Decreased expression of L‐selectin (CD62L) on lymphocytes in enzootic bovine leukaemia

Expression of L‐selectin was determined by single‐ and two‐colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L‐selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV‐infected animals comprised lymphocytotic and non‐lymphocytotic cows. L‐selectin was expressed on 90–98 % of granulocytes in all tested animals. The percentage of PBMC expressing L‐selectin was lower in cattle with persistent lymphocytosis than in non‐lymphocytotic or BLV‐free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L‐selectin was significantly decreased from 60.2 ± 1.9 % in BLV‐free cattle to 43.8 ± 3.6 and 22.5 ± 5.7 % in non‐lymphocytotic and lymphocytotic cattle, respectively. B‐lymphocytes stained for L‐selectin exhibited about 50 % reduction in L‐selectin expression in BLV‐infected cattle compared with BLV‐free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L‐selectin‐positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV‐free and BLV‐infected cattle. However, L‐selectin expression on T lymphocytes was reduced (about 50 %) in BLV‐infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L‐selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.     

Changes in B cell and T cell subsets in bovine leukaemia virus-infected cattle

Direct immunofluorescence and fluorescence-activated cell sorter techniques were used for the detection of surface immunoglobulin positive (SIg+) cells in peripheral blood lymphocytes (PBL's) of bovine leukaemia virus (BLV) infected cattle with or without persistent lymphocytosis (PL+, PL-) and in BLV-free cattle. The percentage of SIg+ cells was more than twice as high in BLV+PL+ cattle than in BLV-free and BLV+PL- cattle. Bovine T cells, and T cell subsets were identified indirectly by the same techniques using three monoclonal antibodies (MAb's) specific for all T cells (IL-A43), T helper (BoT4) cells (IL-A12) and T cytotoxic (BoT8) cells (IL-A17). The major histocompatibility complex (MHC) determinants of both class II (BoT4) and class I (BoT8) as well as all T cells were significantly reduced in BLV+PL+ compared to BLV-free cattle. The actual decrease in the BoT8 cell subset or the dilution effect that would change effector:target cell ratio suggests that a resultant decrease in cytotoxic activity in BLV+PL+ cattle may play an important role in the progress of BLV infection in cattle.     

Cellular immune response cytokine expression during the initial stage of bovine leukemia virus (BLV) infection determines the disease progression to persistent lymphocytosis

We have established experimental models of bovine leukemia virus (BLV) infection followed by progression to persistent lymphocytosis (PL) positive (BLV+PL+) or PL negative (BLV+PL-) stages of infection. Two out of six BLV infected animals developed PL+ 4 weeks after BLV infection. One other animal became PL+ late in the course of infection and three infected animals stayed PL-. These animals (PL-) exhibited transient lymphocytosis 3-4 weeks after infection and sustained PL- lymphocyte counts up to 24 weeks after infection. Competitive RT-PCR analysis of IFN-gamma mRNA expression revealed that peripheral blood mononuclear cells (PBMC) of animals with PL+ status developed by 4 weeks after infection had augmented IFN-gamma mRNA expression 3-4 weeks after BLV infection. However PBMC of animals that sustained a long-termed PL- lymphocyte count had elevated IFN-gamma mRNA expression 1-24 weeks after infection. Competitive RT-PCR analysis of IL-2 mRNA expression showed an increase in the levels of IL-2 mRNA in PL animals. Interleukin-10 (IL-10) mRNAs expression were elevated both in PL+ and PL- animals from 3 and 12 weeks after infection respectively. We suggest that early and extended expression of cellular response cytokines may delay the progression to PL+ in enzootic bovine leukemia.     

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.     

Decreased expression of L-selectin (CD62L) on lymphocytes in enzootic bovine leukaemia

Expression of L-selectin was determined by single- and two-colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L-selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV-infected animals comprised lymphocytotic and non-lymphocytotic cows. L-selectin was expressed on 90-98% of granulocytes in all tested animals. The percentage of PBMC expressing L-selectin was lower in cattle with persistent lymphocytosis than in non-lymphocytotic or BLV-free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L-selectin was significantly decreased from 60.2 +/- 1.9% in BLV-free cattle to 43.8 +/- 3.6 and 22.5 +/- 5.7% in non-lymphocytotic and lymphocytotic cattle, respectively. B-lymphocytes stained for L-selectin exhibited about 50% reduction in L-selectin expression in BLV-infected cattle compared with BLV-free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L-selectin-positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV-free and BLV-infected cattle. However, L-selectin expression on T lymphocytes was reduced (about 50%) in BLV-infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L-selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.     

