Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4(+) T-cell proliferation and IFN-γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein

Abstract

Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-α secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4(+) T-cell proliferation and IFN-γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4(+) T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells.

FIGURE 1. Memory B cells from RRMS patients and HD exhibit an activated phenotype but express less HLA-DR

(A) Gating strategy used to determine the percentage of peripheral blood memory (CD19+CD27+) and naïve (CD19+CD27−) B cell subsets from live lymphocytes based on forward scatter and side scatter characteristics from a single HD; percentage within each gate indicated in corners of dot plot. (B) Percentage of naïve and memory B cell subsets in the peripheral blood lymphocyte population of HD and RRMS patient cohorts; individual points represent individual subjects, and black bars represent mean values of each group. Representative histograms of (C) CD80 and (E) HLA-DR expression from one HD; isotype control (grey line), naïve B cells (thin black line) and memory B cells (thick black line). The expression of CD80 (D) and HLA-DR (F) on memory and naïve B cell subsets in the peripheral blood lymphocyte population of HD and RRMS patient cohorts were quantified by mean fluorescence intensity (MFI) of the population; individual points represent individual subjects, and black bars represent mean values of each group. **p<0.01, ***p<0.001 multiple comparisons post-hoc analysis with Bonferroni correction after two-way mixed-model ANOVA.

FIGURE 2. Memory and naïve B cells from RRMS patients and HD generate similar opposing cytokine profiles in response to various polyclonal stimuli in vitro

Highly purified, sorted naïve and memory B cell subsets from HD and RRMS patients were stimulated with (A and C) CD40L for 48 hours, or (B and D) dual-staggered stimulation with BCR crosslinking and CD40L for 72 hours. LTα (A, B) and IL-10 (C, D) secretion were measured in cell culture supernatants by ELISA. Individual points represent individual subjects, black bars represent mean values of group. **p<0.01, ***p<0.001; multiple comparisons post-hoc analysis with Bonferroni correction after two-way mixed-model ANOVA. ‡p<0.05 paired t-test.

FIGURE 3. CD4+ T cell proliferation from RRMS patients

A) Example of gating strategy used to obtain the raw percentage of CD3+CD4+ T cell proliferation in co-culture of purified CFSE-labeled T cells and autologous FACS purified CD27+CD19+ memory B cells in the presence of 5 ug/mL of TT, from HD-12. Numbers inside of gates are the percentage of cells in each gate. Example of proliferation histograms from MS-10 (B), and MS-1 (C) of CD3+CD4+ T cell proliferation in cocultures of purified CFSE-labeled T cells and autologous highly purified FACS-sorted naïve B cells (top rows) or memory B cells (bottom rows) along with antigens where indicated (above columns). Raw percentage of CD4+ T cell proliferation was determined by gating on CFSE^lowCD3⁺CD4⁺ cells and listed above the histogram gates. ΔProliferation was calculated by subtracting background (No Ag) proliferation (listed in parenthesis above gate). MS-10 CD4+ T cell viability was 40.6±0.3% and 40.8±1.6% in naïve B-T and memory B-T cell cultures respectively. MS-1 CD4+ T cell viability was 36.9±1.7% and 38.3±2.2% in naïve and memory B-T cell cultures respectively.

FIGURE 4. Memory B cells from RRMS patients elicit greater neuro-antigen specific CD4+ T cell proliferation than memory B cells from HD

Highly purified, ex vivo naïve and memory B cells from cryopreseved HD and RRMS PBMC were incubated with purified autologous T cells, and CD4+ T cell proliferation by CFSE dilution was measured in vitro culture after 5 days in response to (A) TT, (B) MBP, (C) GA and (D) MOG antigens (see Figure 3 for sample histograms). Threshold of CD4+ T cell proliferation considered positive was set at 2% and is depicted by a dashed line in all panels. Individual points represent individual donor responses, and black bars represent mean values for each group. *p<0.05, **p<0.01, multiple comparisons post-hoc analysis with Bonferroni correction after two-way mixed model ANOVA. #p<0.05, chi-squared comparing number of positive responders above threshold. Number of responders/donors is indicated at the bottom of each panel. T cell viability of MS CD4+ T cells is listed in Supplementary Table 1.

FIGURE 5. Memory B cells from RRMS patients elicit greater antigen-specific IFN-γ secretion than memory B cells from HD

Highly purified, ex vivo naïve and memory B cell subsets from the HD and RRMS patient cohorts were incubated with purified autologous T cells, and IFN-γ secretion was measured after 5 days in culture supernatants in response to (A) TT, (B) MBP, (C) GA, and (D) MOG. Individual points represent individual donor responses, and black bars represent mean values for each group. Threshold of IFN-γ production was set at 237 pg/mL and was determined empirically by adding 2 standard deviations to the mean detectible IFN-γ production observed in B-T co-cultures when no antigen was present. The threshold is depicted as a dashed line in all panels. #p<0.05, chi-squared test comparing number of positive responders above threshold. Number of responders/donors is indicated at the bottom of each panel.

FIGURE 6. B cells that bind MBP and GA are more prevalent in the memory B cell pool than the naïve B cell pool in both HD and RRMS patients

PBMC from HD and RRMS patients were incubated with FITC-conjugated antigens as described in the Materials and methods. Naïve and memory B cells were gated as shown in Figure 1A. Examples of gating used to enumerate the percentage of memory B cells or naïve B cells (from RRMS patient MS-20) that bound OVA and GA are provided in panels A and B respectively. Of note, OVA-FITC binding represents both OVA-binding and FITC-binding B cells. The frequency of (C) TT-, (D) MBP-, (E) GA-, and (F) MOG-binding B cells for the RRMS patient and HD cohorts were calculated as described in the Materials and methods and depicted here as individual points on the vertical scatter plots. The average frequency of binding in each group is shown as a solid black horizontal line in each panel. The mean OVA binding frequency was 93 cells per million for HD memory B cells; 119.3 cells per million for HD naïve B cells; 636.5 cells per million for RRMS memory B cells; and 471.5 cells per million for RRMS naïve B cells. *p<0.05, **p<0.01, ***p<0.001; multiple comparisons post-hoc analysis with Bonferroni correction after two-way mixed model ANOVA.