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

Nature Immunology volume 18, pages 321–333 (2017)Cite this article

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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.

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Figure 1: FcμR is expressed by various cell subsets.
Figure 2: The IgM–FcμR interaction occurs with both sIgM and mIgM.
Figure 3: FcμR–mIgM interactions constrain surface expression of the BCR.
Figure 4: Enhanced tonic BCR signaling in Fcmrflx/flxCd19-Cre+ B cells.
Figure 5: Increased production of natural IgM and autoantibodies directed against influenza virus in Fcmrflx/flxCd19-Cre mice.
Figure 6: B-1 cells are responsible for the enhanced secretion of natural IgM in Fcmrflx/flxCd19-Cre+ mice.
Figure 7: Increased turnover and survival of B cells result in B cell lymphocytosis.

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Acknowledgements

We thank A. Spinner for help with flow cytometry; A. Treister for FlowJo software; R. Pohlmeyer for sharing flow cytometry data; J. Yang (University of Freiburg) for the GFP-IgD plasmid; M. Cavallari for help with image analysis; H. Kubagawa (Deutsches Rheuma Forschungszentrum) for BM from mice with global Fcmr deficiency; and the UC Davis Mouse Biology Program for generating Fcmrflx/flxCd19-Cre mice. Supported by the US National Institutes of Health (AI51354, AI85568 and U19AI109962 to N.B.), the UC Davis Graduate Group in Immunology, a Vietnamese Education Fellowship (T.T.T.N.), a UC Davis Chancellor's Fellowship (N.B.), the Excellence Initiative of the German Federal and State Governments (EXC 294), the European Research Council (322972) and the DFG (TRR130 and project 111026 of the German Cancer Aid to M.R.).

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  1. nbaumgarth@ucdavis.edu

Authors and Affiliations

  1. Center for Comparative Medicine, University of California, Davis, Davis, California, USA

    Trang T T Nguyen & Nicole Baumgarth

  2. Graduate Group in Immunology, University of California, Davis, Davis, California, USA

    Trang T T Nguyen, Patricia A Castillo, Charles L Bevins & Nicole Baumgarth

  3. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Kathrin Kläsener & Michael Reth

  4. Department of Molecular Immunology, Institute of Biology III at the Faculty of Biology of the University of Freiburg, Freiburg, Germany

    Kathrin Kläsener & Michael Reth

  5. Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany

    Kathrin Kläsener, Christa Zürn & Michael Reth

  6. Department of Medical Microbiology & Immunology, University of California, Davis, California, USA

    Patricia A Castillo & Charles L Bevins

  7. Department of Anatomy, Physiology & Cell Biology, University of California, Davis, California, USA

    Ingrid Brust-Mascher & Colin Reardon

  8. Comparative Pathology Laboratory, School of Veterinary Medicine, University of California, Davis, California, USA

    Denise M Imai & Nicole Baumgarth

  9. Department of Pathology, Microbiology & Immunology, School of Veterinary Medicine, University of California, Davis, Davis, California, USA

    Nicole Baumgarth

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Contributions

T.T.T.N. and N.B. designed experiments, analyzed data and wrote the manuscripts; K.K., C.L.B. and M.R. provided help with experimental design; T.T.T.N., K.K., C.Z. and P.A.C. performed experiments; I.B.-M. and C.R. helped with STED and confocal microscopy and image analysis; D.M.I. performed pathological evaluation of Fcmrflx/flxCd19-Cre mice; and all authors provided edits to the manuscript.

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Integrated supplementary information

Supplementary Figure 1 FcμR is expressed by various cell subsets, including peripheral B cells.

