Activation of Treg suppressor activity by IDO and effector T cells. (A) Resting splenic Tregs (FACS-sorted CD4⁺CD25⁺) were cocultured with pDCs from TDLNs (CD11c⁺B220⁺) plus OT-I T cells, OVA peptide antigen, and feeder layer (all on the B6 background). After 2 days, the Tregs were harvested, resorted, and added to readout assays to measure suppressor activity (A1 T cells + congenic spleen DCs, CBA background). Graph represents proliferation in the readout assay by tritiated-thymidine incorporation, using either IDO-activated Tregs or the same Tregs activated with αCD3 cross-linking plus recombinant IL-2 (with IDO blocked using 1MT). Bars represent SD of replicate wells. (B) Tregs were activated with IDO⁺ pDCs as in panel A, with or without the cognate OVA peptide antigen for OT-I. (C) Tregs in panel A were activated in the presence of OT-I and OVA, with (○) or without (□) D-1MT to block IDO. Experiments in each panel were repeated 3 to 10 times with similar results.