Microscopy analysis of TRAIL localization in pDCs. (A) Deconvolution overlays of representative 2D red cross axis XY focal plan with XZ/YZ view of focal plane and projection overlay of cell stainings (analyzed by Metamorph software). Unstimulated pDCs (i), HTLV-1–stimulated pDCs (HTLV-1; ii), treated with chloroquine (HTLV-1 + Chloro; iii), or with blocking HTLV-1–infected patient serum (HTLV-1 + Serum; iv) were stained with anti-TRAIL (green), anti-HTLV-1 virus (red), and DAPI (nucleus staining). (B) Deconvolution of representative 2D XY focal plane was treated to allow Z scaling of pixel intensity (analyzed by Metamorph and ImageJ software). Each staining (anti-TRAIL [green], anti-HTLV-1 virus]red], and DAPI [nucleus staining]) was merged together with phase contrast (gray, defined cell surface). This representation was used to enhance the observation of TRAIL localization in the cytoplasm (Cy) or its relocalization to the membrane (Mb). Unstimulated pDCs (i) or HTLV-1–stimulated pDCs (ii) treated with inhibitors (HTLV-1 + Chloroquine; iii) or with blocking HTLV-1–infected patient Serum (HTLV-1 + Serum; iv) restrictively expressed TRAIL (green) in the cytoplasm in contrast to HTLV-1–activated pDCs that expressed TRAIL at the cell surface. Inhibitor of endosomal acidification (HTLV1 + Chloroquine) allowed detection of HTLV-1 particles (red) in the cytoplasm (Cy) in contrast to the cell-surface detection of HTLV-1 virions in HTLV-1–activated pDCs. HTLV-1 virion loading was poorly detected in the presence of serum that directly blocked the virus before the pDC contact.