Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin
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Abstract As the central hub of the metabolism machinary, mTORC2 has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. It has been reported that Rictor is involved in B cell development, especially the peripheral development. But the role of Rictor on BCR signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell specfic Rictor knockout mice (cd19Cre) to investigate how Rictor regulates BCR signaling. We found that for the key positive and negative BCR signaling molecules, pBtk is reduced and pSHIP is enhanced in Rictor KO B cells. This indicates Rictor positively regulates the early events of B cell receptor (BCR) signaling. We found that the cellular F-actin is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing the BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration leads to decreases in the humoral immune response in Rictor KO mice. The inhibition of actin polymerization with Latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, where Rictor regulates BCR signaling via actin reorganization.Keywords:
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The protein kinase Btk has been implicated in the development, differentiation, and activation of B cells through its role in the BCR and TLR signaling cascades. These receptors and in particular, the BCR and either TLR7 or TLR9 also play a critical role in the activation of autoreactive B cells by RNA- or DNA-associated autoantigens. To explore the role of Btk in the development of autoreactive B cells, as well as their responses to nucleic acid-associated autoantigens, we have now compared Btk-sufficient and Btk-deficient mice that express a prototypic RF BCR encoded by H- and L-chain sdTgs. These B cells bind autologous IgG2a with low affinity and only proliferate in response to IgG2a ICs that incorporate DNA or RNA. We found that Btk-sufficient RF(+) B cells mature into naïve FO B cells, all of which express the Tg BCR, despite circulating levels of IgG2a. By contrast, a significant proportion of Btk-deficient RF(+) B cells acquires a MZ or MZ precursor phenotype. Remarkably, despite the complete inability of RF(+) Xid/y B cells to respond to F(ab')2 anti-IgM, RF(+) Xid/y B cells could respond well to autoantigen-associated ICs. These data reveal unique features of the signaling cascades responsible for the activation of autoreactive B cells.
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Abstract The B cell receptor (BCR) is essential for both the initial burst of B cell proliferation in response to antigens, and for the differentiation of plasma cells and memory B cells during germinal center reactions. However, the BCR’s precise role in germinal center selection has been difficult to assess: mechanistic investigations into its modulation during the decision of B cell fate suffer from experimental limitations due to the fundamental role of the BCR in B cell biology. Bruton’s tyrosine kinase (BTK), a member of the TEC family, is a key component of BCR signaling. Using a computational screening method based on small molecule docking outside of the BTK active site, we have identified putative allosteric modulators of BTK. In vitro primary B cell Nur77 upregulation, calcium flux, proliferation and plasma cell differentiation confirmed that these molecular probes alter B cell activation strength, kinetics, and fate only when the BCR is engaged. In vivo immunization using an antigen model reveled that BTK agonism strongly favors B cell differentiation into switched plasma cells. BTK agonism, either at the time of the immunization or at the peak of germinal center formation, enhanced humoral immunity, proving for the first time that transient, augmented BCR signaling during germinal center reaction has a direct impact on B cell differentiation ability. In addition to being powerful tools for basic B cell biology investigation, allosteric agonists of BTK could be developed into antigen-licensed B cell adjuvants for vaccines in at risk populations, as well as into cancer immunotherapy drugs which hyperactivate B cells in tumor microenvironments during checkpoint inhibitor blockade therapies.
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Summary The B-cell receptor (BCR) is essential for B-cell development and a crucial clinical target in immuno-oncology. However, therapeutic success against the BCR and downstream signaling pathways is hampered by enhanced NF-κB activation as a resistance mechanism. Using a multiomic approach, we discover the c-REL proto-oncogenic subunit of the NF-κB family as a key transcription factor regulating BCR subunit levels in B-cell lymphoma. Subsequent ChIP- seq, cell biology experiments, and patient data analysis reveal that OTUD4 is a critical deubiquitinase for inhibiting proteasomal degradation of c-REL and for stabilizing a multi-loop positive feedback of NF-κB to the BCR pathway. Remarkably, OTUD4 downregulation destabilizes c-REL and BCR levels and inhibits cell growth of B cell lymphoma. Thus, we shed light on the malignant potential of c-REL abundance, identify a positive feedback from c-REL to upstream BCR and present OTUD4 as a vulnerability to synergistically target NF-κB and BCR pathways in B-cell lymphoid malignancies.