T、B淋巴细胞功能对流行性牛白血病发生发展的影响

本文回顾了T、B淋巴细胞功能与牛白血病发生发展的相关性。阐明了感染BLV牛B细胞淋巴瘤细胞除了来源于Bla(CD5^ CD11b^ )细胞，也可来源于B1b(CD5^-CD11b^ )及常规B2（CD5^-CD11b^-)细胞。探讨了T细胞功能对牛白血病的发生发展的影响，指出IL－12表达下降和IL－10表达增加可能导致了B细胞淋巴肉瘤的形成，Ⅰ型和Ⅱ型细胞因子的不平衡引起了BLV的发展。同时讨论了B－T细胞的相互作用在BLV感染时疾病阶段性发展过程中的作用。     

FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/macrophages.

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers.     

Effects of bovine leukemia virus infection on milk neutrophil function and the milk lymphocyte profile

The effects of bovine leukemia virus (BLV) on the immune response have been extensively investigated; however, its effects on mammary gland immunity are only speculative. Although BLV has a tropism for B cells, it can affect both adaptive and innate immunities because these systems share many effector mechanisms. This scenario is the basis of this investigation of the effects of BLV on mammary gland immunity, which is largely dependent upon neutrophilic functions. Thus, the present study sought to examine neutrophilic functions and the lymphocyte profile in the milk of naturally BLV-infected cows. The viability of the milk neutrophils and the percentage of milk neutrophils that produced reactive oxygen species (ROS) or phagocytosed Staphylococcus aureus were similar between BLV-infected and BLV-uninfected dairy cows. Furthermore, the expression of CD62L and CD11b by the milk neutrophils and the percentage of milk neutrophils (CH138⁺ cells) that were obtained from the udder quarters of the BLV-infected cows were not altered. Conversely, the median fluorescence intensity (MFI) representing intracellular ROS production and the phagocytosis of S. aureus, the expression of CD44 by the milk neutrophils and the percentage of apoptotic B cells were lower in the milk cells from BLV-infected dairy cows, particularly those from animals with persistent lymphocytosis (PL). The lymphocyte subsets were not different among the groups, with the exception of the percentage of CD5⁻/CD11b⁻ B cells, which was higher in the milk cells from BLV-infected cows, particularly those with PL. Thus, the present study provides novel insight into the implications of BLV infection for mammary gland immunity.     

Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4⁺CD25⁺Foxp3⁺ T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3⁺CD4⁺ cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4⁺ cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4⁺ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4⁺ and CD25⁺ T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4⁺ cells in the CD4⁺ T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4⁺CTLA-4⁺ T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection.     

Experimental infection of calves with bovine leukemia virus (BLV): an applicable model of a retroviral infection

An experimental model of chronic infection with bovine leukemia virus (BLV) was established in young calves within a relatively short time. In the sera of all infected calves, precipitating antibodies were detected within 5 weeks after infection but upon disease progression pattern of cellular profiles varied. Three calves exhibited transient lymphocytosis 3-5 weeks after infection, two became persistent lymphocytotic (PL+) by that time and one stayed non-lymphocytotic (PL-) for 11 weeks and became PL+ after 4.5 months. Eventually all infected calves became PL+ by the end of the experiment, 6-12 months after infection. Increase of total counts of peripheral blood mononuclear cells (PBMC) related to polyclonal expansion of B-cells. The latter was assessed in all infected calves where the expansion of CD5-bearing cells (B+ CD5+) correlated with increase or decrease of total PBMC counts. Other cell populations such as CD4 and CD8 were also affected. Percentages decreased by 5 weeks after experimental infection to about half their original values though actual cell numbers stayed relatively stable. The experimental model we established compared well with field cases of naturally BLV-infected cattle and thus permitted the investigation of the disease at early stages of infection.     

Interferon-gamma and B-cell Activating Factor (BAFF) promote bovine B cell activation independent of TLR9 and T-cell signaling

We previously reported that CD21(+) B cells purified from bovine blood do not respond to CpG-ODN stimulation unless either CD14(+) monocytes or B-cell Activating Factor (BAFF), a cytokine produced by activated monocytes, are present. In this report, we present evidence that CD14(+) monocytes are critical for CpG-specific lymphocyte proliferation within the peripheral blood mononuclear cell (PBMC) population but that this response is not mediated by soluble factors produced by CpG-activated monocytes. We further determine that bovine monocytes stimulated with IFN-γ induce expression of the BAFF gene and that recombinant IFN-γ and BAFF induced robust B cell activation when cultured in the absence of CpG ODN. These data suggest that CpG-stimulated monocytes may indirectly promote B cell activation by promoting release of cytokines and/or other soluble factors from accessory cells which in turn act on CpG-stimulated B cells to promote antigen-independent and T cell independent B cell activation. Understanding the T cell independent signals that induce B cell activation has important implications for understanding B cell development in locations where T cells are limited and in understanding polyclonal B cell activation that may contribute to autoimmune diseases.     

Immunohistochemical analysis of expression patterns of tumor necrosis factor receptors on lymphoma cells in enzootic bovine leukosis

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5+/CD11b+), B-1b (CD5-/CD11b+) and B-2 (conventional B) (CD5-/CD11b-). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII, which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.     

