In vitro-Induced Human IL-10+ B Cells Do Not Show a Subset-Defining Marker Signature and Plastically Co-express IL-10 With Pro-Inflammatory Cytokines

Abstract

Regulatory B cells (Breg) have been described as a specific immunological subsets in several mouse models. Identification of a human counterpart has remained troublesome, because unique plasma membrane markers or a defining transcription factor have not been identified. Consequently, human Bregs are still primarily defined by production of IL-10. In this study, we sought to elucidate if in vitro-induced human IL-10 producing B cells are a dedicated immunological subset. Using deep immune profiling by multicolor flow cytometry and t-SNE analysis, we show that the majority of cells induced to produce IL-10 co-express pro-inflammatory cytokines IL-6 and/or TNFα. No combination of markers can be identified to define human IL-10⁺TNFα^-IL-6^- B cells and rather point to a general activated B cell phenotype. Strikingly, upon culture and restimulation, a large proportion of formerly IL-10 producing B cells lose IL-10 expression, showing that induced IL-10 production is not a stable trait. The combined features of an activated B cell phenotype, transient IL-10 expression and lack of subset-defining markers suggests that in vitro-induced IL-10 producing B cells are not a dedicated subset of regulatory B cells.

Keywords: B cell activation; B cell plasticity; Breg; IL-10; IL-6; cytokine co-expression; immune regulation; t-SNE analysis.

Figure 1

Most in vitro-induced human IL-10⁺ B cells also express TNFα and/or IL-6. (A,B) Total CD19⁺ B cells were isolated from blood and stimulated with CpG (1.25 μM) for 1–4 days followed by restimulation with PMA/Ionomycin/BFA (PIB) (n = 6). Representative dot plots are shown in (A). (C) Induction of IL-10 by TLR stimuli. B cells were stimulated by 1.25 μM CpG, 1 μg/ml R848 and 20 μg/ml poly:IC to activate TLR9, TLR7/8, and TLR3, respectively (n = 9). (D) IL-10 induction in isolated CD19⁺ B cells stimulated for 4 days with 1.25 μM CpG, CD40L-expressing 3T3 cells (used in a ratio of 1 3T3-CD40L cell to 50 B cells), anti-IgM + anti-IgG beads (used in a ratio of 2 beads to 1 B cell) or left unstimulated and restimulated with PIB (n = 9). (E,F) Representative dot plots of IL-10, TNFα, and IL-6 producing B cells after 2 days (E) and 4 days of stimulation (F) with 1.25 μM CpG and PIB. (G) CLSM image of a CpG-stimulated B cell co-expressing TNFα and IL-10. Green: HLA-DR, blue: DAPI nuclear stain, red: TNFα, light blue: IL-10. Right panel: overlay. (H) Relative frequency at different time points of IL-10⁺TNFα⁻IL-6⁻, IL-10⁻TNFα⁺IL-6⁺, and IL-10⁺TNFα⁺IL-6⁺ cells after stimulation with 1.25 μM CpG and PIB (n = 6). Friedman test was performed. *p < 0.05. **p < 0.01. (I,J) Relative distribution of IL-10⁺TNFα⁻IL-6⁻ (white), IL-10⁺TNFα⁻IL-6⁺, IL-10⁺TNFα⁺IL-6⁻ (gray) and IL-10⁺TNFα⁺IL-6⁺ (black) B cells within IL-10⁺ B cells stimulated with 1.25 μM CpG at different time points (n = 7). (I) Averages of IL-10⁺ B cell subpopulations of seven donors are depicted as pie charts. (J) IL-10⁺ B cell subpopulations of individual donors are represented. Friedman test was performed. *p < 0.05. (B–D,H,J) Each dot/line represents 1 donor.

Figure 2

IL-10, TNFα, and IL-6 production by transitional B cells and plasmablasts after B cells were stimulated with CpG. (A,F) Frequency and number of transitional B cells (A) and plasmablasts (F) were quantified 2 and 4 days after B cells were stimulated with 1.25 μM CpG (n = 7). Representative dot plots are depicted. Each dot/line represents 1 donor. (B,G) Frequency of cytokine-producing cells within transitional B cell (B) and plasmablast populations (G) (n = 7). (C–E) Relative distribution of (C) IL-10⁺TNFα⁻IL-6⁻ (light blue), IL-10⁺TNFα⁻IL-6⁺ (blue), IL-10⁺TNFα⁺IL-6⁻ (lavender blue), IL-10⁺TNFα⁺IL-6⁺ (dark blue) B cells within IL-10⁺ transitional B cells; (D) IL-10⁻TNFα⁺IL-6⁻ (pink), IL-10⁻TNFα⁺IL-6⁺ (light red), IL-10⁺TNFα⁺IL-6⁻ (red) and IL-10⁺TNFα⁺IL-6⁺ (bordeaux) B cells within TNFα⁺ transitional B cells; (E) IL-10⁻TNFα⁻IL-6⁺ (white), IL-10⁻TNFα⁺IL-6⁺ (light gray), IL-10⁺TNFα⁻IL-6⁺ (gray) and IL-10⁺TNFα⁺IL-6⁺ (black) B cells within IL-6⁺ transitional B cells stimulated with 1.25 μM CpG at different time points (n = 7). (H) Relative distribution of IL-10⁻TNFα⁺IL-6⁻ (pink), IL-10⁻TNFα⁺IL-6⁺ (light red), IL-10⁺TNFα⁺IL-6⁻ (red) and IL-10⁺TNFα⁺IL-6⁺ (bordeaux) B cells within TNFα⁺ plasmablasts stimulated with 1.25 μM CpG (n = 7). Wilcoxon tests were performed. *p < 0.05.

