The IgM receptor FcμR limits tonic BCR signaling by regulating expression of the IgM BCR

Abstract

The FcμR receptor for the crystallizable fragment (Fc) of immunoglobulin M (IgM) can function as a cell-surface receptor for secreted IgM on a variety of cell types. We found here that FcμR was also expressed in the trans-Golgi network of developing B cells, where it constrained transport of the IgM-isotype BCR (IgM-BCR) but not of the IgD-isotype BCR (IgD-BCR). In the absence of FcμR, the surface expression of IgM-BCR was increased, which resulted in enhanced tonic BCR signaling. B-cell-specific deficiency in FcμR enhanced the spontaneous differentiation of B-1 cells, which resulted in increased serum concentrations of natural IgM and dysregulated homeostasis of B-2 cells; this caused the spontaneous formation of germinal centers, increased titers of serum autoantibodies and excessive accumulation of B cells. Thus, FcμR serves as a critical regulator of B cell biology by constraining the transport and cell-surface expression of IgM-BCR.

Figure 1. The FcμR is expressed by various cell subsets

(a) Fcmr mRNA expressions in different B cell subsets in spleen and peritoneal cavities (perc): marginal zone (MZ), follicular (FO), spleen B-1, and perc B-1 cells. Each symbol represents values from one mouse (n=3 mice). (b) Surface FcμR expression in different B cell subsets in spleen, perc, and inguinal lymph node (pLN) (n=6 mice) (c) Fcmr mRNA expression by different B cell developmental stages (n = 4–10 samples/group, each sample was sorted from BM of 2 mice). (d) Surface FcμR expression by pro- and early pre- B cells, late pre-, immature and mature B cells (n=6 mice). (e) Confocal microscopy of immature B cells stained with FcμR (green) and TGN-38 (blue). White bars indicate 2 μm scale bars. (f) Relative expression of Fcmr mRNA of purified Fcmr ^flx/flxCd19-Cre and control B cells (n=6 mice/group). Data are combined from 3 independent experiments (c) or are representative of at least two independent experiments (a–b, d–f) (mean ± SD in a–d, f). *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test.

Figure 2. IgM FcμR interaction occurs with both secreted and membrane IgM

(a) Shown are histogram plots of splenic B cells from control and Fcmr ^flx/flxCd19-Cre mice incubated or not with biotinylated IgM and stained with streptavidin-PE. (b) Splenocytes from Fcmr ^flx/flxCd19-Cre and control mice (IgH^b) were i.v transferred into C6.IgH^a mice (n=3 mice/group). Shown are 5% contour plots with outliers gated on live CD19⁺ B cells, identifying transferred B cells as IgD^a− and IgD^b+. (c) Overlay histograms comparing IgM^a (in vivo sIgM binding) and IgM^b (membrane IgM) expression of transferred B cells (IgD^a− IgD^b+) from Fcmr ^flx/flxCd19-Cre and control mice 3 days after transfer. Graph summarizes mean fluorescence intensities ± SD of IgM^a and IgM^b staining of transferred B cells. (d) Fold-difference in MFI from IgM^b (mIgM) of Fcmr ^flx/flxCd19-Cre B cells compared to controls. (e,f) Follicular B cells unable to secrete IgM (μ_s ^−/−) were isolated from mixed BM μ_s^−/− (IgM^a) and WT (IgM^b) chimeras. (e) STED microscopy of μ_s^−/− B cells stained for sIgM (IgM^b-green) and mIgM (IgM^a-red). (f) Confocal microscopy of μ_s ^−/− follicular B cells stained for FcμR (green), mIgM (IgM^a, red), and sIgM (IgM^b, blue). White arrows identify co-localization of FcμR with mIgM or sIgM. (g) STED microscopy of immature B cells stained for FcμR (green) and IgM (red). (h) Confocal microscopy of immature B cells stained with FcμR (green), IgM (red) and TGN-38 (blue). Scale bars 2μm. (i–j) Quantified box-and-whisker-plots (min, max, median, quartiles) and microscopic representation of Proximal ligation assay (PLA) of GFP-sorted (i) BM immature (n=250 cells) and (j) splenic mature B cells (n=750 cells) treated with or without saponin prior to PLA, as indicated. Red indicates FcμR-IgM interaction. Green shows staining of Golgi network using labeled lectin-GSII staining (J-top, K-top) or GM-1 staining indicating the cell membrane, using labeled cholera-toxin subunit B (J-bottom, K-bottom). DAPI stained nuclei presented in blue. Scale bar 2μm. Data are representative of at least two independent experiments. *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test (a–c) or Mann-Whitney test (i–j).

