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Responsiveness of B cells is regulated by the hinge region of IgD

Nature Immunology volume 16, pages 534–543 (2015)Cite this article

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An Erratum to this article was published on 18 June 2015

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Abstract

Mature B cells express immunoglobulin M (IgM)- and IgD-isotype B cell antigen receptors, but the importance of IgD for B cell function has been unclear. By using a cellular in vitro system and corresponding mouse models, we found that antigens with low valence activated IgM receptors but failed to trigger IgD signaling, whereas polyvalent antigens activated both receptor types. Investigations of the molecular mechanism showed that deletion of the IgD-specific hinge region rendered IgD responsive to monovalent antigen, whereas transferring the hinge to IgM resulted in responsiveness only to polyvalent antigen. Our data suggest that the increased IgD/IgM ratio on conventional B-2 cells is important for preferential immune responses to antigens in immune complexes, and that the increased IgM expression on B-1 cells is essential for B-1 cell homeostasis and function.

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Figure 1: IgD possesses a higher activation threshold than IgM.
Figure 2: The IgD hinge confers unresponsiveness toward monovalent antigens.
Figure 3: Anergic B cells are responsive to polyvalent antigens.
Figure 4: Monovalent antigens inhibit IgD signaling.
Figure 5: IgM BCR promotes proper B-1 cell development.
Figure 6: B-1a cell–derived IgD BCRs are unresponsive to soluble PtC.
Figure 7: Impaired production of natural IgG in IgM−/− mice.

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  • 21 May 2015

    In the version of this article initially published, the bottom left plot in Figure 3c, the top right plot in Figure 4a, and the third and bottom plots in the left column of Figure 6a were duplicates of other plots in the same figure. Those plots have been replaced and the errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank P. Nielsen for scientific discussion and critical reading of the manuscript; U. Stauffer, N. Joswig and C. Johner for mouse work; A. Würch and M. Knoblauch for help with flow cytometry experiments; H.-P. Pircher (University of Freiburg) and A. Weiss (University of California, San Francisco) for providing MD4 and ML5 mice, respectively; and F.D. Batista (London Research Institute) for providing cDNA encoding for immunoglobulin HC and LC genes of the HH10 BCR. This work was supported by the Deutsche Forschungsgemeinschaft (JU 463/2-1 and TRR130) and the Deutsche Krebshilfe (Project 108935).

Author information

Authors and Affiliations

  1. Institute of Immunology, University Hospital Ulm, Ulm, Germany

    Rudolf Übelhart, Eva Hug, Martina P Bach, Marcus Dühren-von Minden & Hassan Jumaa

  2. BIOSS Center for Biological Signaling Studies, Albert-Ludwigs University of Freiburg, Freiburg, Germany

    Thomas Wossning, Michael Reth & Hassan Jumaa

  3. Department of Molecular Immunology, Biology III, Faculty of Biology, Albert-Ludwigs University of Freiburg, Freiburg, Germany

    Marcus Dühren-von Minden, Michael Reth & Hassan Jumaa

  4. Department of Bioinformatics, Institute for Biochemistry, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

    Anselm H C Horn & Heinrich Sticht

  5. Department of Laboratory Medicine, Medical University of Vienna and Center for Molecular Medicine (CeMM), Austrian Academy of Sciences, Vienna, Austria

    Dimitrios Tsiantoulas & Christoph J Binder

  6. Max-Planck-Institute of Immunobiology and Epigenetics, Freiburg, Germany

    Kohei Kometani & Michael Reth

  7. Laboratory for Lymphocyte Differentiation, RIKEN Center for Integrative Medical Sciences (IMS), Turumi-ku, Kanagawa, Japan

    Tomohiro Kurosaki

  8. Laboratory for Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Tomohiro Kurosaki

  9. Department of Biology, Chair of Genetics, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

    Lars Nitschke

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  1. Rudolf Übelhart
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Contributions

R.Ü. performed experiments, analyzed data and contributed to the design of experiments and the writing of the manuscript; E.H., M.P.B. and T.W. performed experiments and analyzed data; A.H.C.H. and H.S. performed the molecular dynamics simulations and, together with M.D.-v.M., modeled the IgD and IgM BCR variants; K.K. and T.K. contributed to the characterization of the hinge region; D.T. and C.J.B. tested the sera from wild-type and IgM-null mice; L.N. generated the IgD-null mice; M.R. gave advice for the experiments on HH10 and B1-8 BCRs; and H.J. designed the study, supervised the work and wrote the manuscript.

Corresponding author

Correspondence to Hassan Jumaa.

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Integrated supplementary information

Supplementary Figure 1 B1-8 and HH10 BCRs specifically recognize the cognate antigens.

