Figure 3.

Replication-defective BAdv^85C5 vaccine is rapidly internalized by mouse DCs

CD11c⁺ DCs purified from bone marrow of WT-C57BL/6 mice were infected with the Cy3-labeled BAdv^85C5 vaccine or control vector (10⁷ PFU/10⁶ DCs), followed by antibody staining for intracellular localization using confocal microscopy at 4 h post-infection. Cy3-BAdv vector or Cy3-BAdv^85C5 vaccine-infected DCs were incubated for a 4-h infection, followed by fixation and staining using specific antibodies to (A) microtubule associate light chain 3 (LC3) autophagosome marker, (B) lysosome associated membrane protein-1 (LAMP1), (C) galectin3 (Lgals-3), and (D) Lgals-8 followed anti-immunoglobulin G (IgG) Alexa Fluor 488 and DAPI nuclear stain. Panels show Cy3 (red), Alexa Fluor 488 (green), and merged images with or without DAPI nuclear stain. Colocalization (inset) analyzed using confocal microscopy and Nikon N90 microscope with MetaView software. Cytosolic (white arrow) and nuclear localization (yellow arrow) of Cy3-labeled vector or vaccines are indicated. Bar graphs to the right show percentage of virus-containing endosomes colocalizing with antibodies calculated per DC and averaged for 20 DCs in triplicates per experiments done twice. ∗p < 0.01 t test. Isotype stains are shown in Figure S5.