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.

Detection of disease-associated microglia among various microglia phenotypes induced by West Nile virus infection in mice.

West Nile virus (WNV) has emerged as a significant cause of viral encephalitis in humans and horses. However, the pathogenesis of the West Nile encephalitis remains unclear. Microglia are activated by WNV infection, and the pathogenic involvement of their phenotypes is controversial. In this study, we examined the diversity of microglia phenotypes caused by WNV infection by assessing various microglia markers and identified disease-associated microglia in WNV-infected mouse brain tissue. Cells positive for general microglia markers such as Iba1, P2RY12, or TMEM119 were detected in the control and WNV-infected brain tissue. The morphology of the positive cells in brain tissue infected by WNV was different from that of control brain tissue, indicating that WNV infection induced activation of microglia. The activated microglia were classified into various phenotypes by investigation of specific marker expression. Among the activated microglia, disease-associated microglia that were positive for CD11c and weakly positive for TMEM119 were detected close to the WNV-infected cells. These results indicate that WNV infection induces activation of diverse microglia phenotypes and that disease-associated microglia may be associated with the pathogenicity of WNV infection in the mouse brain.

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.

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.

Suppressive modulation of host immune responses by Dermanyssus gallinae infestation.

The poultry red mite (Dermanyssus gallinae, PRM) is a blood-sucking ectoparasite in chickens and is one of the most serious threats to poultry farms. Mass infestation with PRMs causes various health problems in chickens, resulting in significant productivity reduction in the poultry industry. Infestation with hematophagous ectoparasites, such as ticks, induces host inflammatory and hemostatic reactions. On the other hand, several studies have reported that hematophagous ectoparasites secrete various immunosuppressants from their saliva to suppress host immune responses to maintain blood sucking. Here, we examined the expression of cytokines in peripheral blood cells to investigate whether PRM infestation affects immunological states in chickens. In PRM-infested chickens, anti-inflammatory cytokines, IL-10 and TGF-ß1, and immune checkpoint molecules, CTLA-4 and PD-1, were highly expressed compared to noninfested chickens. PRM-derived soluble mite extracts (SME) upregulated the gene expression of IL-10 in peripheral blood cells and HD-11 chicken macrophages. In addition, SME suppressed the expression of interferons and inflammatory cytokines in HD-11 chicken macrophages. Moreover, SME induces the polarization of macrophages into anti-inflammatory phenotypes. Collectively, PRM infestation could affect host immune responses, especially suppress the inflammatory responses. Further studies are warranted to fully understand the influence of PRM infestation on host immunity.

Regulation of programmed death ligand 1 expression by interferon-γ and tumour necrosis factor-α in canine tumour cell lines.

Expression of programmed death ligand 1 (PD-L1) on tumour cells provides an immune evasion mechanism by inducing suppression of cytotoxic T cells. Various regulatory mechanisms of PD-L1 expression have been described in human tumours, however, little is known in canine tumours. To investigate whether inflammatory signalling is involved in PD-L1 regulation in canine tumours, the effects of interferon (IFN)-γ and tumour necrosis factor (TNF)-α treatment were examined in canine malignant melanoma cell lines (CMeC and LMeC) and an osteosarcoma cell line (HMPOS). The protein level of PD-L1 expression was upregulated by IFN-γ and TNF-α stimulation. Upon IFN-γ stimulation, all cell lines showed an increase in expression of PD-L1, signal transducer and activator of transcription (STAT)1, STAT3 and genes regulated by STAT activation. Upregulated expression of these genes was suppressed by the addition of a JAK inhibitor, oclacitinib. Contrastingly, upon TNF-α stimulation, all cell lines exhibited higher gene expression of the nuclear factor kappa B (NF-κB) gene RELA and genes regulated by NF-κB activation, whereas expression of PD-L1 was upregulated in LMeC only. Upregulated expression of these genes was suppressed by the addition of an NF-κB inhibitor, BAY 11-7082. The expression level of cell surface PD-L1 induced by IFN-γ and TNF-α treatment was reduced by oclacitinib and BAY 11-7082, respectively, indicating that upregulation of PD-L1 expression by IFN-γ and TNF-α stimulation is regulated via the JAK-STAT and NF-κB signalling pathways, respectively. These results provide insights into the role of inflammatory signalling in PD-L1 regulation in canine tumours.

Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in sheep.

Sheep have been used as a large animal experimental model for studying infectious diseases. However, due to a lack of staining antibodies and reagents, immunological studies on sheep have not progressed. The immunoinhibitory receptor programmed death-1 (PD-1) is expressed on T lymphocytes. The interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We previously reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections using anti-bovine PD-L1 monoclonal antibodies (mAbs). Furthermore, we found that blocking antibodies against PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy of cattle. However, the immunological role of the PD-1/PD-L1 pathway in chronic diseases of sheep remains unknown. In this study, we identified cDNA sequences of ovine PD-1 and PD-L1 and examined the cross-activity of anti-bovine PD-L1 mAbs against ovine PD-L1 as well as the expression of PD-L1 in ovine listeriosis. The amino acid sequences of ovine PD-1 and PD-L1 share a high degree of identity and similarity with homologs from ruminants and other mammalian species. Anti-bovine PD-L1 mAb recognized ovine PD-L1 on lymphocytes in the flow cytometric assay. Furthermore, an immunohistochemical staining confirmed the PD-L1 expression on macrophages in the brain lesions of ovine listeriosis. These findings indicated that our anti-PD-L1 mAb would be useful for analyzing the ovine PD-1/PD-L1 pathway. Further research is needed to determine the immunological role of PD-1/PD-L1 in chronic diseases such as BLV infection through experimental infection of sheep.

Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies.

Coagulase-positive Staphylococci express protein A, which binds to host antibodies, to evade the immune system. Taking advantage of its specific binding to antibodies, protein A from Staphylococcus aureus, which is called SpA, is commonly used as an affinity chromatography ligand for human therapeutic antibodies. However, among four canine IgG subclasses (A, B, C, and D), only IgG-B binds to SpA strongly and establishing an efficient and robust purification scheme for canine therapeutic antibodies whose IgG subclass is A, C, or D remains difficult and depends on finding a suitable substitute to SpA. S. pseudintermedius, a major coagulase-positive Staphylococci found in dogs, expresses spsQ gene which is orthologous to S. aureus spa. We hypothesized that to serve S. pseudintermedius to better adapt to the dog immune system, SpsQ would bind to canine IgGs stronger than SpA, making it a better affinity chromatography ligand for canine therapeutic antibodies. To characterize SpsQ, we first determined the spsQ nucleotide sequence from S. pseudintermedius isolates. Based on the identified sequence, we prepared recombinant proteins containing the immunoglobulin-binding domains of SpA (r-SpA) and SpsQ (r-SpsQ) and determined their binding capacity for each canine IgG subclass. The binding capacity of r-SpsQ for IgG-B was almost as high as that of r-SpA. Interestingly, while both r-SpsQ and r-SpA showed no binding to IgG-C, the binding capacity of r-SpsQ for IgG-A and IgG-D was significantly higher than that of r-SpA. Finally, we performed affinity chromatography using r-SpsQ- or r-SpA-immobilized resin and revealed that the recovery rates of IgG-A and IgG-D using r-SpsQ were significantly higher than those using r-SpA. Our findings indicate that SpsQ has a strong potential to be used as an affinity chromatography ligand for canine therapeutic antibodies of subclass A, B, and D.

In vitro evaluation of Lactiplantibacillus plantarum HOKKAIDO strain, effective lactic acid bacteria for calf diarrhea.

