In the present study, we monitored Foxp3+ T cells in bovine leukemia virus (BLV)-infected cattle. By flow cytometric analysis, the proportion of Foxp3+CD4+ cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3+CD4+ cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN-γ mRNA expression, suggesting that Foxp3+CD4+ T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection.
adult T-cell leukemia/lymphoma
bovine leukemia virus
cytotoxic T-lymphocyte antigen-4
enzootic bovine leukemia
hepatitis C virus
human immunodeficiency virus
human T-lymphotropic virus
mean fluorescence intensity
peripheral blood mononuclear cell
quantitative real-time PCR
simian immunodeficiency virus
regulatory T cells
Bovine leukemia virus (BLV), which is a delta retrovirus that chronically and persistently infects bovine B cells, is the etiological agent of EBL . In Japan, a growing number of cases of EBL have been reported in recent years, a worrisome trend given that there is no effective treatment and vaccination against BLV . Most BLV-infected cattle do not display clinical signs of the disease and are referred to as AL. However, approximately 30% of naturally infected cattle show PL, characterized by benign expansion of circulating B cells. Less than 5% of infected cattle develop EBL after extended latency periods of 5–10 years . Although the pathogenesis of BLV infection clearly involves several host factors [3, 4], many aspects of BLV pathogenesis are still poorly understood.
Treg play a critical role in the maintenance of the host immune system by suppressing the proliferation of, and cytokine production by, pathogenic T cells . A subset of CD4+CD25+ T cells expresses transcriptional regulator Foxp3, which is critical in the development and functioning of Treg cells in both mice and humans . In healthy individuals, CD4+CD25+Foxp3+ T cells negatively regulate excessive T-cell responses in autoimmunity, transplant rejection and viral infections [7, 8]. Furthermore, CD4+CD25+ T cells express CTLA-4, a CD28 homologue and potent negative regulator of immune responses, on their cell surface. CTLA-4 binds to its ligands, CD80(B7-1)/CD86(B7-2), and is expressed on antigen-presenting cells (APC) [9, 10]. As a result, CTLA-4 expressing Treg induce tryptophan-dependent IDO and production of immunosuppressive kynurenine in APC, rendering such effector T cells prone to death by apoptosis [9, 10]. In contrast, it has been recently reported that Treg, including CD4+CD25+Foxp3+ T cells, are involved in the immune evasion mechanisms of tumors and several pathogens that cause chronic infections. In case of viral infections, the kinetics of Treg closely correlated with increased virus load and disease progression. More specifically, upregulation of Foxp3 or CTLA-4 in Treg contributes to immune exhaustion, which results in increased viral load and disease progression in chronic infection with HIV , SIV , HTLV , and HCV . Likewise, intratumoral CD4+CD25+Foxp3+ T cells mediate suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma . Thus, expansion of Treg is one of the mechanisms involved in the downregulation of immune responses during the progression of chronic diseases, as well as in facilitating immune evasion by several pathogens causing chronic infections and tumors. In the present study, to determine the possible role of bovine Treg in chronic infectious diseases, we monitored CD4+Foxp3+ T cells in BLV-infected cattle at different disease stages.
