Fig. 1.
Fig. 1. HTLV-IgG specifically binds to activated primary CD4+ T cells. / The HTLV-I SU immunoadhesin was generated and the binding assays were performed as described in “Materials and methods.” (A) PHA-activated IL-2–dependent primary CD4+ T cells (106) isolated from adult PBMCs were incubated with HTSU-IgG (80 ng) or, as a negative control, a similar immunoadhesin containing the SU protein of an unrelated retrovirus (SUA-IgG; 400 ng). The cells were then incubated with an FITC-conjugated antibody specific for rabbit IgG, and the amount of binding was determined by FACS as described in “Materials and methods.” The left peak represents the amount of binding to SUA-IgG; the right peak represents binding to HTSU-IgG. (B) HTSU-IgG was preincubated with anti–HTLV-I SU human monoclonal antibodies and binding to activated CD4+ T cells determined. Supernatant containing 50 ng/mL HTSU-IgG was incubated with either 2 μg/mL (■) or 10 μg/mL (▪) human mAb directed against HTLV-I SU (PRH-1, PRH-4A, PRH-7A, PRH-11A) or an isotype control directed against a 64-kDa protein of cytomegalovirus (R04)28 for 1 hour on ice before performing the binding assay. The MFI was determined for each of the samples, the value of the MFI of the control (SUA-IgG) subtracted from the MFI of HTSU-IgG samples, and the percent inhibition determined using the following formula: 100-(MFI of experimental/MFI of media-only control × 100). (C) Cell-free HTLV-I and HTLV-II were harvested from the supernatant of producer cell lines (MT-2 and 729pH6neo, respectively), and the relative amount of viral particles determined using a p19 ELISA kit as previously described.26 Stimulated adult CD4+ T cells were preincubated for 30 minutes on ice with a high concentration (50 ng/mL of p19/mL) of HTLV-I or a low concentration (0.2 ng of p19/mL) of HTLV-I or HTLV-II. As controls, the cells were incubated with media without viral particles or with high and low infectious doses of an unrelated mouse retrovirus (amphotropic murine leukemia virus) (gift of Sandra Ruscetti, National Cancer Institute-Frederick). HTSU-IgG (400 ng/mL) was then added to each sample, and binding assays were performed. ■ indicates low concentration of virus; and □, high concentration.

HTLV-IgG specifically binds to activated primary CD4+ T cells.

The HTLV-I SU immunoadhesin was generated and the binding assays were performed as described in “Materials and methods.” (A) PHA-activated IL-2–dependent primary CD4+ T cells (106) isolated from adult PBMCs were incubated with HTSU-IgG (80 ng) or, as a negative control, a similar immunoadhesin containing the SU protein of an unrelated retrovirus (SUA-IgG; 400 ng). The cells were then incubated with an FITC-conjugated antibody specific for rabbit IgG, and the amount of binding was determined by FACS as described in “Materials and methods.” The left peak represents the amount of binding to SUA-IgG; the right peak represents binding to HTSU-IgG. (B) HTSU-IgG was preincubated with anti–HTLV-I SU human monoclonal antibodies and binding to activated CD4+ T cells determined. Supernatant containing 50 ng/mL HTSU-IgG was incubated with either 2 μg/mL (■) or 10 μg/mL (▪) human mAb directed against HTLV-I SU (PRH-1, PRH-4A, PRH-7A, PRH-11A) or an isotype control directed against a 64-kDa protein of cytomegalovirus (R04)28 for 1 hour on ice before performing the binding assay. The MFI was determined for each of the samples, the value of the MFI of the control (SUA-IgG) subtracted from the MFI of HTSU-IgG samples, and the percent inhibition determined using the following formula: 100-(MFI of experimental/MFI of media-only control × 100). (C) Cell-free HTLV-I and HTLV-II were harvested from the supernatant of producer cell lines (MT-2 and 729pH6neo, respectively), and the relative amount of viral particles determined using a p19 ELISA kit as previously described.26 Stimulated adult CD4+ T cells were preincubated for 30 minutes on ice with a high concentration (50 ng/mL of p19/mL) of HTLV-I or a low concentration (0.2 ng of p19/mL) of HTLV-I or HTLV-II. As controls, the cells were incubated with media without viral particles or with high and low infectious doses of an unrelated mouse retrovirus (amphotropic murine leukemia virus) (gift of Sandra Ruscetti, National Cancer Institute-Frederick). HTSU-IgG (400 ng/mL) was then added to each sample, and binding assays were performed. ■ indicates low concentration of virus; and □, high concentration.

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