CD40L stimulation of DCs results in functional IDO expression. (A) CD40L stimulation of DCs induces IDO protein expression. Monocyte-derived DCs were matured with LPS or CD40L. After 48 hours, the cells were extensively washed and lysed in sample buffer. Cell lysates were analyzed by Western blotting for IDO content, and β-actin as loading control. One representative experiment of 3 is shown. (B) IDO is efficiently induced by various sources of CD40L. DCs were either left unstimulated or stimulated with J558-CD40L–transfected cells, sCD40L (500 ng/mL; Immunex, Seattle, WA), or activated CD4⁺ T cells. After 48 hours, the cells were extensively washed and lysed in sample buffer. Cell lysates were analyzed by Western blotting for IDO content, and β-actin as loading control. One representative experiment of 3 is shown. (C) CD40L-induced IDO protein is enzymatically functional. Monocyte-derived DCs were either unstimulated or matured with CD40L in the presence or absence of 1-methyl-tryptophan (MT). After 48 hours, cells were harvested, extensively washed in cold PBS, and freeze-dried. Subsequently, IDO enzymatic activity in the samples was evaluated by testing the capacity to degrade tryptophan into kynurenine. Results represent mean plus or minus SEM from 3 independent experiments (*P < .05). (D) HPLC analysis of kynurenine and tryptophan levels in culture supernatants. Monocyte-derived DCs were either unstimulated or matured with CD40L in the presence or absence of 1-methyl-tryptophan (MT). After 48 hours, supernatants were harvested and IDO enzymatic activity in the samples was evaluated by measuring tryptophan and kynurenine by HPLC. Results are expressed as kynurenine-tryptophan ratio and represent mean plus or minus SEM from 3 independent experiments (*P < .05). (E) CD40L-induced IDO induces apoptosis of preactivated T cells. Monocyte-derived DCs were stimulated with CD40L or LPS in the presence or absence of MT. After 48 hours, cells were cocultured with anti-CD3/anti-CD28 preactivated CD4⁺ T cells in the presence or absence of MT for 24 hours. Subsequently, T-cell apoptosis was assessed by annexin V/propidium iodine (PI) staining and evaluated by flow cytometry. The frequency of apoptosis in T cells stimulated with anti-CD3/CD28 Ab alone is 3% to 5%. Results represent mean plus or minus SEM from 3 independent experiments (*P < .05). (F) CD40L-induced IDO reduces proliferation of preactivated T cells. Monocyte-derived DCs were stimulated with CD40L or LPS in the presence or absence of MT. After 48 hours, cells were cocultured with anti-CD3/anti-CD28 preactivated CD4⁺ T cells in the presence or absence of MT for 3 days. Subsequently, T-cell proliferation was evaluated by [³H]TdR incorporation. The cpm for preactivated CD4⁺ T cells in the absence of DCs was 2515 plus or minus 389 without MT, and 1694 plus or minus 183 in the presence of MT. Data are presented as mean cpm plus or minus SD of triplicate cultures. Results are representative of 3 independent experiments (*P < .05). (G) CD40L stimulation of BDCA1⁺ and BDCA4⁺ cells also results in IDO protein expression. BDCA1⁺ and BDCA4⁺ DCs were freshly isolated from peripheral blood and stimulated with CD40L-expressing J558 cells. After 48 hours, the cells were extensively washed and lysed in sample buffer. Cell lysates were analyzed by Western blotting for IDO content. Representative blots from 4 independent experiments are shown.