(A) Leukocytes were recovered from the CNS of isotype- (left panels), anti–mIL-17 mAb– (middle panels), and h6H12-treated (right panels) mice at 24 days after antigen immunization and analyzed by flow cytometry. First row, representative flow cytometry demonstrating CD45⁺CD4⁺ cells in the CNS (live cell gate). Second row, CCR6 expression of CD4⁺ T cells (CD45⁺CD3⁺ gate). Third row, IL-17 and IFN-γ intracellular staining of CD4⁺ T cells isolated from CNS and stimulated for 5 hours with PMA and ionomycin. Fourth row: CCR6 and FoxP3 intracellular staining of CD4⁺ T cells isolated from CNS. Fifth row: CCR6 and IFN-γ intracellular staining of CD4⁺ T cells isolated from CNS and stimulated for 5 hours with PMA and ionomycin. All flow cytometric analyses were done on pooled CNS from 2 mice (6 mice per group). Results are representative of at least 2 independent experiments and the percentage of cells is shown. (B) Quantitative analysis of infiltrating T cells (first row), CCR6⁺CD4⁺ T cells (second row), IL-17– and IFN-γ–producing CD4⁺ T cells (third row), CCR6⁺ Tregs (fourth row), and CCR6⁺ Th1 cells (fifth row) isolated from whole CNS samples showing total cell number (left columns) and percentage (right columns). Black (isotype; n = 6); light gray (anti–IL-17 mAb, n = 6); dark gray (h6H12 Fc-KO, n = 6). Data are shown as mean ± SEM. For box-and-whisker plots, the whiskers represent the minimum and maximum values, the box boundaries represent the 25th and 75th percentiles, and the middle line is the median value. Statistical significance was determined by 1-way ANOVA with Dunnett’s multiple comparisons test relative to control isotype–treated group. *P < 0.05, **P < 0.01, ***P < 0.001.