Uptake of antigen does not alter the immature phenotype of resident pulmonary APCs. C57BL/6 mice were given an intranasal administration of CFSE-labeled L. major parasites or FITC-OVAin PBS, or PBS alone, and 18 hours later their lungs were isolated and cell suspensions were generated for FACS staining. The cells were stained with a combination of biotinylated anti-CD11c and PE-conjugated anti–B7-1, anti–B7-2, anti-CD40, anti–MHC class II, or a relevant isotype control, followed by RED670-conjugated streptavidin. (a) Dot plots show the distribution of CD11c versus CFSE (after gating on live cells) on lung cells isolated from mice that had received either PBS or CFSE-labeled parasites. Lung cells were analyzed to compare the activation/maturation markers on the CD11cbright cells that had taken up CFSE–L. major (b) or FITC-OVA (c) antigen. Analysis gates were set on CFSE+/FITC+ cells versus CFSE–/FITC– cells to compare the phenotype of cells that had taken up versus cells that had not taken up antigen. Histograms show the expression of different surface markers on CFSE+/FITC+ (heavy lines) versus CFSE–/FITC– cells (thin lines) after gating on CD11cbright cells. The staining with relevant isotype control Ab’s is also shown (dotted lines). The staining profile for CD11cbright cells that received intranasal PBS only colocalized with that of the CFSE–/FITC– populations (data not shown).