PBMCs from treatment naïve, IFN-β treated and GA treated as well as healthy controls were analyzed using flow cytometry for the following: the frequency (A and B) and absolute # (C) of total CD19+ B cells; the frequency (D and E) and absolute # (F) of CD19+CD24++CD38++ transitional B cells; the frequency (G) of transitional-1 and -2 (T1 and T2) B cells; the frequency (H and I) and absolute # (J) of CD19+CD38-IgM-IgD- class-switched memory B cells; and the frequency (K and L) and absolute # (M) of CD19+CD27++CD38++ plasmablasts. Statistical significance was determined by one-way anova (***p<0.001, **p<0.01, *p<0.05).

IL-10 expression in B-cell subsets. (A) Representative analysis of intracellular IL-10 staining in transitional (CD19+CD24++CD38++), naïve (CD19+CD24+CD38+), and memory (CD19+ CD38-) B cells. (B) Frequency of IL-10+ cells in transitional, naïve and memory B cells from 5 IFN-β-treated MS patients and 5 healthy volunteers. Statistical significance was determined by one-way anova. (C) IFN-β (1000U/ml) increases IL-10 secretion in PBMCs from healthy volunteers stimulated with anti-Ig, anti-CD40 and CpG (B-stim). IL-10 was measured in culture supernatants by ELISA and statistical significance was determined by a Student’s T-test. The error bars represent S.E.M.

(A) IFN-β treatment increases BAFF in serum from mice with EAE. Serum BAFF concentrations were determined at EAE day 16 in IFN-β (N=5) or vehicle (N=5) treated mice by ELISA. The error bars represent S.E.M. and two-tailed Student’s t-tests were used to determine significance (**p<0.01).
(B) IFN-β treatment increases splenic B cell numbers in mice with EAE. Absolute B cell numbers were determined at EAE day 16 in mice receiving IFN-β (N=6) or vehicle (N=6) treatment mice by manual hemocytometer counts and FACS analysis. Two-tailed Student’s t-tests were used to determine statistical significance (**p<0.01) and error bars represent S.E.M.
(C–G) IFN-β alters B cell subsets in spleens from mice with EAE. Absolute numbers of (C) CD19+IgMhiIgDloCD21-CD23- transitional 1 and CD19+IgMhiIgDloCD21-CD23+ transitional 2 B cells, (D) CD19+IgDloCD21hi and CD19+IgD+IgMhiCD21hi marginal zone B cells, (E) CD19+IgD+CD21int Follicular B cells, (F) CD19+CD23loCD5+CD1DhiIgMhi regulatory B cells, and (G) CD138+B220loCD19loCD267int plasma cells were assessed in spleens from EAE mice treated with IFN-β (N=3) or vehicle (N=3) by flow cytometry. Two-tailed Student’s t-test were used to determine statistical significance (*p<0.05, **p<0.01) and error bars represent S.E.M. (H) IFN-β elevates of transitional B cells is confirmed by measurement of AA4.1+CD19+ B cells in peripheral blood from EAE mice. Representative FACS plots of blood cells from EAE mice treated with IFN-β or vehicle at EAE day 16.
(I) Peripheral B cell numbers are inversely correlated with EAE severity despite MOG autoantibody production. Negative correlation between EAE scores and the percentage of CD19+ cells in the spleens EAE mice. Using linear regression we determined the slope was non-zero with the indicated R2 and the statistical significance was indicated as a p value. Data are representative of IFN-β treated and vehicle treated mice (N=10 total) from 2 pooled experiments.

(A) IFN-β treatment increases serum anti-MOG IgM and IgG autoantibodies in EAE although these antibodies do not correlate with EAE severity. Serum from EAE treated with IFN-β (N=8), vehicle (N=8) or healthy control mice (N=3) were analyzed for anti-MOG IgM and IgG by sandwich ELISAs. Two-tailed Student’s t-tests were used to determine statistical significance (*p<0.05, **p<0.01). (B) Linear regression between serum anti-MOG antibodies and EAE score are also displayed. R2 and p values are displayed. These data are representative of two pooled experiments.

B cells are required for therapeutic efficacy of IFN-β treatment EAE. EAE was induced in WT (A) and muMT (B) mice and treated with IFN-β (10,000U/dose) or vehicle every other day starting at day 6 until day 20 (treatment initiation and cessation indicated by arrows). WT Veh N=15, WT IFN-β N=14, muMT Veh N=6, muMT IFN-β N=6. Statistics were determined by a Mann-Whitney (*p<0.05) and error bars represent S.E.M. These data are combined from three independent experiments. (C) Histology of spinal cords isolated from WT and muMT mice treated with IFN-β or vehicle. Mice were sacrificed at EAE day 13 and tissue harvested and stained with both H&E and Luxol fast blue. (D) Spinal cord infiltrating CD4+ T-cells and CD19+ B cells were assessed by flow cytometry. WT Veh n=6, WT IFN-β n=5, muMT Veh n= 3, muMT IFN-β= 3. Statistical significance was determined by one-way anova. (E) IFN-β treatment elevates IL-10 in spleen cells from wild type mice. Spleen cells from WT and muMT mice treated with IFN-β or vehicle were cultured with 1 ug/ml of MOG35-55 for 72 hrs and IL-10 was measured in the culture supernatants by ELISA. Statistics were determined by one-way anova.

Adoptive transfer of B cells from IFN-β treated EAE does not protect mice from EAE. Effect of adoptively transferred B cells on EAE. 20×106 B220+ cells from either IFN-β (N=7) or vehicle treated mice (N=5) were transferred into recipient mice on EAE day 6 (injection of B cells indicated by arrow). EAE progression was compared to mice that did not receive B cells (N=3).