(a) Overlay histograms comparing surface FcμR expression in B cells, CD4 T cells, CD8 T cells, Gr-1+ (Granulocytes); CD11b+ and F4/80+ (Macrophages); CD11c+ (Dendritic Cells) from Fcmr flx/flxCd19-Cre and controls. (b) 5% contour plots with outliers of representative spleen samples from wild-type mice analyzed by flow cytometry after gating on live non-doublets. Gating shows FcμR+ of indicated cell subsets. Numbers in black indicate frequencies of FcμR+ cells among total live cells, and those in blue indicate frequencies of FcμR+ cells among CD19+, CD4+, CD8+, Gr-1+, CD11b+, and CD11c+ cells, respectively. Small contour plots are FMOs. (c) Overlay histograms show surface FcμR expression in different B cell subsets in spleen, peritoneal cavities (perc), and inguinal lymph nodes (pLN): spleen marginal zone (MZ), spleen follicular B (FO), spleen B-1, peritoneal cavity (perc) B-1, perc B-2, and peripheral lymph node (pLN) FO B cells. (d) Overlay histograms comparing surface FcμR expression in B cell precursors, including late pre B cells, immature B cells, and mature B cells.

Supplementary Figure 2 FcμR does not control BM B cell output.

(a) Shown are frequencies (left) and numbers (right) of B cell precursors in Fcmr flx/flxCd19-Cre and control BMs (n=4 mice/group). (b,c) Fcmr flx/flxCd19-Cre and control mice (n = 3-4 mice/group) were sublethally irradiated (350rd) and peripheral B cell pools were analyzed 12 days after irradiation. Shown are the frequencies and numbers ± SD of (b) transitional (CD93+) and (c) total (CD19+) B cells in BM and spleen as assessed by multicolor flow cytometry. Data in (a) are representative of at least two independent experiments (mean ± SD in a-c). Data did not show significant differences by unpaired two-tailed Student’s t-test (p > 0.05).

Supplementary Figure 3 B cells rapidly lose surface-bound sIgM.

(a) Shown are histogram plots of splenic B cells from control and Fcmr flx/flxCd19-Cre mice after exposure to biotinylated sIgM (0h) and 0.5h after culture at 37oC. Binding was revealed by staining with streptavidin-Qdot605. (b,c) Mean fluorescent intensities (MFI) ± SD of sIgM binding to (b) B cells and (c) T cells in control and Fcmr flx/flxCd19-Cre mice at indicated times after culture as in (a). (d) To reveal sIgM binding to B cells in vivo, splenic B cells unable to secrete IgM (sIgM–/–) were isolated from mixed bone marrow sIgM–/– (IgMa) x wild type (IgMb) chimeras by autoMACS (n=3 mice). Graphs show MFI ± SD of sIgM binding (IgMb) to IgMa (sIgM–/–) B cells ex vivo and at indicated times during culture. Staining is compared to non-staining control (FMO “fluorescence minus one”) and staining for IgMb on B cells from sIgM–/– (IgMa) mice. n.s. not significantly different, *p<0.05, **p<0.005, ***p<0.0005 by unpaired t-test (a-b) and paired t-test (c).

Supplementary Figure 4 Monoclonal antibodies to IgMa (DS.1) and IgMb (AF6-78.25) are highly specific and bind only to their antigenic target.

(a) STED microscopy of IgHa and IgHb B cells stained for IgMa (red) and IgMb (green). (b) Binding of IgM using serum from wild type IgHa (red) and IgHb (green) wild type mice on ELISA plates coated and revealed with anti-IgMa (left) and IgMb (right). Graph showing mean O.D. per group (n=3-4 mice/group). (c) Overlay flow cytometry histograms showing IgMa (red) and IgMb (green) staining of B cells from congenic IgHa (left) and IgHb (middle) C57BL/6 mice, respectively. Graphs (right and far right) summarizing mean fluorescence intensities (MFI) ± SD of IgMa and IgMb staining for IgHa and IgHb B cells, respectively (n=3 mice/group). Data are compared to “fluorescence minus one” control stains. n.s. not significantly different by unpaired two-tailed Student’s t-test

Supplementary Figure 5 FcμR interacts with mIgM (BCR) in the TGN at the immature B cell stage but rarely in mature FO B cells.

