Figure 1: GITR ligation induces Th9 cells under iTreg-polarizing conditions.

Naive CD4⁺ T cells were FACS sorted from WT B6 (a,b) and OT-II (c,d) Foxp3gfp reporter mice and activated under iTreg-polarizing conditions. Anti-GITR agonistic antibody (DTA-1, 10 μg ml⁻¹) or His-GITR Ligand (200 ng ml⁻¹, along with anti-His mAb) were used to ligate the GITR receptor on activated CD4⁺ T cells. Three days later, cells were harvested and analysed for Foxp3 and cytokines expression by flow cytometry. (a,c) Colour FACS plots depicting Foxp3- and IL-9-producing cells. Numbers in the quadrants indicate the percentage of cells. Data are representative of five independent experiments. (b,d) DTA-1 titrations and their impact on induction of Foxp3⁺ Tregs and Th9 cells, and graphs depict the percentage of cells that express Foxp3 (left) or IL-9 (right). Data are pooled from five independent experiments with triplicate cultures. (e) Expression of other cytokines, as indicated in the x axis, by WT naive CD4⁺ T cells polarized under iTreg conditions for 3 days with or without GITR ligation. Data are representative of three independent experiments.