BLV-infected cattle (non-pregnant adult female cattle, Holstein breed) were diagnosed at the Veterinary Teaching Hospital, Graduate School of Veterinary Medicine, Hokkaido University, between 2007 and 2012. BLV infection was tested by nested-PCR, and the provirus load was further confirmed by qRT-PCR as described previously . Animals were regarded as having PL if at least two consecutive lymphocyte counts were more than 10,000/µL . PBMC isolation and syncytium formation assay were conducted according to the procedure described previously . To assess the degree of immunosuppression in infected cattle, IFN-γ mRNA expression in the freshly isolated PBMC (1 × 106 cells) was quantified by qRT-PCR as described previously . To analyze the Foxp3 expressing cells, dual-color flow cytometric analysis was carried out using anti-bovine CD4 antibody (CACT138A; VMRD, Pullman, WA, USA) as described previously [16, 18]. Purified PBMC (1 × 106 cells) were pre-incubated with 10%-inactivated FBS-PBS containing EDTA (0.5 mg/mL) for 20 min at room temperature. The PBMC were incubated with CACT138A or isotype control mouse immunoglobulin (Ig)G1 (Enzo Life Sciences, Farmingdale, NY, USA) for 30 min at 4°C. Then, the cells were washed with 10%-inactivated FBS-PBS and stained with phycoerythrin (PE)-conjugated goat anti-mouse IgG (Beckman Coulter, Inc., Fullerton, CA, USA). Furthermore, the treated cells were incubated with FOXP3 Fix/Perm solution (BioLegend, San Diego, CA, USA) for 20 min at room temperature, and 120 min at 4°C. The cells were washed with Cell Staining Buffer (two times: BioLegend) and 10-fold diluted FOXP3 Perm buffer (BioLegend). After the washing, the cells were incubated with FOXP3 Perm solution for 15 min at room temperature and 30 min at 4°C. Following that, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated mouse Foxp3 rat monoclonal antibody (eBioscience, San Diego, CA, USA) for 30 min at 4°C. FITC-conjugated rat IgG2aκ (eBioscience) was used as isotype control. Fluorescence of the cells was measured on an EPICS XL flowcytometry system (Beckman Coulter, Inc.), and the data were analyzed using the EPICS EXPO32 ADC software (Beckman Coulter, Inc.). Mean values were compared using Student's t-test or Welch's t-test. Correlation coefficient was done using Spearman's correlation coefficient. Differences between groups were considered significant if probability values of P < 0.05 or P < 0.01 were obtained.
Foxp3 is a regulatory T-cell-specific transcription factor that functions as the master regulator of the development and function of Treg . To assess whether immunosuppressive mechanisms during progression of BLV infection were involved in the expansion of Treg, we monitored Foxp3+CD4+ cells recognized as Treg by flow cytometry analysis. In AL cattle, the percentage of Foxp3+cells within the CD4+ cell population was not significantly different from the BLV-uninfected cattle (Fig. 1). In contrast, the percentage of Foxp3+ cells within the CD4+ cells in PL cattle was significantly higher than in control (P < 0.01) and AL cattle (P < 0.01; Fig. 1), although the MFI for Foxp3 was not different among the different disease states (data not shown). In BLV-infected cattle, significant positive correlations were observed between the proportion of Foxp3+CD4+ cells with the number of leukocytes (P < 0.01; Fig. 2a), virus titer (P < 0.01; Fig. 2b) and virus load (P < 0.01; Fig. 2c), suggesting that Treg increased with BLV-disease progression. It has been shown that IFN-γ, a key cytokine for virus clearance, is downregulated during BLV infection [3, 16, 18]. This phenomenon is characterized as immune suppression that facilitates disease progression during BLV infection through an unknown mechanism. Thus, we investigated the correlation between the proportion of Foxp3+CD4+ cells and IFN-γ expression in PBMC from BLV-infected animals. As observed in the previous study [16, 18], IFN-γ level in PL cattle was significantly lower than that of control cattle (data not shown). Interestingly, increase in the proportion of Foxp3+CD4+ cells in BLV-infected cattle correlated with downregulation of IFN-γ mRNA expression (P < 0.05, Fig. 2d). Specifically, PL cattle with a high proportion of Foxp3+CD4+ cells had low levels of IFN-γ (Fig. 2d). These results prompted the notion that decreased levels of IFN-γ during virus proliferation and progression of BLV-induced disease could be due to the expansion of Foxp3+CD4+ cells.
