The severity of EAE is exacerbated in BM cell–transplanted CD25-deficient mice. (A) Schematic showing design of the experimental BMT model. (B) Gating strategy for FACS analysis of peripheral blood cell populations in reconstituted mice 8 wk after lethal irradiation (900 rad) and BM cell (5 × 10⁶) transplantation. Cells were first gated for singlets (forward scatter height vs. area [FSC-H vs. FSC-A]) and lymphocytes (side scatter area [SSC-A] vs. FSC-A). The lymphocyte populations were further analyzed for the expression of CD45.1 and CD45.2 markers. CD3 or CD4 surface expression was then determined in this gated population. (C) Quantification of CD45.1⁺ and CD45.2⁺ cells after BMT (n ≥ 8 per each group). (D) BM cells were transplanted into either WT (BM^WT→WT, n = 8) or CD25^−/− (BM^{WT→CD25−/−}, n = 7) recipient mice, and EAE was induced. Clinical scores of EAE representing disease severity are depicted. *, P < 0.05 compared with EAE-induced BM cell–transplanted WT mice. Error bars indicate the mean ± SEM. (E) Disease incidence in WT (BM^WT→WT) and CD25^−/− (BM^{WT→CD25−/−}) mice was measured for the indicated length of time after immunization. (F and G) Intracellular production of IFN-γ and IL-17A from CD4⁺ T cells in the CNS (F) or iLNs (G) of EAE-induced mice. Representative flow cytometric plots (left) and compiling data (right) are depicted. *, P < 0.05 compared with EAE-induced BM cell–transplanted WT mice. Error bars indicate the mean ± SEM. (H) The levels of cytokine production were measured by ELISA in culture supernatants from antibody-restimulated splenocytes. *, P < 0.05; **, P < 0.01 compared with the EAE-induced BM cell–transplanted WT mice. Error bars indicate the mean ± SEM. Data are representative of three independent experiments.