Isolated receptor binding domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ T cells

Abstract

Background: We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding.

Results: As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1RBD or H2RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1RBD as well as H2RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell.

Conclusion: Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes.

Figure 1

Antibody and H_RBD binding following transfection of the Glut-1 and Glut-3 glucose transporters. (A) 293T cells were transfected with Glut-1 or Glut-3 expression vectors and assayed for binding to the mAb1418 and C-term anti-Glut-1 polyclonal Ab. The former stainings were performed on whole cells as well as permeabilized cells to determine cell surface and total binding, respectively. All stainings using the anti-Glut-1 pAb were performed on permeabilized cells as the recognized epitope is intracellular. Staining was performed at 4°C. Specific binding and background fluorescence due to the secondary conjugated Ab are indicated in solid line and filled histograms, respectively. (B) Control and transfected 293T cells were incubated with rFc-tagged H1_RBD and H2_RBD fusion proteins for 30 min at 37°C followed by incubation with a FITC-conjugated αrabbit-Fc antibody at 4°C. Direct binding to H2_RBD was demonstrated by incubation of cells with an EGFP-tagged envelope (H2_RBD-EGFP). Binding is shown in solid line histograms whereas control immunofluorescence is shown in filled histograms.

Figure 2

Endogenous Glut-1 expression in diverse cell types is not reflected by mAb1418 reactivity but correlates with binding of the HTLV-1 and HTLV-2 Env RBDs. (A) 293T, Jurkat and primary human erythrocytes were stained with mAb1418 and control binding with the secondary FITC-conjugated antibody is shown in all histograms (filled). Intracellular Glut-1 levels in permeabilized 293T and Jurkat cells were monitored with mAb1418 as well as the C-term polyclonal Glut-1 antibody. Expression of the HTLV-1 and HTLV-2 receptor was monitored by incubation of cells for 30 min at 37°C with the rFc-tagged H1_RBD and H2_RBD fusion proteins as well as the H2_RBD-EGFP fusion protein. Binding is shown in solid line histograms whereas control immunofluorescence is shown in filled histograms. (B) Total Glut-1 protein levels in cell extracts from 293T, Jurkat and human erythrocytes were monitored by immunoblotting with an anti-C-term Glut-1 antibody.

Figure 3

TCR stimulation results in Glut-1 expression and concomitant glucose uptake in CD4 and CD8 lymphocytes: Induction of H1_RBD and H2_RBD binding. CD4+ and CD8+ T lymphocytes were isolated by negative selection and stimulated via the TCR using αCD3/αCD28 mAbs. (A) Non-activated and TCR-activated T cells were used for binding assays with mAb1418 followed by incubation with a FITC-conjugated αmouse IgG. Intracellular Glut-1 levels were monitored in permeabilized cells using the C-term Glut-1 polyclonal antibody followed by incubation with a FITC-conjugated sheep αrabbit IgG antibody. Filled histograms depict binding in the presence of the secondary FITC-conjugated antibody alone. Expression of the HTLV-1 Env receptor was detected by a 30 min incubation of the non-activated and TCR-activated cells with rabbit rFc-tagged H1_RBD fusion protein at 37°C and binding was revealed by a 20 min incubation at 4°C with a FITC-conjugated sheep αrabbit IgG antibody. Binding to the H2_RBD domain fused directly to EGFP (H2_RBD-EGFP) was detected following a 30 min incubation at 37°C. (B) Glucose uptake was assayed by incubating non-activated and TCR-activated CD4 and CD8 T cells (1 × 10⁶) with 2-deoxy-D [1-³H]glucose (2 μCi) for 45 min at 37°C. Uptake for each cell population is expressed as mean counts per minute (CPM) for triplicate samples, error bars indicate SD.

Figure 4

Glucose uptake in CD4+ T cells is abrogated by the cytochalasin B Glut-1 inhibitor. (A) H2_RBD-EGFP binding to Jurkat T cells was assessed following incubation in the absence or presence of the Glut-1 inhibitor cytochalasin B (CytB,100 μM) or the related cytochalasin D (CytD, 100 μM) molecule for 30 min. Filled and solid line histograms depict control and H2_RBD-EGFP binding, respectively. (B) Glucose uptake was assayed by incubation of control, cytochalasin B-, and cytochalasin D-treated Jurkat cells in the presence of 2-deoxy-D [1-³H]glucose (5 μM) for 10 min at room temperature. (C) Glucose uptake in quiescent CD4 T cells, CD3/CD28-activated CD4 T cells and cytochalasin B-treated CD3/CD28-activated CD4 T cells was assessed as described in the legend of figure 3. Uptake for each condition is expressed as mean counts per minute (CPM) for triplicate samples, error bars indicate SD.