Figure 3. FcμR-mIgM interactions constrain BCR surface expression

(a) Overlay histogram flow cytometry plots comparing total surface IgM staining (membrane plus secreted IgM binding), on splenic CD19⁺ B cells from Fcmr ^flx/flxCd19-Cre and control mice. (b) Overlay histograms comparing IgM (membrane IgM) expression on B cells (IgD^a− IgD^b+) from Fcmr ^flx/flxCd19-Cre and control mice 3 days after transfer into IgH^a mice. (c) Mean fluorescence intensities ± SD of staining for Igα (CD79a) in total spleen B cells from Fcmr ^flx/flxCd19-Cre and control mice (n=4 mice/group). (d) Mean relative expression of membrane IgM (BCR) mRNA ± SD of purified Fcmr ^flx/flxCd19-Cre and control B cells (n=4 mice/group). Data are representative of at least two independent experiments. (e) Model of the GFP-μm heavy chain (GFP-μmHc) and the assembled chimeric IgM-BCR containing the CD79a and CD79b signaling subunits, the mcherry-δmHc and the assembled chimeric IgD-BCR expressed in S2 cells. (f) Shown are flow cytometry histogram plots of Drosophila S2 cells stained for IgM or IgD. The cells expressed GFP, GFP-μmHc, mcherry-δmHc, IgM-BCR ± FcμR, IgD-BCR ± FcμR. (g) Box-and-whisker-plots (min, max, median, quartiles) summarize MFI of surface IgM (left) and surface IgD (right) expression by S2 cells after transfection. (h,i) Shown are IgM (left) and IgD (right) re-expression by transfected S2 cells after photobleaching (FRAP) (n=50 cells). The cells expressed the IgM-BCR as GFP-coupled chimera described in (e) and the IgD-BCR GFP-chimera with or without the FcμR. (h) Mean ratios of fluorescence staining of the mobile fraction (Mf) ± SD. (i) Box-and-whisker-plots (min, max, median, quartiles) summarize the half-time recovery. Data are representative of at least two independent experiments (a–d, h–i). Data are combined from five independent experiments (f–g). n.s. not significantly different, *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test (c,d,i), Wilcoxon matched-pairs signed rank test (g), or Mann-Whitney test (h, ***p<0.0001 for IgM-BCR, and n.s for IgD-BCR).

Figure 4. Enhanced tonic BCR signaling in Fcmr^−/− B cells

(a–c) MFI ± SD of ex vivo staining for expression of (a) PI3K-p85 and (b) PI3K-p110 by flow cytometry. (c) Overlay histograms (left) and graph summarizing MFI ± SD of phospho (p)Akt expression by total B cells from Fcmr ^flx/flxCd19-Cre mice and Fcmr ^flx/flx Cre-negative controls (right). (d) pAkt expression levels in total spleen B cells, FO B (B-2) and B-1 cells. (e) Overlay histograms of phospho (p)Btk expression by total B cells from indicated mice (left). Graph summarizing MFI ± SD of phospho (p)Btk expression. (f) pBtk expression in total spleen B cells, FO B (B-2) and B-1 cells from Fcmr ^flx/flxCd19-Cre and control mice (n=3–4 mice/group). (g) 5% contour plots with outliers gated on lived splenic B-1 cells from Fcmr ^flx/flxCd19-Cre and control mice, showing the correlation of surface IgM and pAkt expression. (h) mRNA expression levels of FOXO target genes Rag1, Aicda, Cdkn1b, and Bcl2l11 in Fcmr ^flx/flxCd19-Cre and control B cells after normalization to Gapdh (n=4 mice/group). (i) FO B cells isolated from spleens of Fcmr ^flx/flxCd19-Cre and control mice labeled with CFSE and stimulated with anti-IgM for 72h. Numbers are rounds of cell divisions. (j) pAkt expression levels (ΔMFI= mean fluorescent intensity (MFI) – MFI fluorescence minus one (FMO) control) after 72h culture with/without anti-IgM. (k) FO B-2 cells isolated from Fcmr ^flx/flxCd19-Cre and control spleens stimulated as indicated for 72h. Frequencies of live cells as determined by 7-AAD staining. (l) Frequencies of live (7-AAD⁻) B-1 cells isolated from Fcmr ^flx/flxCd19-Cre and control spleens and stimulated as indicated for 48h (n=3 samples pooled from 2 mice (k–l)). Each data set is representative of at least two independent experiments (mean ± SD in a–f, h, j–l). *p<0.05, **p<0.005 by unpaired two-tailed Student’s t-test.

