Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens
2016; Springer Nature; Volume: 35; Issue: 21 Linguagem: Inglês
10.15252/embj.201694177
ISSN1460-2075
AutoresChristoph Volkmann, Naema Brings, Martin Becker, Elias Hobeika, Jianying Yang, Michael Reth,
Tópico(s)CAR-T cell therapy research
ResumoArticle15 September 2016Open Access Source DataTransparent process Molecular requirements of the B-cell antigen receptor for sensing monovalent antigens Christoph Volkmann Christoph Volkmann BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Naema Brings Naema Brings BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Martin Becker Martin Becker BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Elias Hobeika Elias Hobeika BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Institute of Immunology, University Hospital Ulm, Ulm, Germany Search for more papers by this author Jianying Yang Corresponding Author Jianying Yang [email protected] orcid.org/0000-0001-8197-5413 BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany University of Strasbourg Institute for Advanced Study (USIAS), University of Strasbourg, Strasbourg, France Search for more papers by this author Michael Reth Corresponding Author Michael Reth [email protected] BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Christoph Volkmann Christoph Volkmann BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Naema Brings Naema Brings BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Martin Becker Martin Becker BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Elias Hobeika Elias Hobeika BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Institute of Immunology, University Hospital Ulm, Ulm, Germany Search for more papers by this author Jianying Yang Corresponding Author Jianying Yang [email protected] orcid.org/0000-0001-8197-5413 BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany University of Strasbourg Institute for Advanced Study (USIAS), University of Strasbourg, Strasbourg, France Search for more papers by this author Michael Reth Corresponding Author Michael Reth [email protected] BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Author Information Christoph Volkmann1,2,‡, Naema Brings1,2,‡, Martin Becker1,2, Elias Hobeika1,2,3, Jianying Yang *,1,2,4,5 and Michael Reth *,1,2 1BIOSS Centre for Biological Signalling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany 2Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany 3Institute of Immunology, University Hospital Ulm, Ulm, Germany 4Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany 5University of Strasbourg Institute for Advanced Study (USIAS), University of Strasbourg, Strasbourg, France ‡These authors contributed equally to this work *Corresponding author. Tel: +49 761 5108 427; E-mail: [email protected] *Corresponding author. Tel: +49 761 203 97663; E-mail: [email protected] The EMBO Journal (2016)35:2371-2381https://doi.org/10.15252/embj.201694177 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract How the B-cell antigen receptor (BCR) is activated upon interaction with its cognate antigen or with anti-BCR antibodies is not fully understood. We have recently shown that B-cell activation is accompanied by the opening of the pre-organized BCR oligomers, an observation that strengthens the role of receptor reorganization in signalling. We have now analysed the BCR oligomer opening and signalling upon treatment with different monovalent stimuli. Our results indicate that monovalent antigens are able to disturb and open the BCR oligomer, but that this requires the presence and activity of the Src family kinase (SFK) Lyn. We have also shown that monovalent Fab fragments of anti-BCR antibodies can open the BCR oligomers as long as they directly interact with the antigen-binding site. We found that monovalent antigen binding opens both the IgM-BCR and IgD-BCR, but calcium signalling is only seen in cells expressing IgM-BCR; this provides a molecular basis for IgM- and IgD-BCR functional segregation. Synopsis B-cell receptors (BCRs) form oligomers on resting B cells, inconsistent with the cross-linking model that suggests antigen-mediated B-cell receptor dimerization as initiating intracellular signal transduction. Work with monovalent antigens shows how they can trigger signalling despite being unable to induce cross-linking of receptor monomers. Fab-based proximity ligation assays directly monitor antigen-induced BCR conformation changes at 10–20 nm distances. Monovalent antigens can disturb and open the BCR oligomer, dependent on the presence of the Src family kinase Lyn. Monovalent Fab fragments from anti-idiotypic antibodies also open the BCR oligomer, as long as they directly bind to the antigen-binding site of the BCR. Monovalent antigens open both IgM-BCR and IgD-BCR, but calcium signalling is only triggered in cells expressing IgM-BCR. Introduction