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Abstract The Tec family kinase, Bruton’s Tyrosine Kinase (BTK), is one of the key intracellular signaling components proximal to the B cell receptor (BCR). In this study, we utilized an orally-accessible BTK inhibitor (BTKi) to study the functional effects blocking this signaling pathway has on antigen-driven B cell activation in vivo. We report that BTK is required for anti-IgD driven B cell activation in vivo as evidenced by suppression of the surface activation markers CD86 and mRNA transcripts (e.g. c-Myc, Bcl-xL, CCL3, CD98, EBI2, EGR1, EGR2 and IRF4) otherwise rapidly induced upon engagement of the BCR with antigen. Using the T-dependent protein PE and SRBCs as model antigens, we tested for a requirement of BTK in generating antigen-specific IgM and IgG antibody titers and germinal centers, and find that BTK is required for germinal center maintenance. Lastly, we tested for a role of BTK in long-lived PE-specific memory B cell re-activation and report that BTKi significantly suppressed the induction of PE-specific IgM and IgG titers upon antigen re-challenge. The use of BTKi allowed us to bypass the impact a genetically inactivated BTK gene has on B cell development and to assess BTK inactivation in both a wild-type setting and in a temporal manner. These results provide insight into the role of BTK in BCR-driven B cell activation in various antigen naïve and experienced B cell populations.
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B-cell receptor (BCR)-mediated signals provide the basis for B-cell differentiation in the BM and subsequently into follicular, marginal zone, or B-1 B-cell subsets. We have previously shown that B-cell-specific expression of the constitutive active E41K mutant of the BCR-associated molecule Bruton's tyrosine kinase (Btk) leads to an almost complete deletion of immature B cells in the BM. Here, we report that low-level expression of the E41K or E41K-Y223F Btk mutants was associated with reduced follicular B-cell numbers and significantly increased proportions of B-1 cells in the spleen. Crosses with 3-83 mu delta and VH81X BCR Tg mice showed that constitutive active Btk expression did not change follicular, marginal zone, or B-1 B-cell fate choice, but resulted in selective expansion or survival of B-1 cells. Residual B cells were hyperresponsive and manifested sustained Ca(2+) mobilization. They were spontaneously driven into germinal center-independent plasma cell differentiation, as evidenced by increased numbers of IgM(+) plasma cells in spleen and BM and significantly elevated serum IgM. Because anti-nucleosome autoantibodies and glomerular IgM deposition were present, we conclude that constitutive Btk activation causes defective B-cell tolerance, emphasizing that Btk signals are essential for appropriate regulation of B-cell activation.
