Phenotypic and functional characterization of lung tissue APCs. C57BL/6 mice received an intranasal dose of L. major parasites in PBS, or PBS alone, and 18 hours later the mice were sacrificed and perfused with PBS and their lungs were removed to generate cell suspensions. (a) Lung tissue cells were stained with a combination of biotinylated anti-CD11c and PE-conjugated anti-CD11b, followed by RED670-conjugated streptavidin. FACS dot plot shows a representative profile of CD11c versus CD11b staining of lung tissue cells (after gating on total live cells) following either PBS or L. major administration. Boxes show the FACS-sort regions usually set for the isolation of CD11cbright (CD11bdim) versus CD11cdim (CD11bbright) populations. (b) Sorted populations of antigen-loaded CD11cbright and CD11cdim cells were irradiated and set up in culture at various numbers in the presence of L. major–specific CD4+ T cells. Bar graph shows the proliferation (mean ± SEM of triplicate wells) of the responder T cells after 4 days of culture in the presence of stimulators from mice that had received an intranasal delivery of either PBS alone or antigen (Ag) (L. major parasites). (c) Lung cells were stained with a combination of biotinylated anti-CD11c and PE-conjugated anti–F4/80, -CD14, -CD8α, -FcR, or –I-Ab, followed by RED670-streptavidin. DEC205 staining was performed using PE–anti-CD11c plus biotinylated anti-DEC205 and RED670-streptavidin. Histogram plots show positive staining (heavy lines) relative to staining with irrelevant Ab’s after gating on CD11cbright cells.