FcγRIIB regulates autoantibody responses by limiting marginal zone B cell activation
Ashley N. Barlev, Susan Malkiel, Izumi Kurata-Sato, Annemarie L. Dorjée, Jolien Suurmond, Betty Diamond
Ashley N. Barlev, Susan Malkiel, Izumi Kurata-Sato, Annemarie L. Dorjée, Jolien Suurmond, Betty Diamond
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Research Article Autoimmunity Immunology

FcγRIIB regulates autoantibody responses by limiting marginal zone B cell activation

Abstract

FcγRIIB is an inhibitory receptor expressed throughout B cell development. Diminished expression or function is associated with lupus in mice and humans, in particular through an effect on autoantibody production and plasma cell (PC) differentiation. Here, we analyzed the effect of B cell–intrinsic FcγRIIB expression on B cell activation and PC differentiation. Loss of FcγRIIB on B cells in Fcgr2b–conditional KO (Fcgr2b-cKO) mice led to a spontaneous increase in autoantibody titers. This increase was most striking for IgG3, suggestive of increased extrafollicular responses. Marginal zone (MZ) B cells had the highest expression of FcγRIIB in both mice and humans. This high expression of FcγRIIB was linked to increased MZ B cell activation, Erk phosphorylation, and calcium flux in the absence of FcγRIIB triggering. We observed a marked increase in IgG3+ PCs and B cells during extrafollicular PC responses in Fcgr2b-cKO mice. The increased IgG3 response following immunization of Fcgr2b-cKO mice was lost in MZ-deficient Notch2 Fcgr2b–double KO mice. Importantly, patients with systemic lupus erythematosus (SLE) had a decrease in FcγRIIB expression that was strongest in MZ B cells. Thus, we present a model in which high FcγRIIB expression in MZ B cells prevented their hyperactivation and ensuing autoimmunity.

Authors

Ashley N. Barlev, Susan Malkiel, Izumi Kurata-Sato, Annemarie L. Dorjée, Jolien Suurmond, Betty Diamond

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Figure 4

The effect of FcγRIIB on B cell signaling in MZ B cells.

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The effect of FcγRIIB on B cell signaling in MZ B cells. (A) Simplified ...
(A) Simplified schematic diagram of key signaling molecules downstream of the BCR and FcγRIIB. (B–D) Representative examples and summary of FO and MZ B cells from control mice; cells were stimulated with 3 μg/mL intact anti-IgM (αIgM) or equimolar concentrations (2 μg/mL) of Fab′2 anti-IgM for 10 minutes, followed by Phosphoflow analysis of the phosphorylation of signaling molecules. (E) SHIP1 phosphorylation was analyzed by capillary Western blotting. (F and G) Representative examples and summary of p-Erk expression in FO and MZ B cells from control and Fcgr2b-cKO mice; cells were stimulated with intact anti-IgM as described in B–D. (H–J) PBMCs from healthy donors were stimulated with 15 μg/mL intact anti-IgM or equimolar concentrations (10 μg/mL) of Fab′2 anti-IgM for 60 minutes, after which the phosphorylation of signaling molecules was analyzed by Phosphoflow. (H) Representative example of Syk and Erk phosphorylation in MZ-like B cells. (I and J) Comparison of intact versus Fab′2 anti-IgM in naive and MZ-like B cells. The median percentage of inhibition calculated per donor is indicated. (K–M) Calcium flux following 75 μg/mL intact anti-IgM or equimolar concentrations (50 μg/mL) of Fab′2 anti-IgM. Peak calcium flux and the AUC were calculated using FlowJo. (K) Representative examples of calcium flux in naive and MZ-like B cells. (L and M) Comparison of the peak and AUC of calcium flux following intact versus Fab′2 anti-IgM in naive and MZ-like B cells. inh, inhibition. Data are shown as the median, with each symbol representing an individual mouse (n = 4–7 mice per group for B–D; n = 8 mice per group for G; n = 6 mice per group for H–J; n = 5 mice per group for L and M; data for each were pooled from 2–3 independent experiments). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA with Bonferroni’s post hoc test.