pDCs capture and internalize Ag in vivo. (A,B) Mice were injected intravenously with various numbers of fluorescent red beads. Two hours later, CD11c⁺ cells enriched from spleen were stained with anti-CD11c and BST-2 (PDCA-1) mAbs and analyzed by flow cytometry. The percentage of cells that had captured beads was determined by gating red-positive cells for pDCs (CD11c^low BST-2⁺ cells, left histogram) and cDCs (CD11c^hi BST-2⁻ cells, right histogram). One representative experiment of 4 is depicted in each case. (A) Analysis of capture by DCs from the mouse injected with 10⁹ beads is shown. (B) Percentage of bead-positive cells was determined among cDCs (▲, ) and pDCs (●, ) and plotted against the number of red beads injected per mouse. (C) Purified pDCs isolated from (10⁹) red bead-injected mice were stained with anti-BST-2 (120G8, green) mAb and analyzed by fluorescent microscopy (original magnification ×630). Three-dimensional acquisition was done to obtain different slices of the cells. Representative DIC (differential interference contrast), shadow views (projection of each slice in the same image), and 3-dimensional reconstitutions are shown. (D,E) Mice were injected intravenously with various doses of OVA-A488. Two hours later, splenocytes were labeled with anti-CD11c, BST-2 (PDCA-1), and CD3ϵ mAbs and analyzed by flow cytometry. (D) Histograms show OVA-488 fluorescent intensity for both pDCs and cDCs (gated as in panel A) from mice injected with PBS (filled histogram) or 0.2 mg (thin histogram) or 9 mg (thick histogram) OVA. One representative experiment of 3 is depicted. (E) Mean OVA-A488 fluorescence was determined for T cells (○, black line), cDCs (▲, ), and pDCs (●, ) and is plotted against the dose of OVA-488 injected. T cells were gated as CD3ϵ⁺ cells. (F) Purified pDCs isolated from mice injected with PBS or 3 or 9 mg OVA-A488 (green) were stained with anti-BST-2 (120G8, red) and CD11c (yellow) mAbs and Hoechst 44 432 (light blue) and then analyzed by fluorescent microscopy (original magnification ×630). One representative experiment of 3 is depicted.