In the absence of IDO, activated T cells drive conversion of Tregs to a TH17-like phenotype. (A) Sorted CD4⁺GFP⁺ Tregs from Foxp3^GFP knockin mice were activated in cocultures with pDCs and OT-I as in Figure 1, with or without OVA and 1MT as indicated. After 2 days, cocultures were stained for intracellular IL-17 after a 4-hour stimulation with phorbol myristate acetate/ionomycin plus brefeldin A. The top dot plot represents an example of a representative gate for the Foxp3^GFP-positive CD4⁺ Tregs. The bottom plots represent the gated Treg population in each treatment group. (B) Tregs (CD4⁺CD25⁺) were sorted from homozygous-null Rorγt ^gfp/gfp mice lacking functional RORγt, or from WT controls, and activated in cocultures with and without 1MT as shown. After 2 days, cultures were stained for CD4 to identify Tregs versus IL-17. (The only CD4⁺ cells in cocultures were the original sorted Tregs.) (C) Tregs from Rorγt-null mice, or control wild-type Tregs, were activated in cocultures for 2 days, with (□) or without (◊) 1MT. Tregs were sorted and functional suppressor activity measured against A1 T-cell readout as in Figure 1. (D) Foxp3^GFP Tregs were sorted and activated in cocultures with 1MT and OVA. Plots represent 4-color staining from a representative sample, gated on GFP⁺CD4⁺. (E) Foxp3^GFP Tregs were activated as in panel A and stained for IL-17 versus the cytokines shown. Plots represent the gated CD4⁺GFP⁺ cells. Experiments were repeated 3 to 12 times with similar results.