Fig. 4

Effects of Ki16425 on hypertrophy of secondary lymphatic organs and population of CD4⁺, CD8⁺, Th1, Th2, Th17, and Treg cells in spinal cords or spleens of EAE^low mice. A, B Spleens and lymph nodes (n = 5 per group) were dissected (A) from sham, EAE^low, EAE^low + Ki16425 (15 and 30 mg/kg), and Ki16425 (30 mg/kg) groups at day 19–20 post-immunization and weighed (B). C–F Lysates or cryosections of lumbar spinal cords (n = 3) from each group were used to analyze the degree of infiltration of T cells with CD3 mRNA level by real-time PCR (C), CD3 protein level by Western blot (D), and CD3 distribution by immunofluorescence stain (E, F). G–I Spinal cords and spleens (n = 3) from each group were used to investigate populations of CD4⁺ and CD8⁺ T cells by flow cytometry. Populations of CD4⁺ and CD8⁺ T cell were dotted (G) and displayed in graphs (H, I). J CD4⁺ subset gating scheme. To determine populations of CD4⁺ subset by flow cytometry, CD4⁺ T cells (1 × 10⁴) were first gated using FSC and SSC properties form spinal cords and spleens from each group. CD4⁺ T cells were used to analyze populations of Th1 (CD4⁺/IFN-γ⁺/T-bet⁺), Th17 (CD4⁺/IL-17A⁺/RORγt⁺), Treg (CD4⁺/CD25⁺/Foxp3⁺), and Th2 (CD4⁺/IL-4⁺) cells on CD4⁺ T cells. K–M, P–R, U–W, and Z–AB Populations of Th1, Th17, Treg, and Th2 cells were dotted (K, P, U, and Z, respectively) and displayed in graphs (L, M, Q, R, V, W, AA, and AB). N, O, S, T, X, Y, AC, and AD Lysate of lumbar spinal cords (n = 3) from each group were analyzed by real-time PCR to determine mRNA expression levels of IFN-γ (N), T-bet (O), IL-17A (S), RORγt (T), Foxp3 (X), TGF-ß (Y), GATA3 (AC), and IL-4 (AD). Quantified data were expressed as mean % cells or fold induction ± SEM. (ANOVA test; ^#p < 0.05 and ^##p < 0.01 versus sham group; *p < 0.05 and **p < 0.01 versus EAE^low group)