B cells play a central role in the adaptive immune response and signalling through the B-cell antigen receptor (BCR) complex that controls B-cell proliferation and differentiation (Reth, 1992). The BCR is composed of a membrane-bound form of immunoglobulin (mIg) comprising two identical heavy chains (HC) and light chains (LC) and a non-covalently associated signalling subunit, the CD79a/CD79b heterodimer, also referred to as Igα/Igβ (Hombach et al, 1990). The exact conformation of the BCR on the B-cell surface is still a matter of controversy. We found that on mature resting B cells, both IgM-BCR and IgD-BCR form auto-inhibited oligomers (Yang & Reth, 2010a). This oligomeric organization of the BCR has now been detected by several methods, including bimolecular fluorescence complementation (BiFC), super resolution microscopy and electron microscopy and by a Fab-based proximity ligation assay (Fab-PLA) (Mattila et al, 2013; Kläsener et al, 2014; Maity et al, 2015). The latter method has also shown that B-cell activation is accompanied by an opening of the BCR oligomers, as suggested by the dissociation activation model (DAM) (Yang & Reth, 2010b). The cytoplasmic tail of the BCR signalling proteins Igα and Igβ each carries an immunoreceptor tyrosine-based activation motif (ITAM) that plays a central role in B-cell activation. The specific binding of an antigen to the BCR results in the activation of two different cytosolic protein tyrosine kinases, namely the Src family kinase (SFK) Lyn and the spleen tyrosine kinase (Syk) (Schmitz et al, 1996). By phosphorylating and binding to the two ITAM tyrosines, Syk plays a crucial role in the initiation and amplification of BCR signalling (Pao et al, 1998). While the binding of Syk to the phosphorylated ITAM tyrosines of Igα and Igβ is necessary to open BCR oligomers upon the exposure to multivalent antigens, Lyn facilitates the opening, but is not absolutely required for this process (Kläsener et al, 2014). In addition, Syk phosphorylates the adaptor protein SLP-65, also known as BLNK (Fu et al, 1998; Wienands et al, 1998). Once phosphorylated, SLP-65 organizes a calcium signalosome complex containing BTK and PLCγ (Takata & Kurosaki, 1996), which in turn generates the second messenger inositol-3 phosphate (IP3) that mediates the release of calcium ions from the endoplasmic reticulum (ER). The depletion of the calcium stores of the ER results in the opening of calcium channels in the plasma membrane and the drastic increase in the intracellular calcium concentration associated with B-cell activation (Baba & Kurosaki, 2016). B lymphocytes can be activated by different forms of antigens. Numerous studies in the mouse have shown that polyvalent antigens are more efficient than monovalent antigens in inducing an antibody response (Puffer et al, 2007). B cells have also been shown to be more efficiently activated by membrane-bound than by soluble antigens (Carrasco & Batista, 2006). However, small soluble antigens such as hen egg lysozyme (HEL) or ovalbumin (OVA) are also able to activate B cells in vivo (Schelling & Silverman, 1968; Benjamin et al, 1980). In general, polyvalent antigens such as NIP15-BSA are more potent than monovalent antigens in inducing BCR signalling in cultured B cells, as indicated by the increased ITAM phosphorylation or calcium response (Kim et al, 2006; Minguet et al, 2010; Mukherjee et al, 2013; Avalos & Ploegh, 2014; Ubelhart et al, 2015). Nevertheless, several groups have shown that monovalent antigens can activate B cells, also in vitro (Kim et al, 2006; Mukherjee et al, 2013; Avalos et al, 2014). B cells can also be activated by exposure to diverse anti-Ig antibodies. In fact, the classical cross-linking model of B-cell activation is based on the observation that only bivalent F(ab')2 fragments, but not monovalent Fab fragments, of anti-Ig antibodies can efficiently activate the BCR (Woodruff et al, 1967). The cross-linking model proposes that the surface of resting B cells carries signalling inert BCR monomers and the cross-linking of which leads to B-cell activation. However, this model is in conflict with the finding that many monovalent antigens can activate the BCR although they cannot cross-link this receptor (Kim et al, 2006; Mukherjee et al, 2013; Avalos et al, 2014). Using a Fab-based proximity ligation assay (Fab-PLA), we monitored the nanometre conformation of the BCR before and after the exposure of polyvalent or monovalent reagents. We find that on the surface of resting B cells, the IgM-BCR and IgD-BCR form oligomers that are opened upon the exposure of the B cell to either monovalent antigens or an anti-Ig Fab fragment that interacts directly with the antigen-binding site. Results Opening and activation of BCR oligomers by monovalent reagents To study the activation of the BCR by monovalent antigens, splenic B cells specific