Effect of OVA or PBS aerosol challenge on the number of DCs in the BALF.

Mice were immunized on day 0 with 1 × 10⁶ OVA-DCs or PBS-DCs. From day 10 they were challenged daily for 30 minutes on 1, 3, or 7 consecutive days with OVA or PBS aerosols. (A) In OVA-DC/OVA mice (ii and iv), the FSC/SSC plot contains lymphoid cells (L), and granulocytes (G). In contrast, in PBS-DC/PBS mice (i and iii) the majority of cells are large and spontaneously autofluorescent, representing alveolar macrophages (M). A gate was set (iii and iv) on low autofluorescent cells that lacked expression of CD3 and B220. (B) Within the set gate, MHCII^hi CD11c^hi cells represent DCs, whereas CD11c^dim MHCII⁻ cells represent eosinophils (Eo). In our experiments, eosinophils did not express MHCII molecules. The average percentage of MHCII^hiCD11c^hi DCs as a percentage of total cells analyzed is indicated in the plot. (C) Kinetics of increase of DCs following OVA exposure as expressed as the absolute number of MHCII^hiCD11c^hi cells within the BALF (n = 5 animals per group; *P < .05 compared with PBS-DC/PBS group). Data are expressed as mean number of MHCII⁺/CD11c⁺ DCs ± SEM. (D) Top panel: gated CD11c⁺ MHCII⁺ DCs are of myeloid lineage as revealed by strong staining for CD11b (filled histogram); isotype control is indicated by the open histogram. Bottom panel: gated MHCII⁺CD11c⁺ DCs (filled histogram) do not express the eosinophil marker CCR3, whereas gated CD11c^dim granular eosinophils (open histogram) clearly do.