Delayed onset of EAE induction in Gfi1 cKO mice is correlated with the increased Foxp3⁺CD103⁺ population. (A, left) The time course of the disease induction in wild-type and Gfi1 cKO mice after MOG immunization. (right) The dates of disease onset after immunization in an individual mouse, pooled from three independent experiments, are shown. (B) Splenocytes were harvested at various times after immunization as indicated. CD4 T cells were purified and RNAs were made without in vitro stimulation. The expression of IFN-γ, IL-4, IL-17a, and CD103 relative to CD4 was assessed by real-time PCR. Error bars in A and B represent means ± SD. (C) Splenocytes were harvested at day 7 after immunization. Cells were stimulated with platebound anti-CD3/anti-CD28 for 6 h with the presence of monensin during the last 2 h before intracellular staining. The dot plots were gated on the CD4⁺CD44^hi population. (D) Splenocytes harvested from mice 3 wk after immunization were directly stained for CD4, CD103, and Foxp3. The dot plots were gated on CD4⁺ splenocytes. Numbers indicate the percentage of cells in each quadrant. Data are representative of at least two independent experiments.