Calf diarrhea adversely affects growth and sometimes results in mortality, leading to severe economic losses to the cattle industry. Antibiotics are useful in the treatment against bacterial diarrhea, but not against viral, protozoan, and antibiotic-resistant bacterial diarrhea. Therefore, there are growing requirements for a novel control method for calf diarrhea. Probiotics have been considered promising candidates for preventive and supportive therapy for calf diarrhea for many years. A recent study has revealed that Lactiplantibacillus plantarum HOKKAIDO strain (Lp-HKD) reduces intestinal pathology and the severity of diarrhea in bovine rotavirus (BRV)-infected calves. Lp-HKD is known to enhance the function of human immune cells and expected to be used as probiotics for humans. Therefore, it is hypothesized that Lp-HKD modulates antiviral immune response in cattle and provide the clinical benefits in BRV-infected calves. However, the detailed mechanism of Lp-HKD-induced immunomodulation remains unknown. Thus, this study aimed to elucidate the immunomodulatory and antiviral effects of Lp-HKD in cattle. Cultivation assay of bovine peripheral blood mononuclear cells (PBMCs) showed that live and heat-killed Lp-HKD stimulates the production of interleukin-1ß (IL-1ß), IL-6, IL-10, and interferon-γ (IFN-γ) from PBMCs. Stimulation by heat-killed Lp-HKD yielded stronger cytokine production than stimulation by the live Lp-HKD. Additionally, CD14+ monocytes were identified as major producers of IL-1ß, IL-6, and IL-10 under Lp-HKD stimulation; however, IFN-γ was mainly produced from immune cells other than CD14+ monocytes. Depletion of CD14+ monocytes from the PBMCs cultivation strongly decreased cytokine production induced by heat-killed Lp-HKD. The inhibition of toll-like receptor (TLR) 2/4 signaling decreased IL-1ß and IL-6 production induced by live Lp-HKD and IL-1ß, IL-6, and IFN-γ production induced by heat-killed Lp-HKD. Furthermore, live or heat-killed Lp-HKD also activated T cells and their production of IFN-γ and tumor necrosis factor-α. Then, culture supernatants of bovine PBMCs treated with heat-killed Lp-HKD demonstrated antiviral effects against BRV in vitro. In conclusion, this study demonstrated that Lp-HKD activates the functions of bovine immune cells via TLR2/4 signaling and exerts an antiviral effect against BRV through the induction of antiviral cytokines. Lp-HKD could be useful for the prevention and treatment of calf diarrhea through its immune activating effect.

Characterization of cysteine proteases from poultry red mite, tropical fowl mite, and northern fowl mite to assess the feasibility of developing a broadly efficacious vaccine against multiple mite species.

Infestation with poultry red mites (PRM, Dermanyssus gallinae) causes anemia, reduced egg production, and death in serious cases, resulting in significant economic losses to the poultry industry. As a novel strategy for controlling PRMs, vaccine approaches have been focused upon and several candidate vaccine antigens against PRMs have been reported. Tropical (TFM, Ornithonyssus bursa) and northern (NFM, Ornithonyssus sylviarum) fowl mites are also hematophagous and cause poultry industry problems similar to those caused by PRM. Therefore, ideal antigens for anti-PRM vaccines are molecules that cross-react with TFMs and NFMs, producing pesticidal effects similar to those against PRMs. In this study, to investigate the potential feasibility of developing vaccines with broad efficacy across mite species, we identified and characterized cysteine proteases (CPs) of TFMs and NFMs, which were previously reported to be effective vaccine antigens of PRMs. The open reading frames of CPs from TFMs and NFMs had the same sequences, which was 73.0% similar to that of PRMs. Phylogenetic analysis revealed that the CPs of TFMs and NFMs clustered in the same clade as CPs of PRMs. To assess protein functionality, we generated recombinant peptidase domains of CPs (rCP-PDs), revealing all rCP-PDs showed CP-like activities. Importantly, the plasma obtained from chickens immunized with each rCP-PD cross-reacted with rCP-PDs of different mites. Finally, all immune plasma of rCP-PDs reduced the survival rate of PRMs, even when the plasma was collected from chickens immunized with rCP-PDs derived from TFM and NFM. Therefore, CP antigen is a promising, broadly efficacious vaccine candidate against different avian mites.