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Article18 April 2018Open Access Transparent process Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells Xiaocui He Xiaocui He orcid.org/0000-0002-0106-057X BIOSS Centre For Biological Signaling 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 Kathrin Kläsener Kathrin Kläsener orcid.org/0000-0002-5969-2553 BIOSS Centre For Biological Signaling 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 Joseena M Iype Joseena M Iype BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Martin Becker Martin Becker orcid.org/0000-0002-1751-1056 BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Palash C Maity Palash C Maity BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Marco Cavallari Marco Cavallari BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Peter J Nielsen Peter J Nielsen Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Jianying Yang Jianying Yang orcid.org/0000-0001-8197-5413 BIOSS Centre For Biological Signaling 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 Michael Reth Corresponding Author Michael Reth [email protected] orcid.org/0000-0002-1025-7198 BIOSS Centre For Biological Signaling 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 Xiaocui He Xiaocui He orcid.org/0000-0002-0106-057X BIOSS Centre For Biological Signaling 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 Kathrin Kläsener Kathrin Kläsener orcid.org/0000-0002-5969-2553 BIOSS Centre For Biological Signaling 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 Joseena M Iype Joseena M Iype BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Martin Becker Martin Becker orcid.org/0000-0002-1751-1056 BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Palash C Maity Palash C Maity BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Marco Cavallari Marco Cavallari BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Peter J Nielsen Peter J Nielsen Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Jianying Yang Jianying Yang orcid.org/0000-0001-8197-5413 BIOSS Centre For Biological Signaling 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 Michael Reth Corresponding Author Michael Reth [email protected] orcid.org/0000-0002-1025-7198 BIOSS Centre For Biological Signaling 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 Xiaocui He1,2, Kathrin Kläsener1,2, Joseena M Iype1,3, Martin Becker1,4, Palash C Maity1,3, Marco Cavallari1, Peter J Nielsen2, Jianying Yang1,2 and Michael Reth *,1,2 1BIOSS Centre For Biological Signaling Studies, Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany 2Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany 3Present address: Institute for Immunology, Uni Hospital Ulm, Ulm, Germany 4Present address: Helmholtz Zentrum München, München, Germany *Corresponding author. Tel: +49 761 203 97663; E-mail: [email protected] The EMBO Journal (2018)37:e97980https://doi.org/10.15252/embj.201797980 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 Expression of the B-cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B-cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine-based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B-cell co-receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co-receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B-cell surface, where it is found in close proximity to CD19 and signals in an ITAM-dependent manner. These data suggest that Igβ and CD19 are part of an alternative B-cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells. Synopsis The B-cell antigen receptor (BCR) signaling component Igβ (CD79b) and the co-receptor CD19 act as an alternative B-cell signaling module that promotes the survival of B lymphoma and normal B cells via integrated ITAM/PI3K signaling. A new strategy for functional signaling studies of B-cell lines by the CRISPR/Cas9 mediated deletion of BCR and B-cell co-receptor genes. The fitness of the human Burkitt lymphoma cell line Ramos requires the expression of the BCR signaling component Igβ and the co-receptor CD19. Only Igβ but not Igα can be transported on the B-cell surface in the absence of other BCR components. The BCR-independent Igβ is found on the B-cell surface in close proximity to CD19 and signals in an ITAM-dependent manner. Introduction The B-cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) comprising two heavy (H) and two light (L) chains and the signal-transducing Igα/Igβ (CD79a/CD79b) heterodimer (Reth & Wienands, 1997). The cytoplasmic tails of Igα and Igβ each contain an immunoreceptor