Globotriaosylceramide (Gb(3)/CD77) is synthesized and surface expressed by bovine lymphocytes upon activation in vitro

Neutral glycosphingolipids (GSLs) are considered activation markers on human lymphocytes, which are fundamental for studying the immune system. For cattle, only a limited number of activation markers has yet been identified. We recently showed that Shiga toxin 1, known to use globotriaosylceramide (Gb(3) syn. CD77) as a cellular receptor, depresses proliferation of activated bovine lymphocytes [Infect. Immunol. 67 (1999b) 2209]. In order to confirm the expression of Gb(3)/CD77 on bovine lymphocytes, we flowcytometrically examined a bovine B-lymphoma cell line (BL-3) and bovine peripheral blood mononuclear cells (PBMC) before and after mitogenic stimulation and biochemically characterized neutral GSLs extracted from PBMC. CD77 was detected on the surface of BL-3 cells and cultured PBMC essentially after mitogenic stimulation. Although expressed by all PBMC subpopulations identified, the portion of CD7+ cells was highest for BoCD8+ cells, followed by B-cells and BoCD4+ cells at day 4 of cultivation. Ceramide trihexoside of stimulated PBMC was structurally determined as Gal(alpha1-4)Gal(1-4)Glc(1-1)ceramide (Gb(3)). Biochemically, Gb(3) was also detected within unstimulated PBMC which contained ceramide monohexoside (CMH) and Gb(3) in a ratio of about 4:1. However, stimulation induced an increase of CMH and Gb(3) by a factor of 2.5 and 10, respectively, implicating that bovine lymphocytes regulate surface expression of Gb(3)/CD77 predominantly by quantitative changes in the Gb(3) metabolism. This report presents Gb(3)/CD77 as the first GSL identified on bovine immune cells and highly recommends this activation dependent antigen as a useful tool to investigate lymphocyte activation within the bovine immune system.     

Detection of T-lymphocytes in the peripheral blood and lymphoid tissues of cattle using a variant of the E-rosette test and monoclonal antibodies.

A simple and efficient variant of the E-rosette test based on addition of dextran to the incubation media is described. This variant 1) does not include B cells 2) involves some CD2+ null cells as described 3) is not inhibited by anti-CD5 antibody 4) correlates with CD2 expression 5) detects specific changes in the relative proportion of T-lymphocytes under the different conditions. In a group of calves the mean percentage of RFC in the peripheral blood lymphocytes was 53.59 +/- 8.70 and in cows there was 72.57 +/- 3.85. The proportion of RFC detected in bovine leukemia virus (BLV)--infected cows with lymphocytosis was less than one third of that in BLV--negative animals and vice versa in B (MHC class II+) lymphocytes.     

Expression of Kit, the receptor for stem cell factor, in bovine peripheral blood

The expression of Kit, the receptor for stem cell factor (SCF), on bovine peripheral blood cells (PBCs) was examined by using monoclonal antibodies against the bovine Kit protein. Flow cytometric analysis showed that approximately 1.5% of PBCs expressed Kit. In cytospin preparations, the morphology of most Kit+ PBCs was similar to that of large lymphocytes. Subsets of Kit+ PBCs coexpressed CD3, IgM, and/or CD11b but not CD14 or G1. SCF did not induce the proliferation of Kit+ PBCs in vitro. These results indicate that Kit is expressed on subsets of lymphocytes in bovine peripheral blood, but the ligand of Kit, SCF, does not directly induce the proliferation of this cell population.     

Use of a feline cell line in the syncytia infectivity assay for the detection of bovine leukemia virus infection in cattle

This report describes a modified syncytia infectivity assay (SIA) for the direct detection of bovine leukemia virus (BLV) in blood lymphocytes of cattle, using transformed feline (CC81) cells as the indicator system. The data show that the syncytia present in cultures of CC81 cells inoculated with BLV-infected cells are specific and arise through a mechanism similar to that responsible for the phenomenon of "late" polykaryocytosis described in other virus systems. The susceptibility of the CC81 cells to the syncytia-inducing effect of BLV-infected cells is comparable with that of early passages of bovine embryonic spleen cells, which were previously used as the indicator system in the SIA. Unlike the bovine embryonic spleen cells, CC81 cells retain their susceptibility to syncytia induction for long periods of cultivation. Furthermore, the syncytia induced in the CC81 cultures are larger and easier to identify. Thus, the CC81 cells can be used advantageously as the indicator system when the SIA is applied to the detection of BLV-infected lymphocytes. The results of the SIA for the detection of infective BLV agreed closely with those of the radioimmunoassay for the detection of BLV antibodies in randomly examined cattle. On the other hand, many cattle in early stages of infection were positive in the radioimmunoassay several months before they reacted in the SIA. The detection of BLV in blood lymphocytes provides a useful method for the diagnosis of BLV infection in cattle when serologic tests cannot be used, eg, calves that may have passively acquired maternal antibodies and cattle given BLV vaccines.