Figure S2.
Differential tissue accumulation of T cells and B cells in the brain, meninges, and periphery of young versus aged mice at homeostasis and during SJL/J EAE. (A) Active EAE was induced in young (8 wk) SJL/J mice using an emulsion of CFA and PLP139-151. Mice were followed for 20 days after immunization and sacrificed at acute (D14, n = 3) or postacute (D20, n = 4) time points at which point dura, LM, and brains were subjected to flow cytometry analysis. Data represent one experimental repeat. (B) Fold change expressed as ratio of absolute number of cells detected during active EAE compared with age- and sex-matched naïve (n = 2) or CFA-immunized controls (n = 3). (C) Representative flow cytometry gating strategy as pertaining to Figs. 2, 3, and 5 in a representative young naive SJL/J mouse. Two populations of CD45+CD11b-CD3−CD19+B220+ cells were identified in the naïve dura and femur/tibia BM: mature B220highCD24lowIgM+IgD+ B cells (blue) and immature/developing B220lowCD24highIgD− B cells (red). Further analysis of the surface expression of MHCII and IL7R was analyzed among populations of IgM+IgD+B220high (blue) and B220low (red) B cells. Gating for class-switched B220highCD24lowIgM−IgD− B cells (purple) is additionally shown. (D) Representative flow plot showing IgG versus IgA in the young versus aged LM of representative A/T SJL/J mice at the post-acute time point of EAE. (E) Proportion and absolute numbers of IgG+ class-switched B cells at the post-acute time point of A/T SJL/J EAE in young (8 wk, n = 12) and aged (11 month, n = 12) mice. Data represent one experimental replicate. (F) Single-cell suspensions from the spleens of young (8 wk, n = 6) and aged (11 month, n = 6) SJL/J mice were subjected to flow cytometry analysis to assess the distribution of B cell populations at homeostasis. Error bars indicate mean ± SEM in A and B or SD in E and F. In B, a mixed-effects analysis followed by Dunnett’s multiple comparison test was conducted to assess statistical significance from the dura within time points, whereas in E and F, a Student’s t test (absolute number/counts) or Mann−Whitney test (proportion) was conducted to assess statistical significance. In all cases, statistical significance was denoted as * = P < 0.05, ** = P < 0.005, *** = P < 0.0005, **** = P < 0.0005, and no label = not significant. Of note, in B, the label for statistical significance is labeled in the same color as the group for which there is significance. The two variables compared in the mixed-effects analysis were days postadoptive transfer (time) and tissue type (B: dura versus LM and brain).

Differential tissue accumulation of T cells and B cells in the brain, meninges, and periphery of young versus aged mice at homeostasis and during SJL/J EAE. (A) Active EAE was induced in young (8 wk) SJL/J mice using an emulsion of CFA and PLP139-151. Mice were followed for 20 days after immunization and sacrificed at acute (D14, n = 3) or postacute (D20, n = 4) time points at which point dura, LM, and brains were subjected to flow cytometry analysis. Data represent one experimental repeat. (B) Fold change expressed as ratio of absolute number of cells detected during active EAE compared with age- and sex-matched naïve (n = 2) or CFA-immunized controls (n = 3). (C) Representative flow cytometry gating strategy as pertaining to Figs. 2, 3, and 5 in a representative young naive SJL/J mouse. Two populations of CD45+CD11b-CD3−CD19+B220+ cells were identified in the naïve dura and femur/tibia BM: mature B220highCD24lowIgM+IgD+ B cells (blue) and immature/developing B220lowCD24highIgD− B cells (red). Further analysis of the surface expression of MHCII and IL7R was analyzed among populations of IgM+IgD+B220high (blue) and B220low (red) B cells. Gating for class-switched B220highCD24lowIgM−IgD− B cells (purple) is additionally shown. (D) Representative flow plot showing IgG versus IgA in the young versus aged LM of representative A/T SJL/J mice at the post-acute time point of EAE. (E) Proportion and absolute numbers of IgG+ class-switched B cells at the post-acute time point of A/T SJL/J EAE in young (8 wk, n = 12) and aged (11 month, n = 12) mice. Data represent one experimental replicate. (F) Single-cell suspensions from the spleens of young (8 wk, n = 6) and aged (11 month, n = 6) SJL/J mice were subjected to flow cytometry analysis to assess the distribution of B cell populations at homeostasis. Error bars indicate mean ± SEM in A and B or SD in E and F. In B, a mixed-effects analysis followed by Dunnett’s multiple comparison test was conducted to assess statistical significance from the dura within time points, whereas in E and F, a Student’s t test (absolute number/counts) or Mann−Whitney test (proportion) was conducted to assess statistical significance. In all cases, statistical significance was denoted as * = P < 0.05, ** = P < 0.005, *** = P < 0.0005, **** = P < 0.0005, and no label = not significant. Of note, in B, the label for statistical significance is labeled in the same color as the group for which there is significance. The two variables compared in the mixed-effects analysis were days postadoptive transfer (time) and tissue type (B: dura versus LM and brain).

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