Figure 6
Figure 6. HTLV-1 transported across the tight epithelial monolayer is able to infect productively human DCs. (A) Scheme depicting the experimental procedure. Briefly, Caco-2 cells were grown as usual on Transwell filters until differentiation, at which time filters were turned upside down and DCs were seeded on the inversed filter. After 4 hours, filters were turned again and HTLV-1–infected lymphocytes were added as usual to the apical pole to initiate triculture. TER was checked regularly to confirm the tightness of the cellular barrier. (B,D) Western blot and FACS analysis of the presence of the viral p24 protein within DCs. After 4 days of coculture, DCs were harvested by detaching from the filter by centrifugation (200g) and examined for the p24 viral protein. (B) Western blot assay showing a signal for the p24 protein in DCs (lane c) compared with the whole HTLV-1–infected lymphocytes cell lysate (lane a) or DCs cocultured with Caco-2/uninfected lymphocytes (lane d) and the positive control treated with EDTA (lane b). (D) Flow cytometric analysis revealed 40% DCs positive for the p24 capsid protein (green curve) after coculture underneath the Transwell with Caco-2 and HTLV-1–infected lymphocytes. (C) Localization of HTLV-1 p24 protein within DCs by immunofluorescence. DCs were labeled in green with Cell Tracker vital dye before coculture. After 3 days of contact, immunofluorescence directed against the p24 protein was performed (red). Shown is the lower side of the filter. Immunoreactivity (red) was detected either in the dendrites (left) or in the cell body (right) of DCs. Original magnification was 250×. Observation was with a Zeiss AxioVision fluorescence microscope equipped with an Apotome device. Image acquisition with a Zeiss Axiocam camera. (F) Production of viral p19 protein by DCs harvested from tricultures. Four days after contact, DCs were harvested by centrifugation (200g) from basal side of the Transwell filters, seeded in fresh medium for 4 days, and viral p19 protein was measured in the medium at days 1 and 3 after culture by ELISA assay and compared with control.

HTLV-1 transported across the tight epithelial monolayer is able to infect productively human DCs. (A) Scheme depicting the experimental procedure. Briefly, Caco-2 cells were grown as usual on Transwell filters until differentiation, at which time filters were turned upside down and DCs were seeded on the inversed filter. After 4 hours, filters were turned again and HTLV-1–infected lymphocytes were added as usual to the apical pole to initiate triculture. TER was checked regularly to confirm the tightness of the cellular barrier. (B,D) Western blot and FACS analysis of the presence of the viral p24 protein within DCs. After 4 days of coculture, DCs were harvested by detaching from the filter by centrifugation (200g) and examined for the p24 viral protein. (B) Western blot assay showing a signal for the p24 protein in DCs (lane c) compared with the whole HTLV-1–infected lymphocytes cell lysate (lane a) or DCs cocultured with Caco-2/uninfected lymphocytes (lane d) and the positive control treated with EDTA (lane b). (D) Flow cytometric analysis revealed 40% DCs positive for the p24 capsid protein (green curve) after coculture underneath the Transwell with Caco-2 and HTLV-1–infected lymphocytes. (C) Localization of HTLV-1 p24 protein within DCs by immunofluorescence. DCs were labeled in green with Cell Tracker vital dye before coculture. After 3 days of contact, immunofluorescence directed against the p24 protein was performed (red). Shown is the lower side of the filter. Immunoreactivity (red) was detected either in the dendrites (left) or in the cell body (right) of DCs. Original magnification was 250×. Observation was with a Zeiss AxioVision fluorescence microscope equipped with an Apotome device. Image acquisition with a Zeiss Axiocam camera. (F) Production of viral p19 protein by DCs harvested from tricultures. Four days after contact, DCs were harvested by centrifugation (200g) from basal side of the Transwell filters, seeded in fresh medium for 4 days, and viral p19 protein was measured in the medium at days 1 and 3 after culture by ELISA assay and compared with control.

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