IL-12 production of early life DCs in response to stimulation by CpG ODN. (A-C) Purified splenic CD11c⁺ DCs were stimulated in various conditions and supernatants were harvested after 24 hours and analyzed by ELISA for IL-12p70 production. (A) DCs (10⁵) from 7-day-old (□) and adult (▪) BALB/c mice were stimulated with 10 μg/mL CpG ODN in serum-free HL-1 medium containing GM-CSF in the absence or presence of IL-4 (5 ng/mL), rat IFN-γ (10 ng/mL) or IL-10 (2 ng/mL). (B) Indicated numbers of DCs from adult (▪; 3.75 × 10⁴) or 1-day-old (□; 3.75 × 10⁴) BALB/c mice were cocultured with 10 μg/mL CpG ODN or control ODN (Cntl ODN) in HL-1 medium containing GM-CSF and IL-4. (C) DCs from adult (▪) or 7-day-old (□) C57BL/6 mice were stimulated as indicated. (D) Purified 7-day-old or adult splenic CD11c⁺ DCs were stimulated under the same conditions as in panel B and stained for CD11c and IL-12 in combination with CD8α, CD11b, or B220. Isotype control antibody staining for CpG-ODN–stimulated DCs and IL-12 staining for Cntl ODN–stimulated DCs are shown as control (top). To analyze IL-12 production by different subpopulations of DCs, IL-12 positive cells are gated as indicated (boxes) and DCs from 7-day-old (middle) or adult (bottom) mice are compared. Numbers in panel C indicate the frequency of DCs in each quadrant. Data are from one of 3 experiments with similar results.