Aiolos Overexpression in Systemic Lupus Erythematosus B Cell Subtypes and BAFF-Induced Memory B Cell Differentiation Are Reduced by CC-220 Modulation of Cereblon Activity

Abstract

BAFF is a B cell survival and maturation factor implicated in the pathogenesis of systemic lupus erythematosus (SLE). In this in vitro study, we describe that soluble BAFF in combination with IL-2 and IL-21 is a T cell contact-independent inducer of human B cell proliferation, plasmablast differentiation, and IgG secretion from circulating CD27+ memory and memory-like CD27-IgD- double-negative (DN) B cells, but not CD27-IgD+ naive B cells. In contrast, soluble CD40L in combination with IL-2 and IL-21 induces these activities in both memory and naive B cells. Blood from healthy donors and SLE patients have similar circulating levels of IL-2, whereas SLE patients exhibit elevated BAFF and DN B cells and reduced IL-21. B cell differentiation transcription factors in memory, DN, and naive B cells in SLE show elevated levels of Aiolos, whereas Ikaros levels are unchanged. Treatment with CC-220, a modulator of the cullin ring ligase 4-cereblon E3 ubiquitin ligase complex, reduces Aiolos and Ikaros protein levels and BAFF- and CD40L-induced proliferation, plasmablast differentiation, and IgG secretion. The observation that the soluble factors BAFF, IL-2, and IL-21 induce memory and DN B cell activation and differentiation has implications for extrafollicular plasmablast development within inflamed tissue. Inhibition of B cell plasmablast differentiation by reduction of Aiolos and Ikaros may have utility in the treatment of SLE, where elevated levels of BAFF and Aiolos may prime CD27+ memory and DN memory-like B cells to become Ab-producing plasmablasts in the presence of BAFF and proinflammatory cytokines.

Copyright © 2017 by The American Association of Immunologists, Inc.

Figures

FIGURE 1.

BAFF induces plasmablasts from memory B cells, but not from naive B cells. CD27− naive B cells (A and C) and CD27+ memory B cells (B and D) were isolated from human PBMCs, left untreated, or cultured with indicated stimuli for 5 d. (A and B) Representative dot plots in indicated conditions. Total CD19+ B cells [(A) naive and (B) memory] were gated and analyzed for differentiation by expression of CD20 and CD38; the number in the plot is the percent of plasmablast among CD19+ B cells. (C and D) Graphs show the percent of plasmablast among CD19+ B cells in the indicated conditions (mean ± SEM). Dot plots are representative of multiple donors. Statistical comparisons were made with one-way ANOVA followed by Dunnett’s multiple-comparison test. Naive B cells: n = 12; memory B cells: n = 6. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

FIGURE 2.

Plasmablasts induced by anti-IgM, CD40L, or BAFF are actively dividing B cells. (A) Representative histogram for dividing cells by CellTrace dilution (left). The numbers in histograms are percent of dividing cells among CD19+ B cells. Graphs shown (right) are the mean percent ± SEM of the number of dividing cells in naive (top) and memory (bottom) B cells after 5 d stimulation with the indicated conditions. (B) Representative dot plot for gating of plasmablast and nonplasmablasts. CD19+ B cells were gated as plasmablast (CD20loCD38hi) and nonplasmablast (CD20hiCD38lo), and analyzed for proliferation by CellTrace dilution. (C) Graphs are shown as the mean percent ± SEM of dividing cells in nonplasmablast (left) and plasmablast (right) from naive (top) and memory (bottom) B cells under the indicated conditions on day 5. Representative histograms from the data are shown below the graphs. Statistical comparisons were made with one-way ANOVA followed by Dunnett’s multiple-comparison test. Naive B cells: n = 12; memory B cells: n = 6. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

FIGURE 3.

BAFF induces IgM and IgG production from memory, but not naive, B cells. Naive (A) and memory (B) B cells were cultured as described in Fig.1. IgM and IgG in culture supernatant from indicated conditions were measured by ELISA on day 5. Graphs are shown as mean concentration ± SEM. n = 8 for naive, n = 9 for memory B cells. One-way ANOVA compared with IL-2/IL-21–only condition. *p < 0.05, **p < 0.005, ***p < 0.001.

FIGURE 4.

DN B cells display memory phenotype responses to BAFF. Normal PBMCs were analyzed by flow cytometry to assess the difference between naive and DN B cells. (A) Gating scheme and the representative plots for the four subpopulations: DN, naive, SwM, and NSM. (B) Shown are mean percent ± SEM of B cells expressing IgG (left) or IgM (right) in each population (n = 10). (C) Mean fluorescence intensity (MFI) ± SEM of indicated surface molecules is shown. Statistical comparisons were made with one-way ANOVA followed by Dunnett’s multiple-comparison test (n = 10 donors). (D) CD27−IgD+ naive and DN B cells were sorted based on CD27 and IgD expression and cultured with differentiation mixture used in previous experiments for 5 d. Shown are representative plots on day 5. (E) The plasmablast differentiation by IL-2, IL-21, and CD40L (top) or by IL-2, IL-21, and BAFF (bottom) from naive or DN B cells is shown by percent ± SEM in each population. Student t test between naive and DN B cells (n = 5). (F) Culture supernatants were collected on day 5, and IgG and IgM secretion were measured by ELISA. Student t test between naive and DN B cells (n = 5). *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

FIGURE 5.

