IDO protein expression and activity increase with maturation of human moDCs. (A) IDO protein expression was assessed by flow cytometry in immature, CD83^neg moDCs and in mature, CD83⁺ moDCs. Data are representative of 1 of 10 experiments. (B) IDO protein expression was assessed by Western blot in cell extracts from immature and mature moDCs. Untreated and IFN-γ–treated HeLa cells served as negative and positive controls, respectively. A representative blot from 1 experiment is shown along with pooled densitometry data from 4 separate experiments (mean ± SD; P = NS) showing relative IDO expression between groups. Densitometry values for each group were normalized to Rho-GDI (internal control). Relative IDO expression indicates fold increase above baseline IDO expression in untreated HeLa cells. (C) Supernatants from equal numbers of immature and mature moDCs were resuspended in tryptophan-enriched medium and tested after 4 hours by HPLC for production of the tryptophan catabolite, kynurenine, as an index of IDO activity (average of triplicate means ± SEM, n = 4 independent experiments). *P < .01 vs immature moDCs. (D) Kynurenine concentrations were determined by a spectrophotometric assay at 1 and 4 hours, using culture parameters identical to those in panel C (average of triplicate means ± SEM, n = 3 independent experiments). *P < .001 vs immature moDCs.