Distribution of B220 ^high and B220 ^low B cells in the different meningeal layers in young versus aged SJL/J EAE mice at different time points of disease. Clinical scores were tracked in mice in which EAE was induced by adoptive transfer of encephalitogenic T cells into young and aged SJL/J mice (Fig. 2 A). (A, B, and D–F) At various time points throughout the disease, mice were euthanized, and the dura, LM, brain, skull BM, and femur BM were collected and analyzed by flow cytometry. IgM⁺IgD⁺B220^high, class-switched B220^high, and B220^low B cells were evaluated at naïve (day 0, n = 7–8), late onset (day 9, n = 4–6), acute (day 11, n = 6–14), and post-acute (day 25, n = 11–13) time points. Results are expressed as an absolute number of cells in the whole tissue (A, D, and E), as percentage of the parent CD19⁺B220⁺ population (E), as fold change from naïve mice (B), or as a ratio (F). At the same time points, the supernatant of whole tissue dissections of the dura and LM were collected. (C) Concentrations of BAFF and CXCL13 (in pg/ml) were measured in the supernatant using the Ella microfluidics platform, and results were expressed as a fold change from naïve mice. Results are data from two repeat experiments. Error bars indicate mean ± SEM. A two-way ANOVA followed by Sidak’s multiple comparison test (A, C, D, E, and F) or mixed-effects analysis followed by Dunnett’s multiple comparison test (B) was conducted to assess statistical significance (* = P < 0.05, ** = P < 0.005, *** = P < 0.0005, **** = P < 0.0005, and no label = not significant). The two variables compared in the mixed-effects analysis, or two-way ANOVA, were days postadoptive transfer (time) and either age (A and D–F: young versus aged mice) or tissue type (B: dura versus LM and brain; C: dura versus LM). Of note, statistical significance in graphs plotting immune cell populations as a fold change from naïve in B was evaluated using Dunnett’s by comparing the brain and LM with the dura within each time point.