Evidence that IDO acts via the GCN2-kinase pathway in pDCs to block IL-6 up-regulation. (A) Hypothesized pathway for IDO-induced translational regulation of NF-IL-6 transcription factor. (B) pDCs were isolated from TDLNs of tumors grown in genetically defined hosts (IDO1-KO, GCN2-KO, or WT). pDCs were then used in activation cocultures with OT-I and Tregs, as Figure 2. After 2 days, cocultures were stained for CD11c versus IL-6. (C) Foxp3^GFP Tregs were cocultured as in Figure 2A, using pDCs from TDLNs of WT, IDO1-KO, or GCN2-KO hosts. All cultures were without 1MT. After 2 days, cultures were harvested and stained for intracellular IL-17 versus CD4. (D) Analysis of the inhibitory LIP isoform of NF-IL-6 in T-REX cells stably transfected with inducible IDO cDNA. IDO was either uninduced or induced by treatment with doxycycline (20 ng/mL) as indicated. Induced cells were treated with 50, 25, and 10 μM of the IDO inhibitors L-1MT or methyl-thiohydantoin-tryptophan (MTHT), as indicated. Graph documents production of kynurenine by functional IDO (error bars show SD of triplicate wells). The top Western blot represents expression of IDO after induction; the bottom Western blot represents induction of the 21-kDa LIP isoform of NF-IL-6, and the higher molecular weight LAP isoforms. All experiments were repeated 3 to 4 times with similar results.