cDCs from IK^L/L Rag^−/− mice are functional. Spleen cells from C57BL/6, IK^+/+ Rag^−/−, or IK^L/L Rag^−/− mice were stained and analyzed using flow cytometry. (A) CD11c^high CD317⁻ cells were gated and analyzed for the expression of CD8 and CD11b markers. Numbers indicate the percentage of events within the indicated gate. (B) Cumulative data of numbers of CD11c^high cells (first row), CD11c^high CD8⁺ cDCs (second row), and CD11c^high CD8⁻ cDCs (third row) from the spleen (first column) or LN (second column). Each dot corresponds to results obtained from individual mice, and bars represent the mean. ***P < .001. (C-D) Purified CD11c^high spleen cells from C57BL/6, IK^+/+ Rag^−/−, and IK^L/L Rag^−/− mice were cultured in medium alone or in the presence of LPS (2 μg/mL), CpG (10 μg/mL), or PIC (10 μg/mL) for 18 hours. The concentration of IL-12p40 in the supernatants was determined by ELISA (C), and cells were labeled and analyzed by flow cytometry (D). I-A^b and CD86 expression on CD11c^high-gated cells from the indicated mouse strains is expressed as the mean fluorescence intensity (MFI). One representative experiment of 4 is shown. (E-F) A total of 10⁴ purified CD11c^high spleen cells from the indicated mouse strains were incubated for 2 hours in the presence of various concentrations of OVA. Cells were washed twice and cocultured with 2 × 10⁴ LN cells from OT-I (E) or OT-II (F) transgenic mice for 36 hours. T-cell proliferation was measured by ³H-labeled thymidine incorporation during the final 6 hours of culture. Proliferation of OT-I and OT-II LN cells in the absence of DCs is shown (white diamonds). Results are expressed as the mean cpm ± SD for duplicate wells. One representative experiment of 3 is shown.