Generation of TH17-like cells in TDLNs in vivo. (A) Foxp3^GFP mice with B16-OVA tumors were treated with OVA-Lv vaccine, oral D-1MT, and adoptive transfer–sorted OT-I cells, as shown. On day 11, TDLNs were harvested and stained for IL-17. Percentages in the right-upper quadrants of each plot give the fraction of the Foxp3^GFP-positive cells that coexpressed IL-17. Percentages below give total Foxp3^GFP-positive cells in each LN. (B) Mice were treated as in panel A and TDLN cells stained for CD11c versus B220 versus intracellular IL-6. Plots represent total LN cells; inset represents gated CD11c⁺ population from the (+)1MT group. (C) Wild-type B6 mice with B16-OVA tumors were treated with control (vehicle only) or oral D-1MT plus OVA-Lv vaccine. All mice received coadoptive transfer of 10⁶ CD8⁺ OT-I cells mixed with 10⁶ sorted Foxp3⁺ Tregs (CD4⁺GFP⁺Thy1.1⁺) from OT-II^{Foxp3-GFP Thy1.1} mice. On day 11, TDLNs were harvested and stained for CD4/Thy1.1/IL-17 versus Foxp3^GFP by 4-color FACS. Each left plot represents the population of transferred Tregs (CD4⁺Thy1.1⁺) as a percentage of total TDLN cells; right plots represent GFP versus IL-17 expression in the gated GFP⁺ Tregs (the percentage gives the fraction of Foxp3^GFP-positive cells that coexpress IL-17). (D) Bone marrow chimeras (RORγt-null marrow into wt B6 hosts, or control wtB6→wtB6) received B16-OVA tumors, and mice were treated as in panel A with either control (vehicle only) or oral D-1MT plus OVA-Lv vaccine. All mice received OT-I adoptive transfer on day 7. Plots indicate representative IL-17 up-regulation in gated CD4⁺CD25⁺ population in TDLNs from each treatment group on day 11. Experiments were repeated 3 to 8 times with similar results.