BM-DCs from ICSBP^–/– mice are impaired in Ag presentation in vitro and in vivo. (A) BM-DCs of WT and ICSBP^–/– mice were incubated for 40 minutes with 1 mg/mL OVA-FITC and then stained for CD11c and CD86 expression. Density plots represent the uptake of the OVA and the relative expression of CD86 in CD11c⁺-gated cells. Data are representative of 3 separate experiments. Three days later, the proliferation of CFSE⁺ cells in draining popliteal LN was evaluated by FACS. (B) WT and ICSBP^–/– BM-DCs were magnetically sorted with anti-CD11c Microbeads and incubated with 1 mg/mL OVA for 40 minutes. Cells were washed, cultured overnight with 0.5 μg/mL LPS, and then assayed for their ability to stimulate the proliferation of OVA-specific OT-II CD4⁺ T cells. Thymidine incorporation was measured at the fourth day of coculture. Data represent the mean cpm of triplicate wells ± SD of 1 of 4 independent experiments. (C) BM-DCs were treated as in panel B and then analyzed by FACS for CD86 and I-A expression. (D) Sorted CD11c⁺ BM-DCs of WT and ICSBP^–/– mice were loaded with OVA as in panel B. OVA-loaded DCs were injected in the footpads of WT recipients previously reconstituted with 5 × 10⁶ CFSE-labeled OT-II CD4⁺ T cells. Three days later, proliferation of CFSE⁺ cells in draining popliteal LN was evaluated by FACS. Results are representative of 2 separate experiments.