p15Ink4b-deficient BM-DCs have decreased immunostimulatory functions. (A) BM-DCs were cultured at 37°C in the presence of FITC-labeled OVA antigen. Cells incubated at 4°C for 30 minutes with FITC-labeled OVA antigen were used as a negative control. Representative histograms at the 30-minute time point are shown. (B) BM-DCs from p15Ink4b^wt/wt-LysMcre (wt/wt) or p15Ink4b^fl/fl-LysMcre (fl/fl) mice were harvested at the indicated times after culture in the presence of OVA. Mean fluorescence intensity values are indicated. (C) BM-DCs from wt/wt or fl/fl mice were mixed with CFSE-labeled allogeneic T lymphocytes (ratio 1:10 and 1:100) and cocultured for 5 days. T-cell proliferation was assessed using flow cytometry. Numbers indicate the percentage of CFSE^low T cells. Data are shown as means ± SD from 3 independent experiments. (D-E) BM-DCs were activated with LPS (100 ng/mL) in the presence of GolgiStop (BD Biosciences) for 6 hours, stained for surface molecules that were CD11c⁺CD11b⁺, and then analyzed for the expression of TNF-α, IL-12 (D), and IL-10 (E). Cells were fixed and permeabilized according to the BD Cytofix/Cytoperm (BD Biosciences) protocol. Relative mean fluorescence intensity values from 2 independent experiments (n = 4) are plotted. Data are shown as means ± SD. *P < .05; n.s. indicates not significant.