(A) Lymphocytes from the dLNs of B6.SJL mice (CD45.1⁺) that were previously immunized with MOG_35–55 in CFA were rechallenged with the MOG_35–55 peptide in the presence of BSA or rhCTRP4 under Th17 polarization conditions. The intracellular IL-17A was analyzed via flow cytometry (left), and the production of IL-17A in the supernatants was measured by ELISA (right). (B) After PMA/ionomycin stimulation for 5 hours, the representative FACS plots and the frequency of CD4⁺IL-17A⁺, CD4⁺IFN-γ⁺, CD4⁺IL-17A⁺IFN-γ⁺, and CD4⁺IL-17A⁺GM-CSF⁺ before the time of adoptive transfer were determined. (C) Ex vivo–expanded MOG-specific CD4⁺ T cells under Th17 polarization conditions in the presence of BSA or rhCTRP4 were analyzed for the expression of indicated genes by quantitative PCR. The values were normalized against gapdh. (D–G) Ex vivo–expanded MOG-specific CD4⁺ pretreated with BSA or rhCTRP4 were transferred into irradiated congenic recipients (CD45.2) to induce EAE. (D) Clinical scores of EAE progression were monitored daily. (E) The mononuclear cells isolated from brain and spinal cord at disease peak stage were analyzed by flow cytometry. Absolute numbers of CD4⁺ T cells of donor and recipient mice in CNS were analyzed. Gated CD45.1⁺CD4⁺IL-17⁺ T cells were analyzed for the production of IFN-γ and GM-CSF. Representative contour plots (F) show the percentages and absolute numbers of CD4⁺IL-17⁺IFN-γ⁺, CD4⁺IL-17⁺GM-CSF⁺ donor cells in the CNS (G). Data are represented as mean ± SEM and are from 1 of 3 independent experiments with similar results. Two-way repeated-measures ANOVA was used for D. Statistical significance was determined using unpaired Student’s t test or Mann-Whitney U test for A–C, E, and G. *P < 0.05; **P < 0.01; ***P < 0.001.