tyrosine-based activation motif (ITAM) with two conserved tyrosines that are crucial for the development and maintenance of mature B cells (Reth, 1989; Torres et al, 1996; Kraus et al, 2001, 2004; Reichlin et al, 2001). Upon antigen ligation and opening of the BCR, the spleen tyrosine kinase (Syk) phosphorylates and interacts with the ITAM tyrosines of Igα and Igβ (Rolli et al, 2002). The resulting ITAM/Syk complex amplifies the BCR signal and connects the BCR to several downstream signaling pathways, leading to the activation, proliferation, and differentiation of B cells (Johnson et al, 1995; Kurosaki, 1999; Deane & Fruman, 2004; Werner et al, 2010). A prominent substrate of Syk is the SLP65/BLNK adaptor protein which, upon phosphorylation, organizes the B-cell calcium signalosome and thus is required for calcium mobilization in activated B cells (Jumaa et al, 1999; Kulathu et al, 2008). Another important signaling hub in B cells is the CD19 co-receptor molecule, which forms a complex on the plasma membrane together with the tetraspanners, CD81 (TAPA-1) and leu-13, and the complement receptor CD21 (CR2; Fearon & Carroll, 2000). The long cytosolic tail of CD19 contains nine tyrosines most of which are phosphorylated by the src-family kinase Lyn upon B-cell activation. Once phosphorylated, these tyrosines serve as binding partners for the adaptor proteins Grb2 and Vav, the phosphoinositide-3 kinase (PI3K), and phospholipase C-γ (Fearon & Carroll, 2000). Signal transduction from CD19 thus involves PI3K signaling and cytoskeleton rearrangements. Mature B cells co-express two different classes of BCR, namely the IgM-BCR and the IgD-BCR, which reside in different protein islands on the plasma membrane (Yang & Reth, 2010; Maity et al, 2015). On resting B cells, CD19 complex and the CD81-tetraspanin complex are localized inside IgD-class-specific protein islands (Mattila et al, 2013; Maity et al, 2015). On activated B cells, however, the CD19 complex is found in close proximity to the open IgM-BCR and thus gains access to ITAM signaling (Klasener et al, 2014). The IgM/CD19/CD81/CD21 complex is also stabilized by its co-ligation with complement-bound antigens that can reduce the threshold for BCR signaling 10- to 100-folds (Carter & Fearon, 1992). The mouse CD19-deficient B cells have defects in proliferation and maturation in peripheral lymph tissues and spleen, and have a defective T-cell dependent antigen response (Rickert et al, 1995). The expression of a pre-BCR and a BCR is required for B-cell development and the maintenance of mature B cells. Mice with a deletion of any one of the H chain, Igα, or Igβ genes display a developmental block at the pro-B-cell stage (Kitamura et al, 1991; Gong & Nussenzweig, 1996; Torres et al, 1996; Minegishi et al, 1999; Reichlin et al, 2001; Meffre & Nussenzweig, 2002; Pelanda et al, 2002). The deletion of the H chain gene or the mutation of the ITAM tyrosines of Igα in mature B-cell results in apoptosis and disappearance of B cells from the periphery in a few days (Lam et al, 1997; Kraus et al, 2004). These data show that the proper expression of the BCR is required for the fitness and survival of mature B cells and it was therefore suggested that the unengaged BCR emits a maintenance or tonic survival signal. However, some mature B cells which loose BCR expression after an inducible deletion of the Igα gene survive in mice for more than 20 days, suggesting alternative signals for the survival of these B cells (Levit-Zerdoun et al, 2016). Expression of, and signaling from, the pre-BCR or BCR is also required for the continuous growth of several B-cell lymphomas (Gauld et al, 2002; Kuppers, 2005; Lenz & Staudt, 2010; Stevenson et al, 2011). For instance, most B cells of chronic lymphocytic leukemia (B-CLL) carry an auto-aggregated BCR, which promotes continuous ITAM and PI3K signaling and is required for the expansion and survival of these tumor cells (Duhren-von Minden et al, 2012). Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCL) carry mutations of the BCR and its signal components, resulting in chronic active NFκB signaling and increased tumor survival (Davis et al, 2010). Burkitt lymphoma (BL) is an aggressive B-cell tumor that is derived from germinal center B cells and is transformed by a translocation of the c-Myc oncogene into the H chain locus (Dalla-Favera et al, 1982). Knock-down experiments and the application of specific inhibitors suggest that the survival and expansion of BL cells require ITAM and PI3K signaling (Schmitz et al, 2012, 2014). Furthermore, a recent study on a BL mouse model suggests that the expression of the BCR is required for the fitness of these tumor cells (Varano et al, 2017). Here, we have used the CRISPR/Cas9 method to delete genes that code for the receptor and signaling components of the BCR in the human BL cell line Ramos. A competitive growth assay showed that the fitness of the Ramos B cells requires the expression of the BCR co-receptor CD19 and of the BCR subunit Igβ. We found that Igβ is expressed in a mIg-independent fashion on the B-cell plasma membrane where it is found in close proximity to CD19. We suggest that Igβ and CD19 are part of an alternative receptor module for tonic ITAM and PI3K signaling that promotes the survival of normal and tumor B cells. Results The fitness of Ramos cells depends on the mIg-independent expression of Igβ To study the role of the BCR complex in supporting the growth of B-cell lymphomas, we used the CRISPR/Cas9 gene-editing tool to generate Ramos B-cell mutants lacking all (BCR-null) or some of the four BCR components, H and L chains, Igα and Igβ, (Fig 1A). We then compared the proliferation of the BCR-null line with that of wild-type (WT) Ramos cells (Fig 1B). Surprisingly, the BCR-null cells expanded in culture with similar kinetics to that of WT Ramos B cells, indicating that they were not compromised in their in vitro growth (Fig 1B). We next compared the two Ramos cell lines in a growth competition assay (Fig 1C) in which we labeled WT Ramos B cells with GFP and mixed them in a 1:1 ratio with either unstained (GFP−) WT or BCR mutant Ramos cells. A FACScan analysis verified that BCR-null Ramos cells did not carry any BCR (neither the IgM-BCR nor the IgD-BCR) on their cell surface (Fig 1D). Interestingly, in the growth competition assay, the BCR-null Ramos cells gradually disappear from the culture within 8 days, thereby suggesting that they are less fit than WT Ramos B cells (Fig 1E). Figure 1. Fitness of Ramos cells depends on the Igβ subunit of the BCR A schematic diagram showing the route for the generation of single- and multi-BCR components KO from wild-type (WT) Ramos B cells by the CRISPR/Cas9 method. Cell proliferation assay of WT and Ramos-null cells using CytoTell™ UltraGreen. Null = HC/LC/Igα/Igβ tetra KO. A diagram depicting the growth competition assay. WT Ramos cells were retrovirally transduced with pMIG empty vector (EV) to create GFP-labeled WT cells (gWT). BCR component KO cells were mixed together with gWT Ramos cells at about 1:1 ratio at day 0, and the relative amount of GFP− BCR components KO cells was then measured by flow cytometry at different time points. The expression levels of IgM-BCR and IgD-BCR on the surface of WT and Ramos-null cells were determined by flow cytometry. Growth competition between Ramos-null and gWT cells. The competition between GFP− WT and gWT cells serves as a control. The data represent the mean and standard error of a minimum of three independent experiments. The expression levels of IgM-BCR and IgD-BCR on the surface of WT and BCR component KO Ramos cells were determined by flow cytometry. Growth competition of the BCR components KO cells against the gWT cells. Data represent the mean and standard error of a minimum of three independent experiments. One clone is used for each genotype. Download figure Download PowerPoint We next asked whether the loss of one or both of the signaling components of the BCR caused the reduced fitness of the BCR-null B cells. For this, we generated Igα and Igβ single KO as well as Igα/Igβ double KO Ramos B cells and verified in a FACScan analysis that none of these cells carry an IgM-BCR or IgD-BCR on their cell surface (Fig 1F). The loss of the BCR signaling component was also verified by an intra- and extracellular FACScan as well as by Western blot analysis (Appendix Fig S1). When the different single or double KO cells were cultured separately, they expanded as well as the WT Ramos B cells (Appendix Fig S2). However, in the competition with WT Ramos B cells, only the Igβ single or Igα/Igβ double KO cells were lost within 8 days of culture whereas the Igα single KO cells competed well with the WT Ramos B cells (Fig 1G). To exclude that these results are influenced by the generation of variants during the selection and prolonged culture of Ramos KO clones, we repeated the competition experiments with a new set of Ramos KO clones that were culture for only 14 days and obtained the same results (Appendix Fig S3). Together, this analysis suggests that the fitness of the Ramos B