Foxp3 is recognized as the most reasonable maker for Treg, as Foxp3 is a key regulatory gene for the development of Treg . Recent studies have also indicated the involvement of Treg in immune dysfunction in several human diseases. However, there are only a few reports available on the role of Treg in domestic animals. In the present study, we demonstrated the correlation between BLV infection and Foxp3 expression in CD4+ T cells. A deeper understanding of the Treg functions in domestic animals will facilitate the elucidation of events leading to immune dysfunction during the progression of incurable diseases. Here, we have shown that the percentage of cells positive for Foxp3 in CD4+ T cells was found to be increased during disease progression. Interestingly, the percentage of cells positive for Foxp3 among CD4+ T cells significantly correlated positively to the number of leukocytes, virus titer and proviral load. However, the high percentage of cells positive for Foxp3 tended to downregulate IFN-γ, which can be characterized as immune suppression that would facilitate disease progression during BLV infection through yet unknown mechanisms. Notably, the expansion of Treg was prominently observed in PL cattle with the lowest IFN-γ level. These results prompted the notion that decreased levels of IFN-γ during virus proliferation and progression of BLV-induced disease could be due to the expansion of Treg. Further studies are necessary to elucidate the detailed mechanism(s) behind the increased Treg during BLV infection.
BLV is a retrovirus structurally and functionally related to HTLV-1 [1, 4]. HTLV-1-induced diseases have been extensively studied, and the association between HTLV-1 and Treg cells has been well documented as a mechanism by which HTLV-1 induces immune dysfunction . ATL patient-derived Treg have been shown to express Foxp3 [19, 20]. Interestingly, the ATL-derived Foxp3 expressing Treg exhibit immunosuppressive functions, which may contribute to clinically observed cellular immunodeficiency in ATL patients [21-23]. Therefore, it has been hypothesized that ATL cells may be a derivate of Treg cells [13, 21]. In the present study, the expansion of Foxp3+ cells among CD4 cells was prominently observed in PL cattle with the lowest IFN-γ level, similar to that of ATL patients. However, BLV is an oncogenic B-lymphocytotropic retrovirus that infects cattle, inducing a persistent infection with diverse outcomes. Although BLV-tax expression correlated positively with the proportion of Foxp3+CD4+ cells (data not shown), it is difficult to determine whether there is a similar mechanism via Treg for downregulation of antiviral responses after infection with BLV and other related retroviruses . Nevertheless, the proportion of Foxp3+CD4+ cells closely correlated with BLV-induced disease progression in a similar pattern to that reported in ATL patients.
Although the role of Treg in immunosuppression associated with BLV infection is still speculative, overexpression of Foxp3 induced by Treg might be involved in the immunosuppression. Treg-producing transforming growth factor (TGF)-β or interleukin (IL)-10 have regulatory or suppressive properties that inhibit the effects of anti-pathogenic effector cells . Previous reports had indicated that the expression of IL-10 was closely associated with disease progression in BLV-infected cattle [18, 25]. More recently, we reported galectin-9, which induces expansion of CD4+CD25+Foxp3+ regulatory T cells , is correlated with downregulation of IFN-γ in BLV-infected cattle . These findings raise the possibility that expansion of Treg in bovines is associated with T-cell exhaustion during BLV infection. However, Foxp3 expression levels did not correlate with upregulation of TGF-β and IL-10 in BLV-infected cattle, although the TGF-β expression level was higher than that in uninfected cattle (data not shown). As an alternative hypothesis, Treg suppresses immune responses through direct cell–cell contact in a process that is dependent on signaling via CTLA-4 [9, 10]. Indeed, Foxp3+CTLA-4+ expressing HTLV-1-infected cells suppressed proliferation of naïve T cells that were stimulated with anti-CD3 antibody . However, there is no information about bovine CTLA-4 and its associations with disease. At present, the molecular mechanism behind downregulation of expression of IFN-γ by expanding Treg is still unclear. Further studies are necessary to determine the correlation between Treg expansion and T-cell dysfunction in infected cattle.
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), and by a special grant for the Promotion of Basic Research Activities for Innovative Biosciences from the Bio-oriented Technology Research Advancement Institution (BRAIN). We are grateful to Dr Hideyuki Takahashi and Dr Yoshiyuki Goto, National Agriculture and Food Research Organization, BRAIN, for their valuable advice and discussion.
The authors have no conflicts of interest to declare.