B cell subpopulation alterations and Aiolos overexpression in SLE. (A) PBMCs were isolated from buffy coat of HC and SLE donors, and phenotype was analyzed by flow cytometry. The percent of CD27− B cells (left) and IL-21R+ B cells (middle) among B cells were shown as mean ± SEM. The correlation of CD27 and IL-21R expression is shown as a representative histogram (right). (B) PBMCs from HCs and SLE patients were analyzed by flow cytometry. The mean percent ± SEM of four subpopulations in HC and SLE are shown. (C) Aiolos and Ikaros expression in the indicated B cell subpopulations are shown as mean fluorescence intensity (MFI) ± SEM. (D) Expressions of indicated B cell markers in indicated populations are shown as MFI ± SEM. Student t test between HC and SLE B cells (n = 5): *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

FIGURE 6.

CC-220 reduces B cell Aiolos and Ikaros expression in HC and SLE patients independent of IL-21 STAT3 signaling. (A) Chemical structure of CC-220. (B) B cells from HC were stimulated with CD40L and anti-IgM for 24 h. Cells were then treated with DMSO or CC-220 (10 nM) for 1 h at 37°C, then exposed to IL-21 for 15 min and p-STAT3 levels determined in CD19+CD27lo naive B cells (left) or CD19+CD27hi memory B cells (right) by flow cytometry. (C) B cells from HCs were treated with the indicated amounts of CC-220 for 0.25, 2, 6, and 24 h at 37°C, and expression of Aiolos and Ikaros were measured by Western blot. (D) Inhibition of Aiolos (top) and Ikaros (bottom) expression by CC-220 in indicated populations are shown as mean percent ± SEM relative to untreated HC Aiolos or Ikaros expression (n = 10). Statistical comparisons are by one-way ANOVA and Dunnett’s multiple-comparison test compared with HC vehicle control: *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

FIGURE 7.

CC-220 reduces mRNA levels of B cell differentiation transcription factors. Naive and memory B cells were cultured as described in Fig. 1. Cells were harvested on day 5, and mRNA was isolated for quantitation by quantitative RT-PCR. The effects of CC-220 on gene expression related to B cell differentiation in plasmablasts from naive (A) and memory (B) B cells are measured as relative expression to vehicle-treated samples. The graphs shown are mean relative expression to vehicle control ± SEM (n = 7–8), one-way ANOVA followed by Dunnett’s multiple-comparison test: *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

FIGURE 8.

CC-220 inhibits B cell proliferation and plasmablast differentiation. Naive (A) and memory (B) B cells were treated with vehicle or CC-220 (1, 10, and 100 nM) for 1 h before adding indicated stimuli and cultured for 5 d. Proliferation, plasmablast differentiation, and cell viability were assessed 5 d later by flow cytometry. Effects of CC-220 are shown as percent of vehicle control, mean ± SEM. (C) Representative plasmablast differentiation and proliferation plots for naive B cells treated with CC-220. (D) Representative plasmablast differentiation and proliferation plots for memory B cells treated with CC-220. (E) Comparison of CC-220 effects on naive and memory B cell plasmablast differentiation induced by IL-2/IL-21/CD40L. Statistics were Student t test comparison of naive and memory B cells on plasmablast differentiation at the same concentration of CC-220 treatment. (F) Comparison of CC-220 effects on memory B cell plasmablast differentiation induced by IL-2/IL-21/CD40L or IL-2/IL-21/BAFF. Statistics were Student t test comparison of CC-220 effects on plasmablast differentiation at each concentration between IL-2/IL-21/CD40L and IL-2/IL-21/BAFF treatment. (E and F) n = 6. *p < 0.05, **p < 0.005.

FIGURE 9.

Differential potency effects of CC-220 on IgM and IgG production from naive and memory B cells. Naive and memory B cells were cultured for 5 d under the indicated stimulation conditions in the absence and presence of CC-220 and assessed for IgM (A) and IgG (B) production. Graphs shown are plotted as the mean percent of vehicle control (naive or memory B cells in the absence of CC-220) ± SEM. (C) CC-220 potency comparison between memory B cells stimulated with either IL-2/IL-21/BAFF or IL-2/IL-21/CD40L and production of IgM (left) and IgG (right) after 5 d of culture. Statistics were Student t test comparison of naive versus memory B cell IgM or IgG production at the same concentration of CC-220 treatment. n = 8 for naive, n = 9 for memory B cells. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

FIGURE 10.

CC-220 inhibits plasmablast differentiation and Ab production from DN B cells. DN B cells from HC PBMCs were sorted based on CD19+CD27− and IgD− expression, and treated with vehicle or CC-220 (10 nM) for 1 h at 37°C before adding the stimuli indicated and culturing for 5 d. (A) Data shown are the percent of plasmablasts and cell viability ± SEM relative to the total cells present after 5 d of treatment. (B) IgG (left) and IgM (right) production in the culture supernatant from DN cells were measured on day 5. Inhibition by CC-220 is shown as the percent of stimulated cells in the absence of CC-220 treatment ± SEM: IL-2/IL-21 only (black bars), IL-2/IL-21/CD40L (white bars), or IL-2/IL-21/BAFF (gray bars) (n = 5). Student t test: *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

FIGURE 11.

CC-220 inhibits SLE plasmablast differentiation and Ab production from BAFF activated B cells. Total B cells isolated from HCs and SLE patients were treated with CC-220 (10 nM) for 1 h at 37°C before adding IL-2/IL-21/BAFF and culturing for 5 d. (A) Induced differentiated plasmablasts defined as percent CD20loCD38hi cells of total CD19+ B cells. IgG (B) and IgM (C) production after 5 d in culture. (D) Viability of cells posttreatment with CC-220 as a percent of the corresponding vehicle control (HC, SLE). Graphs shown are plotted as the mean percent of the corresponding vehicle control (HC or SLE B cells in the absence of CC-220) ± SEM. n = 13 HCs, n = 7 SLE patients. ** p < 0.005, ****p < 0.0001 for CC-220–treated cells versus their corresponding vehicle-treated cells (HC or SLE). NS, not significant comparing CC-220–treated HC versus CC-220–treated SLE cells.