acid sequences of GSTs from TFMs and NFMs were 80% similar to those of the PRMs. The rGSTs exhibited catalytic activity in conjugating glutathione to the 1-chloro-2,4-dinitrobenzene substrate. Immune plasma against each rGST showed cross-reactivity with rGST from different mite species. Moreover, the survival rate of PRMs fed with immune plasma against the rGST of TFMs and NFMs was significantly lower than that of the control plasma. These results demonstrate the potential application of GST as an antigen for the development of a broad-spectrum vaccine against avian mites.

Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro.

Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1+ CD4+ T cells in blood and lymph node and PD-1+ CD8+ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.