Specific accumulation of CD11b⁺ DCs near allergic airways. (A) Method for inducing allergic airway inflammation involving three i.p. sensitizations with OVA/alum followed by three i.n. administrations of either OVA or PBS. (B) Quantification of CD11b⁺ DCs, CD103⁺ DCs, and AMs as a percentage of live leukocytes based on CD11c surface antibody staining in lungs of PBS- and OVA-challenged mice. (C) Quantification of CD11b⁺ DC and CD103⁺ DC populations as a percentage of live leukocytes gated on CD11c-EYFP^hi cells in PBS- and OVA-challenged mice. (D) Large-scale three-dimensional surveys of >300-µm slices from CD11c-EYFP mice stained with Hoechst shows DC and AM distribution in PBS- and OVA-challenged mice obtained by two-photon microscopy with 910-nm excitation. Insets provide a zoomed in view of the two distinct morphologies. AW, airway. (E) Quantification of CD11c-EYFP⁺ cells near the airways in PBS- or OVA-challenged mice. (F) Measurement of sphericity to separate DCs from AMs. AMs were defined as having a sphericity >0.75. (G) Imaris was used to make surfaces of Siglec-F⁺ CD11c-EYFP⁺ cells from OVA-challenged mice. The sphericity of these cells is graphed, showing that ∼80% of Siglec-F–stained AMs are captured by the sphericity measurement. (H) Color coding of localization based on sphericity with bulk CD11c-EYFP data shown on the left and subdivided data (DCs, purple; AMs, green; airway, white) on the right from an OVA-challenged mouse. (I) Images were subdivided by sphericity to determine whether DCs or AMs account for the accumulation within 75 µm of the airway after OVA challenge. (J) Example images from CD11c-EYFP sections (gated on EYFP⁺ sphericity <0.75) stained with CD11b APC. Bars: (D [top] and H) 100 µm; (D, bottom) 10 µm; (J) 150 µm. (K) Quantification of CD11b⁺ and CD11b⁻ DC distance from the nearest airway for PBS- and OVA-challenged mice. (B, C, and K) Error bars represent SEM. Graphs represent at least five mice per group from at least three independent experiments. Images are representative data from at least three independent experiments.