Supplementary Figure 1: Characterization of T_reg cells activated in vivo.

(a) Schematic of generation of in vivo activated Treg and Teff cells. The time course of DT administration to Foxp3^DTR mice and subsequent rebound of Treg cells are shown. (b) DT-treated mice display splenomegaly and lymphadenopathy (representative of n>5 experiments, each with n>3 DT treated and n>3 untreated Foxp3^DTR mice). (c) DT-treatment results in increased numbers of Foxp3⁺ Treg and Teff cells on day 11. (d) Cell surface effector molecules and proliferation markers are increased in aTreg cells (representative of n=3 experiments, each with n>3 mice). (e) Activated Treg cells isolated from diphtheria toxin treated Foxp3^DTR mice are representative of activated Treg cells in other inflammatory settings. Intra-tumoral Treg and Teff cells were flow cytometry sorted from B16 melanoma tumors established in Foxp3^GFP mice. Gene expression datasets were compared for tumor infiltrating Treg vs. Teff cells (x-axis) and aTreg vs. Teff cells isolated from DT-treated Foxp3^DTR mice (y- axis). Plots show all genes called as present (left), those that are expressed at a lower level in aTreg compared to rTreg cells (center), and those that are expressed at a higher level in aTreg cells compared to rTreg cells (right). Pearson correlation values are shown. Tumor Treg and Teff array analysis was performed using two biological replicates.