Gfi-1 binds to the Il17a/Il17f intergenic region and inhibits IL-17a and IL-17f expression. CD4⁺ T cells from naive mice were activated under neutral conditions for 24 h and infected with (A–C) GFP-RV, (A–C) Gfi-1–GFP-RV, (C) or Gfi-1(P2A)–GFP-RV. (A and C) Infected cells were further primed under Th17 conditions for 4 d. Cytokine production was assessed by intracellular staining after PMA plus ionomycin stimulation. (A) Numbers indicate the percentage of cells in each quadrant (left). Relative IL-17–producing cells (GFP⁺/GFP⁻) pooled from three independent experiments are shown (right). (B) GFP⁺ and GFP⁻ cells were sorted 24 h after infection. Sorted cells were maintained under Th17 conditions for another 2 d. Cells were harvested and total RNAs were isolated. The expression levels of different genes normalized to GAPDH were assessed by real-time PCR after RT. (C) The percentage of IL-17–producing cells from each GFP⁺ group was plotted. (D) Gfi-1–GFP-RV–infected Th17 cells were used for Gfi-1 ChIP (left). Wild-type and Gfi1 cKO Th2 cells were used for LSD1 ChIP (right). The enrichment of individual sites was normalized to a negative control from Gata3-intron3. (E) CD4 T cells from naive wild-type or Gfi1 cKO mice were primed under Th17 conditions for 4 d. Cytokine production was assessed by intracellular staining after PMA plus ionomycin stimulation. Numbers indicate the percentage of cells in each quadrant (left). Data from nine independent experiments are shown (right). Error bars represent means ± SD.