Figure 3

t-SNE clustering on IL-10, TNFα, and IL-6 within B cells stimulated with CpG for 2 days and the expression of co-stimulatory markers. t-SNE 2D scatter plot of 91,000 living single CD19⁺ B cells stimulated for 2 days with CpG and stained with staining mixture 1 containing antibodies against IL-10, TNFα, IL-6, CD86, CD27, CD25, CD24, CD40, CD38, CD80, and CD19 (n = 7). t-SNE settings were as followed: perplexity 70; theta 0.5; eta 200 and iterations 1,000. IL-10, TNFα, and IL-6 were selected as t-SNE parameters. Different colors depict the IL-10⁺ (blue), TNFα⁺ (red) and IL-6⁺ (dark gray) B cells. (A–C) Five IL-10⁺TNFα⁻IL-6⁻ (A), six IL-10⁺TNFα⁺IL-6⁺ (B), and two IL-10⁺TNFα⁻IL-6⁻ and IL-10⁺TNFα⁺IL-6⁺ (C) B cells were gated within the t-SNE 2D scatter plot, selected populations are depicted in a separate t-SNE 2D scatter plot and expression of various markers within these populations (represented as color spectrum of gray and black) were depicted in half-off set histograms.

Figure 4

t-SNE clustering on IL-10, TNFα, and IL-6 within B cells stimulated with CpG for 2 days and the expression of B_reg associated markers. t-SNE 2D scatter plot of 91,000 living single CD19⁺ B cells stimulated for 2 days with CpG and stained with staining mixture 2 containing antibodies against IL-10, TNFα, IL-6, CD48, CD1d, CD5, CD24, CD21, CD38, CXCR5, and CD19 (n = 7). t-SNE settings were as followed: perplexity 70; theta 0.5; eta 200 and iterations 1,000. IL-10, TNFα, and IL-6 were selected as t-SNE parameters. Different colors depict the IL-10⁺ (blue), TNFα⁺ (red) and IL-6⁺ (dark gray) B cells. (A–C) Five IL-10⁺TNFα⁻IL-6⁻ (A), six IL-10⁺TNFα⁺IL-6⁺ (B) and two IL-10⁺TNFα⁻IL-6⁻ and IL-10⁺TNFα⁺IL-6⁺ (C) B cells were gated within the t-SNE 2D scatter plot, selected populations are depicted in a separate t-SNE 2D scatter plot and expression of various markers within these populations (represented as color spectrum of gray and black) were depicted in half-off set histograms.

Figure 5

CD21, CD80, and CD5 are not unique markers for CpG-induced IL-10⁺TNFα⁻IL-6⁻ B cells. Total CD19⁺ B cells were isolated from blood and stimulated with CpG (1.25 μM) for 2 days followed by restimulation with PMA/Ionomycin/BFA (PIB) (n = 7). All live CD19⁺ cells were analyzed using flow cytometry. (A–C) Median expression of CD21, CD80, and CD5 on IL-10⁺TNFα⁺IL-6⁺ (white bar) and IL-10⁺TNFα⁻IL-6⁻ (gray bar) B cells. (B,C) Frequency of CD80⁺ and CD5⁺ B cells within IL-10⁺TNFα⁺IL-6⁺ and IL-10⁺TNFα⁻IL-6⁻ B cells. (D–F) Median expression of CD21, CD80, and CD5 on IL-10⁺TNFα⁺IL-6⁺ (white bar), various cytokine double-positive (IL-10⁻TNFα⁺IL-6⁺, IL-10⁺TNFα⁺IL-6⁻, IL-10⁺TNFα⁻IL-6⁺; as depicted) and IL-10⁻TNFα⁺IL-6⁻ (gray bar) and IL-10⁻TNFα⁻IL-6⁺ B cells (gray bar). (E,F) Frequency of CD80⁺ and CD5⁺ B cells within IL-10⁺TNFα⁺IL-6⁺ (white bar), various cytokine double-positive (IL-10⁻TNFα⁺IL-6⁺, IL-10⁺TNFα⁺IL-6⁻, IL-10⁺TNFα⁻IL-6⁺; as depicted) and IL-10⁻TNFα⁺IL-6⁻ (gray bar) and IL-10⁻TNFα⁻IL-6⁺ B cells (gray bar). Wilcoxon tests were performed. *p < 0.05. (G–I) Median expression of CD21, CD80 and CD5 on IL-10⁺TNFα⁻IL-6⁻ (gray bar), IL-10⁻TNFα⁺IL-6⁻ (gray bar) and IL-10⁻TNFα⁻IL-6⁺B cells (gray bar). (H,I) Frequency of CD80⁺ and CD5⁺ B cells within IL-10⁺TNFα⁻IL-6⁻ (gray bar), IL-10⁻TNFα⁺IL-6⁻ (gray bar) and IL-10⁻TNFα⁻IL-6⁺B cells (gray bar). Representative histograms are shown. Friedman test was performed. *p < 0.05. **p < 0.01. Each dot/line represents 1 donor.

Figure 6

IL-10 expression by in vitro-stimulated human B cells is a transient trait. IL-10⁺ (dark gray) and IL-10⁻ (light gray) B cells were sorted on day 3 and re-cultured in B cell medium for 3 days. After PIB restimulation, IL-10 expression was assessed using intracellular staining (B; n = 3). Representative dot plots are shown (A). Black bar depicts IL-10⁺ B cells before sort. Friedman test was performed. *p < 0.05.