Gfi-1 limits the differentiation of iTreg cells and regulates the CD103⁺Foxp3⁺ subset. (A and B) CD25-depleted CD4⁺ T cells were activated under neutral conditions for 24 h, infected with RV, and further primed under iTreg cell conditions for 4 d. Cells were restimulated with PMA plus ionomycin for 4 h in the presence of monensin during last 2 h. Cytokine production and Foxp3 expression was assessed by intracellular staining. (C) CD25-depleted CD4⁺ T cells were activated under iTreg cell conditions for 4 d. Cell-surface markers were costained with Foxp3. Dot plots were gated on CD4⁺ cells. (D) Lymph node cells were stained directly with CD4, Foxp3, and CD103. Dot plots were gated on CD4⁺ cells. Numbers indicate the percentage of cells in each quadrant (top). Within the CD4⁺Foxp3⁺ population, the percentage of CD103⁺ cells was calculated from the data obtained from four mice per group (bottom). (E) Wild-type CD4 T cells that had been primed under Th2 conditions for 4 d were used for Gfi-1 ChIP (left), and both wild-type Th2 and Gfi1 cKO Th2 cells were used for LSD1 ChIP (right). Enrichment of individual sites from the Cd103 locus was normalized to a negative control from Gata3-intron3. Error bars in B, D, and E represent means ± SD.