Up-regulation of IL-17 is driven by IL-6. (A) Cocultures were performed as in Figure 2, with and without 1MT. After 2 days, cultures were stained for IL-6 versus CD11c (to mark the sorted pDCs). The right graph represents IL-6 measurement by enzyme-linked immunosorbent assay on supernatants of cocultures (error bars show SD of quadruplicate wells). (B) Tregs were sorted from Foxp3^GFP mice and activated in cocultures, with or without 1MT plus neutralizing polyclonal antibody against IL-6. After 2 days, cocultures were stained for CD4 versus IL-17. Plots represent the gated CD4⁺ (Treg) population. (C) Sorted Foxp3^GFP Tregs were activated in cocultures with or without 1MT. Recombinant IL-6 (100 ng/mL) was added as shown. Plots represent the gated CD4⁺ (Treg) population. Plot at right indicates a representative example of coexpression of IL-22 and IL-17 on gated Tregs in IL-6–treated cocultures. (D) IL-6 up-regulation in pDCs requires OVA antigen or CD28→B7 engagement. Cocultures were performed in the presence of 1MT, with and without OVA or recombinant CD28-Ig fusion protein (20 μg/mL), as indicated. After 2 days, cultures were stained for IL-6 versus CD11c (top plots), and for CD4 versus IL-17 versus IL-22 (bottom plots, gated on the CD4⁺ Treg population). All experiments were repeated 3 to 10 times with similar results.