(A) After immunization with MOG peptide, the progression of disease was monitored. Clinical EAE scores and body weights of Ctrp4^–/–(n = 10) or WT (n = 10) mice following disease induction are shown. Statistical significance was determined using 2-way repeated measures ANOVA. (B) Spinal cord sections were stained with H&E or Luxol fast blue (LFB). Histological images are representative of 3 mice in each group. Scale bars: 200 μm. (C and D) Single cells were isolated from the CNS on day 18 after EAE induction and stained with the indicated cell type–specific markers. Summary bar graph shows absolute numbers of CNS-infiltrating immune cells in control and Ctrp4^–/– mice (C). Representative flow cytometry plots of macrophage (CD45^hiF4/80⁺) and microglia (CD45^loF4/80⁺) infiltrated in CNS are shown (D). (E and F) Representative flow cytometry plots showed percentages of IFN-γ⁺CD4⁺, IL-17A⁺CD4⁺, and IFN-γ⁺IL-17A⁺CD4⁺ in CNS of WT and Ctrp4^–/– mice (n = 5 mice per group) on day 18 after EAE induction. Quantified percentages (E) and absolute cell numbers (F) are shown. (G and H) Recall response of antigen-specific T cells from the dLNs of WT and Ctrp4^–/– mice on day 9 after EAE induction. CD4⁺ T cells were expanded with irradiated autologous-presenting cells plus 10 μg/mL (G) or the indicated concentration (H) of MOG peptide for 72 hours and subjected to cell-proliferation assay to determine T cell recall response based on BrdU assay (G) and ELISA assay (H) to quantitate the production of IL-17A and IFN-γ. (I) Flow cytometric analysis of CFSE-labeled CD4⁺ T cells and quantification of intracellular cytokine staining at day 5 after in vitro coculturing with irradiated autologous-presenting cells plus 10 μg/mL MOG peptide. The percentages of IL-17⁺ and IFN-γ⁺ cells were gated on CSFE^loCD4⁺ T cells, and data are presented as representative flow plots. Data are represented as mean ± SEM and are from 1 of 3 independent experiments with similar results. (C–H) Statistical significance was determined using 2-tailed, unpaired Student’s t test or Mann-Whitney U test as appropriate after assessing for distribution. *P < 0.05; **P < 0.001.