Figure 5. Increased influenza-protective natural IgM and autoantibody production in Fcmr ^flx/flxCd19-Cre mice

Concentrations of (a) IgM, (b) IgA, (c) IgG in Fcmr ^flx/flxCd19-Cre and control sera from 3 months old mice (n=10–18 mice/group). (d) Frequencies of IgM and (e) IgG secreting cells in spleens and BMs of these mice (n=4 mice/group). (f) Relative amounts of IgM and (g) IgG anti-dsDNA and relative amounts of anti-dsDNA/mg Ig per μg total Ig in Fcmr ^flx/flxCd19-Cre and control sera (n=10–12 mice/group). (h) Overlay histograms of Fas expression by B cells from indicated mice (left). Graph summarizing results (right) (n=2 mice/group). (i) 5% contour plots with outliners gated on live CD19⁺ B cells. Gate and numbers identify CD38^low PNA⁺ germinal center (GC) B cells. Graphs summarize frequencies ± SD GC B cells of total cells in Fcmr ^flx/flxCd19-Cre and control spleens (n=4–5 mice/group). (j) Relative amounts influenza A/Puerto Rico/8/34-binding IgM in sera from naïve Fcmr ^flx/flxCd19-Cre and control mice (n=15–18 mice/group). (k) Relative amounts of influenza A/PR8-binding and (l) total IgM in sera of Fcmr ^flx/flxCd19-Cre, and control mice one day after A/PR8 infection (n=8, 14 mice/group). Data are representative of at least two independent experiments. (m) Lung viral loads at 1 day after A/PR8 infection. For Figure k, l and m data are combined from three independent experiments (mean ± SD in a–m). *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test.

Figure 6. B-1 cells are responsible for increased natural IgM secretion in Fcmr ^flx/flxCd19-Cre mice

(a) 5% contour plots with outliers gated for live CD19⁺ spleen B cells from control and Fcmr ^flx/flxCd19-Cre mice. B-1 cells were identified as: CD43⁺ IgM^hi CD19^hi and CD5^+/− (B-1a/B-1b). Graph summarizes numbers of B-1 cells, including B-1a and B-1b cells in spleen, (b) peritoneal cavity and (c) BM (n=4 mice/group). (d–g) Generation of chimeras with Fcmr^−/− B-1 (IgH^b) and wild-type B-2 (IgH^a) cells and controls (n=4 mice/group). (d) (left) 5% contour plots with outliers gated for lived CD19⁺ B cells from peritoneal cavities and spleens. For BM, cells were pre-gated for B-1 cells (CD19⁺ IgM⁺ CD43⁺). The transferred B-1 cells were identified as IgM^b+ IgM^a−. Graph provides summary of IgM^b B-1 cell frequencies in indicated tissues. (e) 5% contour plots with outliers gated on IgM^b+ B-1 cells in spleen. Gates and numbers identify frequencies of CD138⁺ of IgM^b+ B-1 cells. FMO, fluorescence minus one control. (f) Graphs summarize the frequencies of IgM^b secreting B-1 (left) and IgM^a secreting B-2 cells (right) in spleen, and BMs. (g) Serum IgM^b levels in the chimera. Data are representative of at least two independent experiments (a–c) (mean ± SD in a–d, f–g). *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test.

Figure 7. Increased B cell turnover and cell survival of B cells result in B cell lymphocytosis

(a) 5% contour plots with outliers of representative spleen samples (n=4 mice/group) from Fcmr ^flx/flxCd19-Cre and control mice analyzed by flow cytometry after gating on live CD19⁺ B cells. Identified subsets: CD21^hiCD23⁻ marginal zone B cells (MZ); CD21^intCD23⁺ follicular B cells (FO) (left). Graph summarizes frequencies (middle) and numbers (right) of MZ and FO in Fcmr ^flx/flxCd19-Cre and control spleens. (b–c) Cell cycle/turnover analysis of CD19⁺ live spleen B cells from Fcmr ^flx/flxCd19-Cre and control mice (n=4 mice/group). (b) 5% contour plots with outliers of live CD19⁺ B cells from indicated mice, identifying cell cycle stages by Ki67 and 7-AAD staining (G0: Ki67⁻ 7AAD⁻, G1: Ki67⁺ 7AAD⁻; G2/S/M: Ki67⁺ 7AAD⁺). Graph summarizes frequencies (n = 4 mice/group). (c) B-2 and B-1 cell turnover was determined by flow cytometry 24h after i.p. injection of BrdU. Shown are frequencies of BrdU⁺ cells (n=4 mice/group). (d) 5% contour plots with outliers gated on 7AAD⁺ (dead cells) among splenic live CD19⁺ CD43⁺ IgM^hi IgD^lo B-1 cells (left). Shown are frequencies of 7AAD⁺ cells (n=4 mice/group; right). (e) Total spleen (left) and B cell counts (right) of indicated mice (n=8 mice/group). (f) Photo of representative spleens from 3-months old control and Fcmr ^flx/flxCd19-Cre mice. (g) H&E staining of spleens from 8-months old C57BL/6 and Fcmr ^flx/flxCd19-Cre mice. 2X scale bar is 500μm, 10X scale bar is 100μm. Data are representative of at least two independent experiments. Data are combined from two independent experiments (f) (mean ± SD in a–e). *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test.