Confocal microscopy of (a) spleen CD19+CD23+ follicular B cells from F1 mice generated by crossing secreted IgM-deficient sIgM–/– (IgHa) with wild type C57BL/6 (IgHb) mice. B cells from sIgM–/– mice were purified based on expression of IgDa and immunostained for secreted IgMb (green), mIgMa (red) and trans-Golgi stain TGN-38 (blue). Far right shows overlap of all three stains, identifying rare co-localization of mIgM but not sIgM with TGN-38. (b) CD19+ B220+ CD43– IgD– late-pre B/immature B cells were purified from C57BL/6 mice and stained for FcμR (green), IgM (red) and TGN-38. Far right shows overlap of all three stains. Note the strong co-localization of IgM with the FcμR in the trans-Golgi network. Scale bar 2μm. (c) Quantification of colocalization coefficients (n=40 cells), presented in a range from 0 (no co-localization) to 1 (total co-localization) (Manders et al., 1993). P, Pearson’s coefficient (R total value of co-localization); t M(1) and t M(2), extend of overlap of signal 1 (channel 1 488nm) with signal 2 (channel 2 PLA 545nm). Each symbol represents an individual cell, horizontal lines indicate the mean ± SD.

Supplementary Figure 6 Fcmr–/– B cells from BM chimeras with knockout of Fcmr show enhanced phosphorylated Akt and Btk.

Fcmr–/– and wild-type bone marrow chimeras were generated by i.v injecting Fcmr–/– (kindly provided by Dr. Hiromi Kubagawa) or wild type bone marrow cells into lethally irradiated C57BL/6 mice. Chimeras were allowed to reconstitute for at least 7 weeks before analysis. (a) Ex vivo pAkt and (b) pBtk expressions in total spleen B cells, follicular (FO) B (B-2) and B-1 cells from Fcmr–/– and control chimeras (n=4 mice/group). *p < 0.05, **p < 0.005, as assessed by comparing mean fluorescence intensities of stained cells (MFI) after subtraction of background MFI (mean ± SD in a-b), using unpaired two-tailed Student’s t-test.

Supplementary Figure 7 The presence of Cd19-Cre does not affect the number of B-1 cells or the PI3K pathway.

(a,b) Box-and-whisker-plots (min, max, median, quartiles) showing (a) ex vivo PI3K expressions and (b) ex vivo phostpho-Akt expressions in total spleen B cells from Fcmr wt/flxCd19-Cre– (Control), Fcmr wt/flxCd19-Cre+(Control), Fcmr flx/flxCd19-Cre– (Control) and Fcmr flx/flxCd19-Cre+ mice (n=4-5 mice/group). (c,d) Box-and-whisker-plots (min, max, median, quartiles) show numbers of B-1 cells, including B-1a and B-1b cells in (c) spleens and (d) peritoneal cavity. *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test.

Supplementary Figure 8 Increased IgM production in chimeras reconstituted with Fcmr–/– B-1 cells and wild-type BM cells.

For lethally-irradiated B-1 chimeras, 2-months old μMT mice were lethally irradiated one day before transfer of 5 x 106 peritoneal lavage cells from Fcmr flx/flxCd19-Cre or control mice (IgHb) as a source of B-1 cells and BM cells from wild-type (IgHa) mice as a source of B-2 cells. Chimeras were rested for at least 7 weeks before analysis (n=4 mice/group). (a) Graphs summarize mean frequencies ± SD of IgMb+ secreting cells derived from B-1 cells and IgMa+ secreting cells derived form BM B-2 cells in spleens, and BMs as assessed by ELISPOT analysis. (b) Mean IgMb, IgMa, and IgM concentrations ± SD in sera of the chimeras, measured by ELISA. *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test.

Supplementary Figure 9 Increases in B-1-cell-derived plasma cells in the absence of FcμR.

(a) Mean frequencies ± SD of IgM+ B-1 cell-derived plasma cells (CD19lowIgMbCD138+) in neonatal chimeras generated with Fcmr–/– or control B-1 cells as measured by multicolor flow cytometry (n=4 mice/group). Neonatal chimeras were created as outlined in Fig. 6d-g. (b) Mean frequencies ± SD of B-1 cell-derived IgM+ plasma cells (CD19lowIgMbCD138+) in BM chimeras generated with Fcmr–/– or control B-1 cells. BM irradiation chimeras were created as outlined in Supplementary Fig. 2 (n=4 mice/group). *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t-test.

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Nguyen, T., Kläsener, K., Zürn, C. et al. The IgM receptor FcμR limits tonic BCR signaling by regulating expression of the IgM BCR. Nat Immunol 18, 321–333 (2017). https://doi.org/10.1038/ni.3677

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