cells is not associated with total BCR but rather with Igβ expression. We next analyzed whether the apparent signaling function of Igβ on Ramos B cell requires the expression of the mIg part of the BCR. We generated HL double as well as HLα and HLβ triple KO Ramos BCR mutant cell lines (Fig 2A) and verified the loss of the corresponding BCR components by a FACScan and a Western blot analysis (Appendix Fig S1D and E). Interestingly, the FACScan analysis using anti-Igβ antibodies showed that the HLα triple KO Ramos B cells still expressed some Igβ protein on their cell surface albeit in reduced amounts in comparison with WT Ramos B cells (Fig 2B). In the competition assay with WT Ramos cells, only the HLβ KO cells lacking Igβ expression gradually disappear from the culture (Fig 2C). The finding that the Igβ protein is expressed in the absence of mIg on the surface of HLα KO Ramos B cell prompted us to test whether anti-Igβ antibodies could stimulate these cells. We thus exposed WT, HL-KO, HLα-KO, and HLβ-KO Ramos cells to the monoclonal anti-Igβ antibody and measured the calcium mobilization in these cells by FACScan (Fig 2D–G). This analysis shows that Ramos cells expressing Igβ in the absence of mIg can be stimulated by anti-Igβ antibody, albeit in a delayed and reduced manner compared with the WT Ramos B cells. The HLβ-KO triple KO Ramos cells did not respond to the anti-Igβ antibody treatment and served as negative control in this experiment (Fig 2G). As a control, we exposed the four different Ramos clones to anti-Igα antibodies and found that only the WT Ramos B cells responded (Appendix Fig S4). Figure 2. BCR-independent Igβ expression determines the fitness of Ramos cells A. A schematic diagram showing the route map for generating single- and multi- BCR component KOs from WT Ramos B cells by the CRISPR/Cas9 method. B. The expression of Igβ on the surface of different BCR component KO Ramos cells was determined by flow cytometry. C. Growth competition of the BCR component KO cells against gWT cells. The data represent the mean and standard error of a minimum of three independent experiments. D–G. Calcium responses of WT and BCR component KO Ramos cells upon the stimulation with anti-Igβ antibodies. HL-KO = HC/LC double KO; HLα KO = HC/LC, Igα triple KO; HLβ KO = HC/LC, Igβ triple KO. One clone is used for each genotype. Download figure Download PowerPoint A functional ITAM is required for Igβ-dependent B-cell fitness The cytoplasmic tail of human Igβ carries two conserved ITAM tyrosines (Y196 and Y207) that are important for the interaction of the BCR signaling components with Syk. To test whether the fitness of Ramos B cells depends on ITAM signaling, we transduced HLβ KO Ramos B cells with either empty vector (EV) or with vectors encoding WT or ITAM-mutated Igβ proteins and verified the expression of the Igβ proteins on the surface of the transduced HLβ KO cells by FACScan (Fig 3A). In co-cultures containing transduced and non-transduced HLβ KO cells, the Igβ-producing Ramos cells were enriched over time whereas the EV transfected control cells did not show this effect (Fig 3B). The growth-promoting effect of Igβ was abolished, however, when one or both ITAM tyrosines were mutated to phenylalanine (Fig 3C). Thus, the fitness of Ramos B cells depends on the ITAM signaling mechanism. This conclusion was also confirmed by the analysis of HLβ KO cells expressing an Igβ-α tail chimera, which expressed the extracellular portion and transmembrane region of Igβ and the cytosolic tail of Igα. ITAM signaling from this chimeric receptor provided an even higher growth advantage, whereas an Igβ tail truncation (Igβtl) could not increase the fitness of HLβ KO Ramos B cells (Fig 3C). Figure 3. Signaling through the ITAM of BCR-independent Igβ contributes to the fitness of Ramos cells A. The expression of Igβ on the surface of HLβ KO cells reconstituted with WT or different Igβ mutants was determined by flow cytometry. B, C. The percentage of GFP-positive transduced cells at different time points after the transduction of HLβ KO cells reconstituted with WT or different mutated form of Igβ is shown. The vectors express GFP as a transduction marker. The data represent the mean and standard error of a minimum of three independent experiments. D. Calcium responses of HLβ KO cells reconstituted with WT or different Igβ mutant constructs upon the stimulation of anti-Igβ antibodies. The data are representative of three independent experiments. E. The expression of Igβ on the surface of αβ KO cells reconstituted