FIG. 1.

Analysis of HTLV-1 binding to B-THP-1 cells. (A) Parental B-THP-1 cells or cells transduced to stably express DC-SIGN (B-THP-1/DC-SIGN) were incubated with an FITC-labeled anti-CD19 Ab in combination with APC-labeled anti-DC-SIGN, Alexa Fluor 647-labeled anti-GLUT-1, or anti-HSPGs, and PE-labeled anti-NRP-1 Abs and were analyzed by flow cytometry. A total of 50,000 events collected for each sample were gated to include the CD19⁺ population. Isotype controls are represented by dotted-line histograms, whereas solid-line histograms represent Ab reactivity with respect to the indicated receptor molecules. (B) A QDot-based binding assay was employed to determine the effect of blocking DC-SIGN and other receptor molecules on HTLV-1 binding to B-THP-1 cells. Target cells (1 × 10⁶) either were left untreated or were treated (30 min, room temperature) with a blocking Ab against DC-SIGN (clone 507; 20 μg/ml), the GLUT-1 inhibitor Cyto B (20 μM), the HSPG inhibitor Hep (20 mU), or NRP-1 ligand (VEGF; 50 ng/ml). Cells subsequently were incubated with Biot-HTLV-1 (125 ng/10⁶ cells) for 45 min on ice, and the binding was quantified as described in Materials and Methods. Cells incubated with QDots or biotin alone were used as negative controls. HTLV-1 binding was estimated as the fluorescence measured at 400 or 605 nm. The results shown represent the mean fluorescence ± standard deviations (T bars) from three independent experiments each performed in duplicate. An asterisk denotes a statistically significant decrease in fluorescence compared to maximum binding without any inhibitor (P ≤ 0.05).