Blood B Cell and Regulatory Subset Content in Multiple Sclerosis Patients

Abstract

Objective: B cell targeted therapies have been effective in slowing multiple sclerosis (MS) disease progression suggesting a direct causal link for this lymphoid subset. A small subset of B cells with regulative properties (Bregs) exists in peripheral blood, and induction of Bregs ameliorates experimental autoimmune encephalomyelitis (EAE), the murine model for MS. Therefore the frequency of B cell subsets and regulatory B cells in particular in peripheral blood of MS patients is of interest.

Methods: The phenotype and frequency of B cell subsets in peripheral blood from 32 MS patients and 34 healthy controls (HC) were examined using flow cytometry.

Results: We found that there is an increase in CD19⁺ cell number in MS 1347 ± 159 cells/μL, (average ± SEM) compared to HC, 935 ± 129 cells/μL and no apparent deficiency in B-cells with a regulatory phenotype. In addition, we observed a loss of correlation between CD19⁺ B cells and total lymphocyte count in MS.

Conclusion: These findings suggest altered blood B-cell homeostasis in MS patients.

Keywords: B cells; Flow cytometry; IL-10; Multiple sclerosis; Regulatory B cells; Rituximab.

Figure 1

Whole blood flow cytometric analysis. A: Forward scatter (FSC) measures cell size and side scatter (SSC) measures cell granularity. Counting Beads and Lymphocyte gates are depicted. B: Lymphocyte gating revealed a CD19⁺ (B cell) population and a CD19⁻ population. C: Bregs are defined as CD19⁺CD5⁺CD1d^+. D: Memory B cells are defined as CD19⁺CD27⁺IgD⁻ and naïve B cells are defined as CD19⁺CD27⁻IgD⁺. E: The CD19⁻ subset from Figure 1B revealed clear CD5 and CD1d populations which were used to establish gating in Figure 1C. F: The CD19⁻ subset also revealed clear CD27 and IgD populations which were used to determine gating in Figure 1D.

Figure 2

Analysis of whole blood samples for HC (n=34), total MS (n=32), no DMD (n=11), interferon-beta (n=12), and glatiramer acetate (n=7). Data for fingolimod was not included due to low sample size (n=2). A: Total number of CD19⁺ B cells. B: Number of CD19⁺CD5⁺CD1d⁺ Bregs. C: Number of CD19⁺CD27⁺IgD⁻ memory B cells. D: Number of CD19⁺CD27⁻IgD⁺ naïve B cells. E: Number of CD19⁺CD27⁻IgD⁺CD5⁺CD1d⁺ naïve Bregs. F: Percent of naïve B cells that are CD5⁺CD1d⁺. Error bars represent mean ± SEM. Significant difference is indicated: * p < 0.05.

Figure 3

Analysis of 11 MS and 15 HC samples for which an absolute lymphocyte count (ALC) was obtained. A: Absolute lymphocyte count versus total number of CD19⁺ supplementary files B cells. Linear regression lines were calculated for MS (p=0.22) and HC (p<0.01). B: Two-way ANOVA of absolute lymphocyte count and CD19⁺ B cells. Significant difference is indicated: ^* p < 0.05.