Cutting Edge: Redox Signaling Hypersensitivity Distinguishes Human Germinal Center B Cells

Abstract

Differences in the quality of BCR signaling control key steps of B cell maturation and differentiation. Endogenously produced H2O2 is thought to fine tune the level of BCR signaling by reversibly inhibiting phosphatases. However, relatively little is known about how B cells at different stages sense and respond to such redox cues. In this study, we used phospho-specific flow cytometry and high-dimensional mass cytometry (CyTOF) to compare BCR signaling responses in mature human tonsillar B cells undergoing germinal center (GC) reactions. GC B cells, in contrast to mature naive B cells, memory B cells, and plasmablasts, were hypersensitive to a range of H2O2 concentrations and responded by phosphorylating SYK and other membrane-proximal BCR effectors in the absence of BCR engagement. These findings reveal that stage-specific redox responses distinguish human GC B cells.

Figure 1. CD20^hi B cells in human tonsil were sensitive to H₂O₂

(A) Contour plots show gating for CD3⁻ cells and CD3⁺ CD20⁻ T cells in human tonsil. (B) Contour plots show phosphorylated SYK (p-SYK) and p-SFK in CD3⁻ tonsillar B cells left unstimulated or stimulated by 3.3 mM H₂O₂ for 2 minutes. Sensitivity to H₂O₂ in a CD20^hi B cell population is indicated (gray arrows). Plots are representative of three tonsils.

Figure 2. Mass Cytometry Revealed H₂O₂ ‘Responder’ Population as CD20^hi, CD38⁺, and IgD⁻

(A) Contour plots show gating for CD19⁺ B cells and CD3⁺ T cells in human tonsil. (B) Contour plots show p-PLCγ in CD19⁺ tonsil B cells left unstimulated or stimulated by 3.3 mM H₂O₂ for 4 minutes. Cells that were sensitive to H₂O₂ stimulation were labeled responder (R) cells and contrasted with non-responder cells (NR). (C) Heat map shows the median fold change CD20 and CD38 in CD3⁺ T cells, R, and NR subsets.

Figure 3. GC B cells were hypersensitive to H₂O₂

(A) Density dot plots show gating for identification of plasmablasts, GC B cells, memory B cells, and naïve B cells in human tonsils. (B) Histogram overlays show p-SFK in each B cell population (shown in A) following 2 minutes of 3.3 mM of H₂O₂ (n=3, representative data shown). Color denotes median fold change in p-SFK expression compared to unstimulated (0 mM of H₂O₂). (C) Plots illustrate the median fold change in p-PLCγ, p-SYK, and p-SRC in H₂O₂-stimulated conditions compared to the unstimulated condition (arcsinh scale). Each point represents the average of three individual tonsil specimens (n=3) stimulated for 2 minutes with the indicated concentration of H₂O₂, except for the 0.04 mM and 0.12 mM H₂O₂ stimulated conditions (where n=2). Red squares represent GC B cells and blue circles represent naïve B cells. Error bars denote the standard deviation for each point.

Figure 4. SHP-1 expression was heterogeneous within B cell populations

viSNE maps show CD45⁺ leukocytes arranged based on marker expression profiles (see gating in Fig. 2A). Color denotes protein expression, as indicated. (A) Gates were drawn around the main populations identified by viSNE, using protein expression to identify each population. CD19⁺ B cells were subdivided into naïve B cells (CD38⁻CD27⁻IgD⁺), GC B cells (CD20^hiCD38⁺), memory B cells (CD38⁻CD27⁺IgD⁻), and plasmablasts (CD20⁻CD38^hi) and compared to CD3⁺ T cells. One representative tonsil of four analyzed is shown. (B) Box and Whisker plots illustrate expression of SHP-1, CD38, and CD20 proteins across three tonsil specimens for naïve B cells, GC=germinal center B cells, M=memory B cells, P=plasmablasts, and T= T cells. Median of each marker is indicated by a black line. Bars denote the minimum and maximum observed MFI of each marker.