with WT and different Igβ mutant constructs was determined by flow cytometry. F, G. The proportion of GFP-positive αβ KO Ramos cells at different time points after their transduction with vectors encoding either WT or different mutated forms of Igβ. The vectors express GFP as a transduction marker. The data represent the mean and standard error of a minimum of three independent experiments. H. The calcium responses of αβ KO cells reconstituted with WT or different Igβ mutant constructs stimulated by anti-Igβ antibodies. The data are representative of three independent experiments. A minimum of two clones were used for both the HLβ KO and the αβ KO. Download figure Download PowerPoint We next exposed some of the transduced HLβ KO Ramos B cells to the monoclonal anti-Igβ antibody and measured the calcium mobilization in these cells by FACScan (Fig 3D). The HLβ KO Ramos B cells expressing WT Igβ or the Igβ-α tail chimera displayed an increased calcium mobilization whereas EV control or Igβ tail-less expressing HLβ KO Ramos B cells did not respond to this stimulus. This analysis shows that the increased fitness of the Ramos B cells is correlated with increased responsiveness toward anti-Igβ antibody stimulation. The above analysis was repeated with αβ KO Ramos B cells with the same results (Fig 3E–H). Thus, it is the expression of Igβ and not the formation of an Igα/Igβ heterodimer that is required for the increased fitness, and the anti-Igβ induced calcium release of Ramos B cells. Taken together, these results suggest that Igβ promotes cellular fitness by a constant signaling through its ITAM tyrosines. Igβ employs the same signaling pathway as the activated BCR Upon exposure to its cognate antigen, the BCR interacts with the kinases Lyn and Syk, both of which support the dissociation and activation of the BCR and mediate downstream signaling (Klasener et al, 2014). To test whether Igβ also uses these classical BCR signaling components, we pre-treated HL-KO Ramos B cells with either DMSO, the Lyn inhibitor PP2, or the Syk inhibitor R406 and then measured their calcium response after anti-Igβ stimulation. We found that the calcium response was most strongly reduced by the Syk inhibitor and to a lesser extent by the Lyn inhibitor (Fig 4A). This result demonstrates that Syk and Lyn are involved in the Igβ-mediated signaling. Indeed when we exposed HL or HLβ KO Ramos B to the oxidant pervanadate, we found that the latter cells lacking Igβ contain reduced pSyk levels (Fig EV1). Figure 4. The BCR-independent Igβ signals though the canonical BCR signaling pathway Calcium responses of HL double KO Ramos cells stimulated by anti-Igβ after treatment of the indicated kinase inhibitors. The data are representative of three independent experiments. Calcium responses of HLSLP65 triple KO Ramos cells reconstituted with a tamoxifen-inducible SLP65 after the stimulation with anti-Igβ and treatment with 4-OHT. Cells without SLP65 production were used as controls. The data are representative of three independent experiments. Growth competition assay of HLSLP65 KO cells with HL-KO cells. HLSLP65 KO cells were mixed together with GFP-expressing HL-KO cells. The relative amount of HLSLP65 KO cells was then measured at the indicated time points by flow cytometry. The data represent the mean and standard error of a minimum of three independent experiments. One clone is used for each genotype. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Flow cytometry analysis of phosphoSykNormalized intracellular phosphoSyk levels in resting and pervanadate treated HL double KO and HLβ triple KO Ramos cells. The data represent the mean and the standard deviation of three independent experiments. P-values were calculated by paired t-test. One clone is used for each genotype. Download figure Download PowerPoint One prominent substrate of Syk is the adaptor protein SLP65/BLNK, a component of the calcium signalosome of B cells (Fu et al, 1998; Wienands et al, 1998). To test whether SLP65 is also required for Igβ signaling, we generated HLSLP65 triple KO Ramos B cells and verified the loss of SLP65 expression by Western blot (Appendix Fig S5). The cells were then transduced with plasmids encoding SLP65ERT2, a tamoxifen-inducible form of SLP65 (Trageser et al, 2009). Upon exposure to anti-Igβ and tamoxifen, the SLP65ERT2-transduced HLSLP65 triple KO Ramos cells showed a calcium flux, whereas cells lacking SLP65ERT2 display no change in the calcium level (Fig 4B). This indicates that SLP65 expression is required for proper Igβ signaling. In agreement with this conclusion, we found that the HLSLP65 triple KO cells show a growth disadvantage when co-cultured with HL-KO B cells (Fig 4C). Together, these results show that Igβ improves the fitness of Ramos B cells by employing the same Lyn, Syk, and SLP65 signaling route as activated B cells. The co-receptor CD19 is required for the Igβ-induced fitness signal All mature B cells express the co-receptor CD19, which requires association with the tetraspanner CD81 for its stable expression on the B-cell surface (Maecker & Levy, 1997). Upon B-cell activation, CD19 and the IgM-BCR move closer together and both receptors cooperate in the amplification of the BCR signal (Fujimoto et al, 2000; Klasener et al, 2014). To test for the role of CD19 in Igβ signaling, we generated by bulk transfection a HLCD19 or HLCD81 triple KO Ramos population (Appendix Fig S6) and verified by a FACScan analysis that these cells had lost CD19 but maintained Igβ expression on their cell surface (Fig 5A and B). After transfection of the HL double KO cells with the CRISPR/Cas9 KO vector for either CD19 or CD81, the surface CD19-negative Ramos B cells gradually disappeared from the culture within 12 days (Fig 5C). Furthermore, the HLCD19 and HLCD81 mutant Ramos cells were also unable to mount a calcium response upon exposure to anti-Igβ antibodies (Fig 5D). This indicates that the expression of CD19 is required for Igβ signaling and the fitness of Ramos B cells. Figure 5. The Igβ-dependent Ramos cell fitness requires CD19 A, B. The expression of CD19 and Igβ on the surface of different Ramos KO cells was determined by flow cytometry. C. The CD19 and CD81 genes in HL-KO Ramos cells were rendered defective by the CRISPR/Cas9 method, and the percentage of the CD19- or CD81-negative cells in the triple KO population was measured by flow cytometry at the indicated time points. The data represent the mean and standard error of three independent experiments. D. Calcium responses of HL-KO, HLCD19 KO, and HLCD81 KO cells stimulated by anti-Igβ antibodies. The data are representative of three independent experiments. E. Expression of CD19 or Igβ on the surface of HLCD19 KO cells transduced with empty vector (EV) or the CD19 vector measured by flow cytometry. F. Percentage of GFP-positive HLCD19KO cells at different time points after their transduction with EV or the CD19 expression vector. The data represent the mean and standard error of three independent experiments. G. Calcium responses of HLCD19 KO cells transduced with EV or CD19 expression vector after their stimulation with anti-Igβ antibodies. The data are representative of three independent experiments. One clone is used for HL-KO. HLCD19KO and HLCD81 KO are batch sorted. Download figure Download PowerPoint We next verified the fitness-promoting role of CD19 by a gain-of-function assay. We retrovirally transduced the HLCD19 triple KO cells with either the EV control or the CD19 expression vector. The latter cells show large amounts of CD19 on their surface while the amount of surface Igβ is the same in EV and CD19-transduced HLCD19 KO cells (Fig 5E). Compared to EV, the CD19 (GFP+)-transduced HLCD19 KO cells were enriched in the cell culture, indicating that CD19 expression promotes Ig
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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.
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Abstract The Tec kinase Bruton’s tyrosine kinase (Btk) represents a key intermediary for B cell receptor (BCR) signaling. Btk mutation produces B cell deficiency in mice with X-linked immunodeficiency (xid), and surface Ig-mediated responses of mature B cells are seriously deranged. The central role that Btk plays in directing downstream events produced by BCR engagement is demonstrated by the complete failure of NF-κB induction and cellular proliferation following anti-Ig treatment of B cells obtained from xid mice. In this study, we report that the block in BCR signaling produced by Btk mutation is reversed by CD40 engagement. Prior treatment with CD40 ligand normalized subsequent responses of xid B cells to BCR cross-linking, so that typical outcomes of BCR signaling such as NF-κB activation and cell cycle progression occurred in a Btk-independent fashion. These results demonstrate that a specific genetic lesion interrupting BCR-mediated intracellular signaling is circumvented through stimulation of CD40.
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LYN
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