for the hapten 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP) were isolated from κ-deficient B1-8 transgenic mice (Sonoda et al, 1997). The B cells were first left in culture medium for a minimum of 2 h and then loaded with the calcium indicator indo-1. The B cells were then exposed to different forms of antigens or anti-BCR antibodies at pM to nM concentrations and their calcium response was monitored by flow cytometry. The multivalent antigen NIP15-BSA induced a strong calcium flux in the splenic B cells (Fig 1A). For monovalent antigens, we employed either 1NIP-pep carrying a single NIP hapten group covalently attached to an 8-amino acid peptide or 1NIP-DNA with the NIP hapten attached to a double-strand 19-nucleotide DNA oligomer (Fig EV1A and B). These monovalent antigens also induced a calcium response (Fig EV1C and E), which was weaker than that observed with the multivalent antigen NIP15-BSA (Fig 1A). Figure 1. Monovalent antigen binding opens BCR oligomers and induces a calcium flux in splenic B cells A–C. Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Fig EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows. D. Representative microscopic images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. E. The Fab-PLA results are quantified by BlobFinder software and presented as box plots. The median values are highlighted as thick lines, and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from the treated samples were compared with data from the resting cells; P-values were calculated by Kruskal–Wallis one-way analysis of variance (ANOVA). Data information: Data are representative of at least three independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Synthetic monovalent antigens activate B1-8 splenic B cells A, B. Schematic illustration of the synthetic monovalent antigens. C–E. Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (C) 1NIP-pep (80 nM) with or without 1% FCS in the medium; (D) 1NIP-pep (80 nM) (with or without the staining of anti-B220); (E) 1NIP-DNA (80 nM) and control DNA (same sequence, without NIP, 80 nM). The addition of the stimuli to the cells is indicated by arrows. Data are representative of three independent experiments. Download figure Download PowerPoint It has previously been reported that divalent anti-Ig antibodies can activate the BCR, whereas monovalent Fab fragments derived from these anti-Ig antibodies fail to do so (Woodruff et al, 1967). To test whether this behaviour also holds true for anti-idiotypic antibodies, we used the monoclonal antibodies Ac146 and Ac38 that bind with similar affinity to different epitopes in the variable region of the B1-8 BCR (Reth et al, 1979). The binding of Ac146 to the B1-8 BCR competes with the free hapten NIP-ε-aminocaproic acid (NIP-cap), suggesting that this antibody directly interacts with the antigen-binding site. In contrast, the binding of Ac38 is only inhibited by larger antigens such as NIP15-BSA, but not by the free hapten, suggesting that Ac38 binds close to, but not directly to, the paratope of the B1-8 BCR (Reth et al, 1979). Both anti-idiotypic antibodies are able to induce a calcium flux in B1-8 splenic B cells, but only the Fab fragment of Ac146 (Ac146Fab), and not the Fab fragment of Ac38 (Ac38Fab), can stimulate B1-8 B cells to mobilize calcium (Fig 1B and C). The purity of our Fab preparations was verified by SDS–PAGE (Appendix Fig S1). B1-8 B cells responded to the Ac38Fab only after further treatment of the B cells with a secondary anti-κ antibody (Appendix Fig S2A), and the same holds true for Fab fragments derived from a monoclonal anti-λ light chain antibody (Appendix Fig S2B). The DAM hypothesis proposes that BCR signalling is initiated by the opening of auto-inhibited BCR oligomers (Yang & Reth, 2010b). The finding that monovalent antigens are able to elicit a calcium flux in B1-8 splenic B cells prompted us to examine whether BCR oligomers open when bound by monovalent reagents. For this, we monitored and quantified the proximity of BCR on the surface of resting and activated B1-8 splenic B cells using Fab-PLA (Kläsener et al, 2014) (Fig 1D and E). As a positive control, we stimulated the B1-8 B cells for 1 min with the polyvalent antigen NIP15-BSA. Compared with resting B cells, the close proximity of BCR monomers was lost for both the IgM-BCR and the IgD-BCR oligomers upon contact with the polyvalent antigen, as previously reported (Kläsener et al, 2014). A reduced BCR proximity was also found for both BCR classes after the exposure of B1-8 B cells to the monovalent reagent 1NIP-pep or Ac146Fab. The Ac38Fab, as well as the anti-λ Fab, however, did not reduce the BCR proximity, indicating that the binding of these reagents did not open the BCR oligomer (Fig 1D and E, Appendix Fig S3). In comparison with polyvalent antigens and anti-BCR antibodies, the monovalent reagents, however, only