Defective MHC-II presentation of s-DCs from ICSBP^–/– mice in vivo. (A) ICSBP^–/– and WT mice were injected intravenously with 1 mg OVA-FITC. Two hours later, s-DCs were isolated by magnetic cell sorting. CD11c⁺ cells were stained with PE-labeled anti-CD8α antibody and analyzed by FACS. Density plots represent the uptake of OVA-FITC and the expression of CD8α in CD11c⁺ s-DCs from mice injected with OVA-FITC or PBS. Numbers in top right and bottom right quadrants are the percentages of FITC⁺ cells in CD8α⁺- and CD8α^–-gated DC subsets, respectively. Data are representative of 2 separate experiments. (B-C) ICSBP^–/– and WT mice were intravenously injected with 1 mg OVA. Two hours later, CD11c⁺ s-DCs were isolated and used as stimulators of 10⁵ OT-II CD4⁺ T cells in vitro. DC were either assayed ex vivo (B) or after overnight culture in the presence or absence of 0.5 μg/mL LPS (C). The cultures were pulsed with ³H-thymidine for 16 hours at the fourth day, and the incorporation into the cellular DNA was determined by liquid scintillation counting. Each point represents the mean counts per minute (cpm) ± SD in triplicate cultures of 1 out of 3 experiments. (D) Phenotype of s-DCs from ICSBP^–/– and WT mice cultured overnight with or without LPS (0.5 μg/mL). (E) Splenic DCs from ICSBP^–/– and WT mice injected with OVA were inoculated in the footpads of WT recipients that had been adaptively transferred with 5 × 10⁶ CFSE-labeled OT-II CD4⁺ T cells the day before. Three days later, proliferation of CFSE⁺ cells in draining popliteal LN was evaluated by FACS. Results are representative of 2 similar experiments.