3D analysis of TRAIL localization by HTLV-1–activated pDCs. (A) Freshly purified CD123⁺BCDA4⁺ pDCs (96%, left) stimulated by HTLV-1 express TRAIL on their membrane (middle and right, red histograms) that is significantly reduced by both HTLV-1–infected patient serum containing HTLV-1 blocking antibody (middle, blue histogram) and chloroquine (right, blue histogram). (B) Unstimulated pDCs (Unst), HTLV-1–stimulated pDC (HTLV-1) treated with chloroquine (HTLV-1 + Chloro) or with blocking HTLV1-infected patient serum (HTLV-1 + Serum) were stained with anti-TRAIL (green) and anti–HTLV-1 virus (red). pDC stainings (green and red) were merged with DAPI (blue)–colored nucleus (Overlay) or with phase contrast (Bright). HTLV-1–activated pDCs (HTLV-1) showed a TRAIL relocalization to cell surface in contrast to unstimulated pDCs (Unst). TRAIL surface localization (green) induced by HTLV-1 was inhibited in the presence of the HTLV-1 blockers (HTLV-1 + Chloro and HTLV-1 + Serum). Inhibition of endosomal acidification (chloroquine) allowed easier detection of HTLV-1 virions (HTLV-1 + Chloro) in contrast to few HTLV-1 particles detected in HTLV-1–stimulated pDCs. Blocking HTLV-1 serum inhibited HTLV-1 entry (HTLV-1 + Serum) and TRAIL localization on the membrane (analyzed by Metamorph software). (C) pDCs in different culture conditions (Unst, HTLV-1, HTLV1 + Chloro, HTLV-1 + Serum) were observed in several plane (Z-stack) by the use of a 3D microscope. Panels shown here were the results of image compilation allowing entire cell observation in a 2D representation to refine plane analysis. Whole 3D cell acquisition was performed and the entire Z-stack was projected in 2D (analyzed by Imaris 6.2 software).