Secreted IgM deficiency leads to increased BCR signaling that results in abnormal splenic B cell development

Abstract

Mice lacking secreted IgM (sIgM ^-/-) antibodies display abnormal splenic B cell development, which results in increased marginal zone and decreased follicular B cell numbers. However, the mechanism by which sIgM exhibit this effect is unknown. Here, we demonstrate that B cells in sIgM ^-/- mice display increased B cell receptor (BCR) signaling as judged by increased levels of phosphorylated Bruton's tyrosine kinase (pBtk), phosphorylated Spleen tyrosine kinase (pSyk), and nuclear receptor Nur77. Low dosage treatment with the pBtk inhibitor Ibrutinib reversed the altered B cell development in the spleen of sIgM ^-/- mice, suggesting that sIgM regulate splenic B cell differentiation by decreasing BCR signaling. Mechanistically, we show that B cells, which express BCRs specific to hen egg lysozyme (HEL) display diminished responsiveness to HEL stimulation in presence of soluble anti-HEL IgM antibodies. Our data identify sIgM as negative regulators of BCR signaling and suggest that they can act as decoy receptors for self-antigens that are recognized by membrane bound BCRs.

Figure 1

sIgM deficiency results in altered splenic B cell development. (a) Representative flow cytometry plots and bar graphs show absolute numbers of FO/T2 (blue), MZ (purple), CD21⁺ CD23⁻ B cells (red), T1 (green) and NF (grey) cells (defined in Fig. S2) of sIgM ^+/+ (light colored bars) and sIgM ^−/− (dark colored bars) mice. (b) Representative flow cytometry plots and dot plots show the frequencies of MZ (purple) and FO/T2 (blue) B cells of sIgM ^+/+ (●) and sIgM ^−/− (▼) mice treated with vehicle and sIgM ^−/− (▲) treated with polyclonal IgM (n = 4–5 mice per group). (c) Absolute numbers of splenic B220^highCD93⁻CD21⁺ CD23⁻ B cells and (d) Blimp-1 mean fluorescence intensity (MFI) in CD21⁺ CD23⁻ B cells of sIgM ^+/+ (light red bar) and sIgM ^−/− (dark red bar) mice analyzed by flow cytometry. (e,f) Representative flow cytometry plots show the percentages of either kappa or lambda light chain positive and bars represent the mean kappa/lambda light chain ratio of (e) B220⁺CD43⁻ splenic cells and (f) mature (B220^high CD43⁻) and immature (B220^low CD43⁻) bone marrow cells within cells that express BCR. (a,c–f) Data shown are from one representative experiment of at least two to three independent experiments with 5–6 mice per group. All results show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Mann-Whitney or unpaired t test or One-Way Anova test followed by Tukey’s test).

Figure 2

sIgM deficiency results in increased B cell receptor signaling in vivo. Representative flow cytometry plots and bar graphs show (a–c) the mean fluorescence intensity (MFI) for pSyk, pBtk levels and Nurr77-GFP expression in FO/T2 (blue), MZ (purple), CD21⁺ CD23⁻ (red), T1 (green) and NF (grey) B cells (as defined in Fig. S2) of sIgM ^+/+ or Nur77-GFP/sIgM ^+/+ (light colored bar) and sIgM ^−/− or Nur77-GFP/sIgM ^−/− (dark colored bar) mice. Data shown are from one representative experiment (a,b) or pooled (c) from two/three independent experiments. (d) Representative flow cytometry plots and dot plots show the frequencies of CD21⁺ CD23⁻ (red), FO (blue) and MZ (purple) B cells within B-2 cells of sIgM ^+/+ (●) and sIgM ^−/− (▼) mice treated with vehicle, and sIgM ^−/− (▲) mice treated with the Btk inhibitor Ibrutinib. All results show mean ± SEM, n = 4–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired or paired t test or One-Way Anova test followed by Tukey’s test).

Figure 3

Ibrutinib treatment limits MZ and favors FO B cell development in sIgM^+/+ mice in vivo. Representative flow cytometry plots and dot plots represent the frequencies of MZ (purple) and FO (blue) B cells within B-2 cells of sIgM ^+/+ mice treated with vehicle (●) or the Btk inhibitor Ibrutinib (▲) for (a) 2 weeks or (b) 3 weeks. All results show mean ± SEM, n = 4–5 mice per group. *P < 0.05, **P < 0.01 (unpaired t test).

Figure 4

Antigen-specific secreted IgM limit self-antigen induced B cell receptor signaling. (a) Quantification of hen egg-white lyoszyme (HEL) specific IgM in the plasma of MD4, RAG1^−/− and wild-type (WT) mice by ELISA. (b) Representative flow cytometry plots and bar graphs represent the mean fluorescence intensity (MFI) for pBtk in purified B-2 (B220⁺ CD43⁻) cells from MD4^+/− mice stimulated with HEL for 3 minutes in presence of either WT (grey), RAG1^−/− (purple) or MD4 plasma (blue). ELISA quantification of (c) HEL-specific and (d) total IgM in MD4 plasma treated with either unconjugated or BSA- or HEL-conjugated microspheres. (e) Representative flow cytometry plots and bar graphs show the MFI for pBtk in purified B-2 cells from MD4^+/− mice stimulated with HEL for 3 minutes in the presence of plasma from MD4 mice that has been treated with either unconjugated or BSA- or HEL-conjugated microspheres. All results show mean ± SEM, ****P < 0.0001 (unpaired t test or One-Way Anova followed by Tukey’s test). n.d.: not detechtable.