Expression and function of IDO in monocyte-derived DCs and myeloid BDCA-1⁺ DCs. mRNA for RT-PCR and cell lysates for immunoblot analysis were obtained from monocytes, imDCs, or maDCs cultured in the presence or absence of PGE2. Similarly, BDCA-1⁺ myeloid DCs were cultured for 18 hours in the presence or absence of PGE2, αCD40, and TNFα, combined as indicated. (A) Identification of IDO regulation on mRNA level by RT-PCR using forward and reverse IDO primers spanning exons 6 to 10. Two additional primer sets spanning exons 1 to 7 and 8 to 10 of IDO were also used and gave the same results. Results shown are representative of at least 3 independent experiments. (B) IDO protein expression in cell lysates from different DCs was determined by at least 5 independent immunoblot analyses using a monoclonal or polyclonal IDO antibody. Shown here are data using the monoclonal antibody. (C) IDO RNA was also assessed in BDCA-1⁺ DCs upon purification and after short-term in vitro culture using the same primer sets. (D) Enzymatic activity of IDO was determined measuring tryptophan levels in supernatants derived from imDCs or maDCs in the presence or absence of PGE2 by reversed-phase HPLC analysis. Except for imDCs stimulated with PGE2 alone (n = 2), at least 10 experiments were performed for the other conditions. Black bars represent PGE2-treated DCs; mean ± standard deviation of all experiments is shown. ^*P < .05. (E) IDO activity as assessed by tryptophan reduction in supernatants of short-term cultured BDCA-1⁺ DCs under the same culture conditions as described for Figure 4C (n = 2). Black bars represent PGE2-treated DCs; mean ± standard deviation of all experiments is shown. ^*P < .05.