Dimethyl fumarate treatment of relapsing-remitting multiple sclerosis influences B-cell subsets

Steven K Lundy, Qi Wu, Qin Wang, Catherine A Dowling, Sophina H Taitano, Guangmei Mao, Yang Mao-Draayer, Steven K Lundy, Qi Wu, Qin Wang, Catherine A Dowling, Sophina H Taitano, Guangmei Mao, Yang Mao-Draayer

Abstract

Objective: To test the hypothesis that dimethyl fumarate (Tecfidera, BG-12) affects B-cell subsets in patients with relapsing-remitting multiple sclerosis (RRMS).

Methods: Peripheral blood B cells were compared for surface marker expression in patients with RRMS prior to initiation of treatment, after 4-6 months, and at more than 1 year of treatment with BG-12. Production of interleukin (IL)-10 by RRMS patient B cells was also analyzed.

Results: Total numbers of peripheral blood B lymphocytes declined after 4-6 months of BG-12 treatment, due to losses in both the CD27+ memory B cells and CD27(neg) B-cell subsets. Some interpatient variability was observed. In contrast, circulating CD24(high)CD38(high) (T2-MZP) B cells increased in percentage in the majority of patients with RRMS after 4-6 months and were present in higher numbers in all of the patients after 12 months of treatment. The CD43+CD27+ B-1 B cells also increased at the later time point in most patients but were unchanged at 4-6 months compared to pretreatment levels. Purified B cells from 7 of the 9 patients with RRMS tested after 4-6 months of treatment were able to produce IL-10 following CD40 ligand stimulation, and the amount corresponded with the combined levels of T2-MZP and B-1 B cells in the sample. None of the patients with RRMS in this study have had a relapse while taking BG-12.

Conclusions: These data suggest that BG-12 differentially affects B-cell subsets in patients with RRMS, resulting in increased numbers of circulating B lymphocytes with regulatory capacity.

Figures

Figure 1. BG-12 treatment reduced total circulating…
Figure 1. BG-12 treatment reduced total circulating B cells and had variable effects on memory B cells
Peripheral blood mononuclear cells (PBMC) isolated from patients with relapsing-remitting multiple sclerosis (RRMS) before and after BG-12 treatment and healthy controls were stained for B-cell markers. (A) Representative forward scatter, side scatter, and anti-CD19 staining from a single patient (RRMS-8) shows standardized gating for lymphocytes, single cells, and total CD19+ B cells that was used for all of the samples. (B) Comparison of %CD19+ B cells for healthy controls and patients with RRMS before treatment. Each dot represents an individual sample and lines are the median interquartile range for each group. The exact p value shown below the title was calculated using Mann-Whitney test. (C) Total B-cell numbers/mL of blood for the patients with RRMS were calculated before and after 4–6 months of treatment by multiplying the %CD19+ cells by the lymphocyte counts reported by the clinical pathology laboratory (table 1). Lines connect individual patients. Exact p value for paired patient data was determined using Wilcoxon matched-pairs signed-rank test. (D) Representative histograms from patient RRMS-8 show gating used to define CD27+ memory B cells. (E) Percentages of CD27+ memory B cells in patients with RRMS before treatment compared with controls. Each dot represents data from one individual and lines are the median and interquartile range for each group. The p value was calculated using Mann-Whitney test. (F) Longitudinal %CD27+ memory B-cell data for individual patients with RRMS (n = 8) before, after 4–6 months, and after more than 1 year of treatment with BG-12.
Figure 2. Increases in CD24 high CD38…
Figure 2. Increases in CD24highCD38high transitional 2 marginal zone precursor (T2-MZP) and CD27+CD43+ B-1 B cells after BG-12 treatment
Peripheral blood mononuclear cells (PBMC) isolated from healthy controls and patients with relapsing-remitting multiple sclerosis (RRMS) before and after BG-12 treatment were stained and gated for CD19+ B cells as shown in figure 1 and then analyzed for coexpression of CD24 and CD38 or CD27 and CD43. (A) Representative staining from patient RRMS-8 shows the gating used to select the CD24highCD38high (T2-MZP) B-cell subset. (B) Representative staining from patient RRMS-8 shows gating used to analyze CD43+ CD27+ B-1 B cells before and after 4 months of BG-12 treatment. (C) Comparison of T2-MZP B cells in controls and patients with RRMS before treatment. Each dot represents an individual sample and lines are the median and interquartile range for each group. The p values shown for group comparisons were calculated using Mann-Whitney tests. (D) Percentages of B-1 B cells in controls and patients with RRMS before treatment. Each dot represents an individual and lines are the median and interquartile range for each group. Exact p value was calculated using Mann-Whitney test. (E) Percentages of T2-MZP B cells in individual patients with RRMS before BG-12 therapy and after 4–6 months and >12 months of treatment. (F) Longitudinal changes in percentage of B-1 B cells for individual patients with RRMS at baseline, 4–6 months, and >12 months BG-12 treatment.
Figure 3. B cells from most patients…
Figure 3. B cells from most patients with relapsing-remitting multiple sclerosis (RRMS) produced interleukin-10 (IL-10) at levels that correlated with the presence of transitional 2 marginal zone precursor (T2-MZP) and B-1 B cells
CD19+ B cells were positively selected by magnetic bead separation before and after 4–6 months of BG-12 treatment. Purified B cells were stimulated with irradiated hCD40L-Fb for 6 days prior to measurement of IL-10 in culture supernatants. Cultures containing hCD40L-Fb alone or B cells with CD40Lneg fibroblasts did not produce IL-10 (data not shown). (A) Mean IL-10 production ± SD in triplicate culture wells. Student t test calculated *p values for the data before and after treatment were p = 0.0018, p < 0.0001, and p = 0.2084 for patients RRMS-1, RRMS-2, and RRMS-10, respectively. (B) Stacked bar graph of percentage of circulating T2-MZP B cells (open bars) and B-1 B cells (hashed bars) within the total CD19+ B-cell population before culture. (C) Comparison of B-cell IL-10 production vs combined levels of T2-MZP and B-1 B cells after 4–6 months treatment for 9 patients. Two patients did not produce IL-10 despite high levels of regulatory B cells (marked with x). Linear regression for the other 7 patients (diamonds) showed a high degree of correlation: Y = 50.3X + 181, R2 = 0.79, F = 18.7, p = 0.0075. §Data point at 11.6% T2-MZP + B-1 B cells represents 2 patients, RRMS-1 and RRMS-2, who produced IL-10 = 1,010 ± 45 and 1,008 ± 99 pg/million cells, respectively.

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