Cell Surface Expression of the Bovine Leukemia Virus-Binding Receptor on B and T Lymphocytes Is Induced by Receptor Engagement

BLV-truncated RBD bind to the BLV receptor, a molecule distinct from Glut1. A, The specificity of B_RBD receptor binding was validated by an envelope interference assay wherein 293T cells were transiently transfected with a control plasmid, the B_RBDrFc expression plasmid (top panels) or the entire BLV Env (bottom panels) and assessed 48 h later for binding to B_RBDeGFP and H_RBDeGFP. Representative histograms showing staining of control-transfected (solid black line), B_RBDrFc-transfected, and BLV Env-transfected (dashed line) 293T cells are presented. Nonspecific background staining is depicted as a filled histogram. The MFI of B_RBDeGFP staining following B_RBDrFc and BLV Env transfection changed from 30 to 15 and 95 to 27, respectively, while the MFI of H_RBDeGFP staining changed from 14 to 17 and 13 to 12, respectively. B, 293T cells were transiently transfected with a control plasmid or a Glut1 expression vector. Thirty-six hours after transfection, cells were stained with either a rFc-tagged HTLV-2 RBD (H_RBDrFc) that binds Glut1 (19 28 31 60 ) (upper panels) or the rFc-tagged BLV RBD (B_RBDrFc, lower panels), followed by incubation with a secondary FITC-conjugated sheep anti-rabbit Ab. Filled histograms depict control binding. C, Binding of H_RBDrFc and B_RBDrFc was monitored on human red cells as described above. Staining profiles are presented in comparison to background binding (filled histogram). Data are representative of results obtained in three independent experiments.

The BLV Env- binding receptor is expressed on diverse cell lines of different species and is down-regulated upon BLV infection. A, B_RBDrFc was used to assess the cell surface expression of the BLV- binding receptor on transformed cell lines derived from the following species; human (293T, TE671, HeLa, JY, and Jurkat), simian (COS-7), porcine (PK15), dog (D17), hamster (A23), mink (CCL64), and mouse (Dunni and NIH3T3). Expression was also monitored on the Sf9 insect cell line. Cells were incubated with the B_RBDrFc for 30 min at 37°C, followed by incubation with a FITC-conjugated anti-rabbit Fc Ab at 4°C. Binding was monitored by flow cytometry. Filled histograms depict background binding in the presence of the secondary FITC-conjugated anti-rabbit Ab alone while specific binding is shown as a solid line. Data are representative of results obtained in at least two independent experiments. B, B_RBDrFc binding was assessed on bovine (TEK, KNS-R, and BL3) and ovine (PO) cell lines (top panels) as well as on the BLV-infected PO cell line and the fetal lamb kidney cell line (FLK-BLV) (bottom panels).

The BLV-binding receptor is expressed on differentiating B cells and is induced on mature B cell following receptor activation. A, Expression of the BLV-binding receptor on differentiating B cells was monitored in mouse BM. Pro-/pre-B cells, immature B cells, and mature B cells were distinguished on the basis of IgM and B220 expression as shown and B_RBDrFc binding in the three subsets is shown. B, B_RBDrFc binding was assessed in freshly isolated (−) B220⁺ spleen and LN cells isolated from C57BL/6 mice as well as 48 h after LPS (20 μg/ml) stimulation (+). Human CD19⁺ lymphocytes were purified by positive selection and B_RBDrFc binding was assessed before (−) and after (+) 3 days of BCR stimulation with an anti-IgM mAb (20 μg/ml).

The BLV-binding receptor is an early marker of TCR stimulation and its expression requires protein synthesis. A, Human umbilical cord CD4⁺ T lymphocytes were purified by negative selection. Expression of CD69, CD25, and the BLV Env-binding receptor was assessed on freshly isolated cells (0 h) and after 2, 4, 6, 8, 16, and 24 h of TCR stimulation with immobilized anti-CD3 and anti-CD28 mAbs (solid line histograms). Expression of these markers at each time point was also evaluated in the presence of the protein synthesis inhibitor CHX (5 μg/ml; dashed histograms). Background fluorescence obtained with the secondary FITC-conjugated Ab alone is shown in each histogram (filled histograms). B, BLV Env-binding receptor expression was also assessed at extended time points after 3, 6, and 11 days of TCR stimulation as well as on T cells undergoing homeostatic proliferation induced by rIL-7 stimulation (10 ng/ml). Data are representative of results obtained in at least two independent experiments.