Normal development and function of Jak3^-/- BMDCs. (A) Normal in vitro generation of CD11c⁺ BMDCs from Jak3^-/- mice. Bone marrow cells from wild-type (left) and Jak3^-/- (right) mice were cultured for 7 days with GM-CSF and IL-4. The resultant cells were stained with anti-CD11c antibody (solid line). Shaded histograms represent samples stained with an isotype control. (B) Jak3^-/- BMDCs have normal expression of costimulatory and MHC molecules. Purified Jak3^-/- (solid line) and wild-type (broken line) BMDCs (> 90% CD11c⁺) were stained with the indicated antibodies after 7 days of culture in GM-CSF and IL-4. (C) Normal micropinocytosis by Jak3^-/- BMDCs. BMDCs from wild-type and Jak3^-/- mice were pulsed with 5 μg/mL FITC-conjugated albumin for 60 minutes. Broken line indicates background uptake of cells incubated at 0°C; solid line, albumin uptake at 37°C. The shaded histograms represent samples in which FITC-conjugated albumin was omitted. (D) Normal induction of CD86 in Jak3^-/- BMDCs. BMDCs from wild-type and Jak3^-/- mice were stimulated with CD40L-bearing cells (NIH3T3CD40L) and LPS (1 μg/mL) for 24 hours (solid line) and analyzed for CD86 expression compared with unstimulated cells (broken line). Shaded histograms are samples stained with an isotype control antibody. (E) Nonspecific inhibition of CD86 expression by WHI-P-154. Wild-type and Jak3^-/- BMDCs were pretreated with dimethyl sulfoxide (DMSO; broken line) or WHI-P-154 (10 μg/mL; solid line) for 30 minutes, stimulated with CD40L and LPS for 24 hours, and stained with anti-CD86. Note that the putative Jak3 inhibitor effectively blocked CD86 expression in Jak3^-/- cells, indicative of nonspecific effects.