weakly increased the phosphorylation of BCR signalling elements such as Igα, ERK and AKT (Fig EV2). Together, these data show that monovalent antigens and anti-Ig Fab fragments that directly engage the antigen-binding site can open the BCR oligomers and activate the calcium signalling pathway inside the B cell. Furthermore, the finding that a reagent such as Ac38Fab which fails to open the BCR oligomer is unable to induce downstream calcium signals supports the DAM hypothesis. Click here to expand this figure. Figure EV2. Monovalent antigens induce weak phosphorylation of AKT, ERK and IgαWestern blot analysis of AKT, ERK and Igα phosphorylation for B1-8 splenic B cells upon 1- and 5-min treatment with different stimuli. Western blot analysis of Syk and β-actin expression functions as a loading control. Data are representative of three independent experiments. Source data are available online for this figure. Download figure Download PowerPoint Lyn is required for sensing monovalent reagents by the BCR We have recently shown that the binding of Syk to the phosphorylated ITAM tyrosines of Igα and Igβ is necessary for the opening of BCR oligomers on B cells exposed to multivalent antigens, while a SFK such as Lyn enhances but is not absolutely required for this process (Kläsener et al, 2014). This is in agreement with earlier reports showing that the BCR can signal independently of SFKs, but strictly requires Syk activity (Takata et al, 1994). Interestingly, it was recently found that in the presence of SFK inhibitors, B cells with a HEL-specific BCR cannot be stimulated by monomeric soluble HEL, whereas multimeric HEL still elicits a response in the absence of SFK activity (Mukherjee et al, 2013). We therefore asked whether Lyn, the dominant SFK in B cells, is involved in sensing monovalent reagents by the BCR. We first interbred B1-8 transgenic and Lyn-deficient mice (Hibbs et al, 1995). B cells isolated from the spleen of Lyn-deficient B1-8 mice (Appendix Fig S4) were exposed to different antigens and antibodies and analysed as described above. The Lyn-deficient B1-8 B cells showed a strong calcium signal upon binding of NIP15-BSA (Fig 2A), although, in comparison with B1-8 B cells, the peak of the response was delayed (Fig EV3A). The 1NIP-pep, however, failed to induce a calcium release in these B cells (Figs 2A and EV3B). Furthermore, the Lyn-deficient B cells responded only to the complete anti-idiotypic antibodies Ac146 and Ac38, but not to Fab fragments derived from these antibodies (Fig 2B and C). Both anti-idiotypic Fab fragments required the binding of secondary anti-κ antibodies to induce a calcium response (Appendix Fig S5). The analysis of the BCR proximity by Fab-PLA confirmed the results of the calcium assay. Only the polyvalent NIP15-BSA, but none of the monovalent reagents, opened the IgM-BCR or IgD-BCR on Lyn-deficient B1-8 B cells (Fig 2D and E). Figure 2. Lyn is indispensable for the opening and signalling of the BCR upon monovalent antigen binding A–C. Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. D. Representative microscopic images showing Fab-PLA results monitoring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. E. Quantified Fab-PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample; P-values were calculated by Kruskal–Wallis one-way ANOVA. Data information: Data are representative of at least three independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Monovalent antigen-induced BCR signalling is Lyn dependent A, B. Calcium flux measured by FACScan for splenic B cells isolated from B1-8 and Lyn-deficient B1-8 mice upon stimulation with (A) NIP15-BSA (30 pM) or (B) 1NIP-pep (80 nM). Stimuli were added at time points indicated by the arrows. Data are representative of three independent experiments. Download figure Download PowerPoint We next treated B1-8 splenic B cells for 45 min with 1 μM of the SFK inhibitor PP2 to inhibit the kinase activity of Lyn. Similar to the Lyn-deficient B cells, the PP2-treated B cells did not open the IgM-BCR and IgD-BCR and hardly mobilize calcium upon the exposure to monovalent reagents (Fig EV4). Together, these results suggest that Lyn plays a crucial role in the sensing of monovalent reagents. Click here to expand this figure. Figure EV4. The kinase activity of Lyn is crucial for monovalent antigen-induced calcium response and BCR opening A, B. Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after the stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM) after 45-min incubation with 1 mM PP2. Arrows indicate the addition of the stimuli to the cells. C. Representative microscopic images showing Fab-PLA results monitoring the BCR proximity for IgM-BCR and IgD-BCR on untreated or treated cells. PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. D. Quantified PLA results presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. P-values were calculated by Kruskal–Wallis one-way analysis of variance (ANOVA). Data information: Data are representative of three independent experiments. Download figure Download PowerPoint The IgD-BCR opens, but fails to signal upon exposure to monovalent reagents B1-8 splenic B cells co-express IgM-BCR and IgD-BCR oligomers on their surface. It is thus not possible to determine the individual contribution of each receptor class to the antigen-induced calcium response of splenic B cells. To address this question, we expressed NIP-specific IgM-BCR or IgD-BCR separately on the surface of the SLP65/Igα-double-deficient pro-B-cell line 3046 transfected with expression vectors for Igα and the Bcr-Abl oncogene (Yang & Reth, 2010a). Although the 3046 cells produce the surrogate light chain components VpreB and λ5, they express after their transfection with λ LC and HC vectors mostly a BCR instead of a pre-BCR on the cell surface (Appendix Figs S6 and S7). As SLP65 is an important part of the calcium signalosome of mature B cells (Fruman et al, 2000), we also transfected the 3046 pro-B cells with a SLP65 expression vector to generate the 3046S cell line. The expression of the mIg chains at similar levels and their similar 1NIP-pep binding capacity on the different mIg transfectants of 3046 and 3046S cells were assessed by flow cytometry (Appendix Fig S7). The cells were then exposed for 1 min to different antigens and antibodies and analysed for BCR oligomer opening and calcium mobilization (Fig 3). The IgM-BCR was opened upon the exposure of 3046SM or 3046M B cells to either the polyvalent antigen NIP15-BSA or the monovalent reagent 1NIP-pep or Ac146Fab, whereas Ac38Fab again failed to alter the BCR conformation (Fig 3A–D). The monovalent reagents that opened the IgM-BCR also induced a calcium response in 3046SM, albeit not in the SLP65-deficient 3046M B cells (Figs 3E–H and EV5A–C). In contrast, the polyvalent antigen NIP15-BSA induced a calcium release in both IgM-BCR-positive 3046 B-cell lines (Fig 3E and G). These data show that the calcium response induced by binding of monovalent reagents to the IgM-BCR is dependent on SLP65. Figure 3. Monovalent antigen binding to IgM-BCR induces calcium signalling in a SLP-65 dependent manner A–D. Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopic images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample, and P-values were calculated by Kruskal–Wallis one-way ANOVA. E–H. Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Data information: Data are representative of a minimum of three independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Monovalent antigen induced calcium signalling only in IgM-BCR-expressing cells A. FACScan analysis of 3046S cells transduced with IgM-BCR components. The GFP-positive gate represents the transduced cells (3046SM). B, C. Calcium flux measured by FACScan for the transduced 3046SM cells upon stimulation with (B) 1NIP-Pep (80 nM) or (C) Ac146 Fab (25 nM). The non-transduced cells in the same tube function as internal control. D–F. Calcium flux measured by FACScan for 3046SM and 3046SD cells upon stimulation with (D) anti-HC (2 μl/ml), or (E) 200 nM or (F) 40 nM of 1NIP-pep. Data information: Arrows indicate the addition of the stimuli to the cells. Data are representative of a minimum of three independent experiments. Download figure Download PowerPoint We next analysed the conformation and signalling function of the IgD-BCR expressed on 3046SD or 3046D B cells (Fig 4). The binding of either NIP15-BSA or the monovalent reagents 1NIP-pep and Ac146Fab to the IgD-BCR on these two B-cell lines resulted in the opening of receptor oligomers, whereas the Ac38Fab failed to do so (Fig 4A–D). Thus, in terms of receptor opening, the IgD-BCR and IgM-BCR behave similarly. The two BCR classes differ, however, in their requirements for calcium signalling. Only the polyvalent NIP15-BSA, but none of the monovalent reagents, induced a calcium release and that held true even in 3046SD B cells expressing the SLP65 adaptor (Figs 4E–H and EV5D–F). These results are in agreement with the recent finding that antigens with low valency are not able to trigger IgD-BCR signalling (Ubelhart et al, 2015). Together, our data show that a calcium signal is always associated with BCR opening, but that this opening does not always result in a calcium response. Figure 4. Monovalent antigen binding opens IgD-BCR oligomers, but fails to induce a calcium response A–D. Proximity between IgD-BCRs on the surface of 3046SD (A, C) or 3046D (B, D) cells before and after a 1-min stimulation, assayed by Fab-PLA. Results are presented as representative microscopic images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample, and P-values were calculated by Kruskal–Wallis one-way ANOVA. E–H. Calcium flux m
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