Essential requirement of the noncanonical NF-κB pathway for CD40L-induced IDO in DCs. (A) Immature DCs were treated with control nonblocking siRNA (siC) or siRNA for the noncanonical NF-κB pathway–associated kinases NIK (siNIK) and IKKα (siIKKα). Subsequently, cells were matured for 2 days with CD40L, extensively washed, and lysed in sample buffer. Cell lysates were analyzed by Western blotting for IKKα and IDO content, and β-actin as loading control. Densitometry was performed on IDO blots; numbers indicate percent reduction of IDO expression by siIKKα. One representative experiment of 3 is shown; densitometry includes data from all experiments (*P < .01; **P < .001). (B) Increased IL-12p70 production by DCs after siRNA-mediated knockdown of the noncanonical pathway. Immature DCs were preincubated with NBD/MUT peptides and matured for 2 days with CD40L in the presence or absence of 1-methyl-tryptophan (MT). After 48 hours, the cells were thoroughly washed and stimulated with CD40L-expressing mouse plasmacytoma cells in the presence or absence of 1-methyl-tryptophan (MT). Supernatants were harvested after 24 hours and secreted IL-12p70 was measured by ELISA. Results are expressed as mean plus or minus SD from one representative experiment of 3 performed in triplicate (*P < .001). (C) Increased IL-6 production by DCs after siRNA-mediated knockdown of the noncanonical pathway. Immature DCs were preincubated with NBD/MUT peptides and matured for 2 days with CD40L in the presence or absence of 1-methyl-tryptophane (MT). After 48 hours, the cells were thoroughly washed and stimulated with CD40L-expressing mouse plasmacytoma cells in the presence or absence of 1-methyl-tryptophan (MT). Supernatants were harvested after 24 hours and secreted IL-6 was measured by ELISA. Results are expressed as mean plus or minus SD from 1 representative experiment of 3 performed in triplicate (*P < .001). (D) IDO-mediated inhibition of preactivated T-cell proliferation is noncanonical NF-κB pathway dependent. Monocyte-derived DCs were stimulated with CD40L in the presence or absence of methyl-tryptophan (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]thymidine incorporation. Data are presented as mean cpm plus or minus SD of triplicate cultures. Results are representative of 3 independent experiments (*P < .05).