Selective constitutive expression of ICSBP in CD8α⁺CD11c⁺ DCs.

(A) Murine splenic CD11c⁺CD4⁺CD8α⁻ (CD4), CD11c⁺CD4⁻CD8α⁺ (CD8), and CD11c⁺CD4⁻CD8α⁻ (DN) cells were sorted, mRNA extracted, and RT-PCR performed using ICSBP- and β-actin–specific probes. PCR products were resolved by electrophoresis on agarose gels and products visualized by ethidium bromide staining. (B) Wild-type (+/+) or ICSBP^−/− (−/−) splenic CD11c⁺ or CD3⁺ cells were purified by positive selection using CD11c- or CD3-conjugated magnetic beads. The resulting cells were then permeabilized, labeled with anti-ICSBP, and analyzed by FACS. (C) Wild-type CD11c⁺ spleen cells were obtained as in panel B and stained with CD8α⁺, permeabilized, and incubated with isotype-control or anti-ICSBP. Contour plots represent CD8α versus ICSBP or control-isotype expression; numbers represent the event frequency of the quadrant. Experiments were performed using at least 5 animals per group and are representative of at least 2 independent experiments.