Down-regulation of DAP12 expression correlates with the acquisition of an IDO-competent phenotype by CD8⁺ and CD8^- DCs. (A) Real-time PCR analysis of mRNA expression for IDO (Indo), DAP12 (Tyrobp), MIG (Cxcl9), and CD14 (Cd14). Purified CD8⁺ and CD8^- DCs were incubated for 4 hours with medium alone or were treated with IFN-γ, IL-6, CTL0-4-Ig, or CD28-Ig, as specified in “Materials and methods.” mRNA levels were quantified by real-time PCR using Gapdh normalization. Data (means of duplicate samples in 1 of 2 experiments) are presented as normalized specific gene transcript expression in the samples relative to normalized transcript expression in the respective control cultures—that is, CD8⁺ or CD8^- DCs maintained in control medium (fold change = 1; dotted line). Indicated in bold are treatments resulting in the acquisition of IDO competence in a specific DC subset. (B) Western blot analysis of DAP12 protein expression. Purified CD8⁺ and CD8^- DCs were incubated overnight with medium alone (C), IFN-γ, or CTL0-4-Ig. DC lysates were resolved on SDS-PAGE. DAP12 expression was investigated with a specific antibody reagent. Loading controls consisted of samples reprobed with tubulin-specific antibody. Results are from 1 of 2 representative experiments.