(a) Flow cytometry analysis shows specific binding of the cognate antigens for cYFP+ triple-deficient cells expressing the indicated

constructs. (b) Cells from a were subjected to calcium measurement. After a 1-min baseline recording, cells were stimulated with

the indicated molecules. (c) The Coomassie gel shows crosslinking of HEL by incubation with the indicated concentration of

glutaraldehyde (GA) for 48 h at room temperature. Results are representative of at least three independent experiments.

Supplementary Figure 2 IgM-BCR induces AKT phosphorylation in response to soluble HEL.

Triple-deficient cells expressing the indicated constructs were treated with s-HEL or c-HEL for various periods of time, fixed, permeabilized and stained for intracellular phosphorylated AKT with specific immunofluorescent antibodies. Representative results of one out of two independent experiments are shown.

Supplementary Figure 3 Structural properties of the linker sequences investigated.

For each linker an overlay of ten representative conformations sampled over the simulation time is shown (generated by

superposition of the N-terminal residues). (a) SG5. (b) SG10. (c) SG17. (d) p34.

Supplementary Figure 4 Western blot analysis under non-reducing conditions of triple-deficient cells expressing the indicated constructs.

The BCR variants were detected using anti-κ light chain antibodies. Actin served as a loading control. Results are representative of two independent experiments.

Supplementary Figure 5 The IgD hinge confers unresponsiveness toward low-valence antigens.

(a) Schematic representation of the different IgD-BCR variants. (b,c) Flow cytometric analyses of cYFP+ triple-deficient cells show

(b) BCR surface expression and (c) specific binding of NIP(2)-peptide. (d) Triple-deficient cells from b and c were subjected to calcium

measurement. After a 1-min baseline recording, cells were stimulated with 2 μM OHT and 10 nM NIP(2)-peptide or NIP(7)-BSA. Results are

representative of at least three independent experiments. (e) Statistical analysis of calcium flux experiments of TKO cells expressing the

indicated BCRs and stimulated with the indicated antigens. Shown is the mean calcium flux peak over the mean baseline signal of four

independent experiments (n=4, mean ± SEM). P values were calculated using Student’s t-test.

Supplementary Figure 6 Proposed model of the respective BCR types with or without antigen.

Whereas (a) monomeric s-HEL is bound by a single IgD molecule through a high-affinity interaction with the

cognate epitope and, presumably, a second non-specific epitope on the same HEL protein, (b) ic4-HEL

complexes lead to efficient ligation of multiple IgD molecules. (c) Low-valence antigens such as NIP(2)-peptide can stably ligate two

rigid IgM BCR molecules, whereas ligation of multiple IgD BCRs requires polyvalent antigen.

Supplementary Figure 7 Soluble low-valence antigens inhibit IgD activation by polyvalent antigens.

(a) Triple-deficient cells expressing either HH10 IgM or the IgD BCR were subjected to calcium measurement. After a 1-min baseline

recording, cells were stimulated with streptavidin-crosslinked HEL-biotin (c-HEL) supplemented with the indicated amounts of soluble

HEL or BSA. Data are representative of four independent experiments. (b) Statistical analysis of the experiment depicted

in a. Shown is the mean calcium flux peak over the mean baseline signal of four independent experiments (n = 4, mean ± s.e.m.).

(c) Shown is a calcium flux experiment using freshly isolated CD43− splenic B cells from MD4 and MD4 × ML5 mice,

before and after administration of the indicated stimuli. Calcium measurements are representative of at least three independent

experiments. (d) Statistical analysis of the experiment depicted in c. Shown is the mean calcium flux peak over the mean baseline

signal of three independent experiments (n = 3, mean ± s.e.m.). P values were calculated using Student’s t-test.

Supplementary Figure 8 IgD-null mice exhibit markedly increased quantities of peritoneal-cavity B-1a cells.

Statistical analysis of flow cytometry stainings with cells isolated from the peritoneal cavities of wild-type and IgD-null mice. Gated for CD19+Mac-1+CD5+ (B-1a), CD19+Mac-1+CD5– (B-1b) and CD19+Mac-1–CD5– (B-2) cells (n = 4 mice per genotype of at least two independent experiments; mean ± s.e.m.). P values were calculated using Student’s t-test.

Supplementary Figure 9 B1a cell–derived BCR induces B cell proliferation when expressed as IgM but not IgD.

(a) Histograms of a representative measurement out of four experiments showing enrichment of triple-deficient cells expressing IgM

and IgD variants of a B-1a cell–derived BCR. The percentage of cYFP+ cells was assessed at day 1 and day 4 after transduction.

(b) A summary of four independent experiments (mean ± s.e.m.). P values were calculated using Student’s t-test.

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Übelhart, R., Hug, E., Bach, M. et al. Responsiveness of B cells is regulated by the hinge region of IgD. Nat Immunol 16, 534–543 (2015). https://doi.org/10.1038/ni.3141

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