The limited availability of IL-12 during neonatal exposure to Ag is related to delayed accumulation in the spleen of the presenting CD8α⁺CD4⁻ DC subset. Newborn BALB/c mice were killed at the indicated day after birth, and splenic CD11c⁺ DCs were isolated on anti-CD11c microbeads. (A) The DCs (100 × 10³ cells per well) were stimulated with 6 μg/ml CpG, and 24 h later, IL-12 was measured by ELISA. Each bar represents the mean ± SD of triplicate wells. (B–E) 1- and 6-d-old DCs (100 × 10³ cells per well) were purified from the spleens of 45 newborns, and 10⁶ CD11c⁺ cells were stimulated with 6 μg/ml CpG for 10 h, after which BFA was added and the culture was continued for 4 h to facilitate intracellular cytokine accumulation. Subsequently, the cells were stained with anti-CD11c, anti-CD4, and anti-CD8 antibodies and permeabilized with 2% saponin. Intracellular IL-12 was detected by staining with anti–IL-12 p70 antibody. B shows the frequency of the subsets, and C–E illustrates flow cytometry plots of intracellular IL-12 on the indicated subset. ND, not detected.