B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling streng... more B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR)1 or hyperactivation above maximum (for example, self-reactive BCR)2,3 thresholds of signalling strength causes negative selection. In 25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), whichmimics constitutively active pre-BCR signalling4,5. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival6. We tested the hypothesis that targeted hyperactivation—above a maximum threshold—will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR–ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patientderived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1)9, we demonstrated that pharmacological hyperactivation of SYKand engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
The recently determined cryo-EM structures of the T cell antigen receptor (TCR) and B cell antige... more The recently determined cryo-EM structures of the T cell antigen receptor (TCR) and B cell antigen receptor (BCR) show in molecular details the interactions of the ligand-binding part with the signaling subunits but they do not reveal the signaling mechanism of these antigen receptors. Without knowing the molecular basis of antigen sensing by these receptors, a rational design of optimal vaccines is not possible. The existence of conserved amino acids (AAs) that are not involved in the subunit interaction suggests that antigen receptors form higher complexes and/or have lateral interactors that control their activity. Here, I describe evolutionary conserved leucine zipper (LZ) motifs within the transmembrane domains (TMD) of antigen and coreceptor components that are likely to be involved in the oligomerization and lateral interaction of antigen receptor complexes on T and B cells. These immunoreceptor coupling and organization motifs (ICOMs) are also found within the TMDs of other ...
B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen r... more B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have studied the cell surface features of Ramos B cells and the spatiotemporal organization of the IgM-BCR using lattice light sheet microscopy in combination with tailored custom-built 4D image analysis. Ramos B cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A proportion of membrane-localized IgM-BCR was found in clusters, which were associated with the ridges and the microvilli. The dynamic ridge network organization and the IgM-BCR cluster mobility were linked and both were controlled by Arp2/3 complex activity. Our results suggest that topographical features of the cell surface govern the distribution and dynamic localization of IgM-BCR clusters to facilitate ...
The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M... more The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M, D, G, A or E for antigen recognition and a disulfide-linked heterodimer between Igα and Igβ (Igα/β, also known as CD79A and CD79B) that functions as the signalling entity. The organizing principle of BCR assembly remains elusive. Here we report the cryo-electron microscopy structures of the intact IgM class BCR at 8.2 Å resolution and its Fab-deleted form (IgM BCRΔFab) at 3.6 Å resolution. At the ectodomain (ECD), Igα and Igβ position their respective Ig folds roughly in parallel with an approximate 2-fold symmetry, which is distinct from structures of Igβ/β homodimers. Unlike previous predictions, the BCR structure displays an asymmetric arrangement, in which the Igα/β ECD heterodimer mainly uses Igα to associate with Cµ3-Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC’) empty. The transmembrane domain (TMD) helices of the two µHCs also deviate from the 2...
We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-C... more We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-CoV-2-derived peptides performed in March and April 2020, respectively. The first set of peptides contained eight peptides predicted to bind to the individual’s HLA molecules. The second set consisted of ten peptides predicted to bind promiscuously to several HLA-DR allotypes. The vaccine formulation contained the new TLR 1/2 agonist XS15 and was administered as an emulsion in Montanide as a single subcutaneous injection. Peripheral blood mononuclear cells isolated from blood drawn before and after vaccinations were assessed using Interferon-γ ELISpot assays and intracellular cytokine staining. We detected vaccine-induced CD4 T cell responses against six out of 11 peptides predicted to bind to HLA-DR after 19 days, following vaccination, for one peptide already at day 12. We used these results to support the design of a T-cell-inducing vaccine for application in high-risk patients, with w...
The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneum... more The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneumonia and other severe disease manifestations known as COVID-19. This health crisis has resulted in a significant effort to stop the spread of this new coronavirus. However, while propagating itself in the human population, the virus accumulates mutations and generates new variants with increased fitness and the ability to escape the human immune response. Here we describe a color-based barcoded spike flow cytometric assay (BSFA) that is particularly useful to evaluate and directly compare the humoral immune response directed against either wild type (WT) or mutant spike (S) proteins or the receptor-binding domains (RBD) of SARS-CoV-2. This assay employs the human B lymphoma cell line Ramos, transfected for stable expression of WT or mutant S proteins or a chimeric RBD-CD8 fusion protein. We find that the alpha and beta mutants are more stably expressed than the WT S protein on the Ramos ...
To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and succ... more To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and successful vaccination against coronavirus disease 2019 (COVID‐19), the kinetics of neutralizing or blocking anti‐SARS‐CoV‐2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS‐CoV‐2 blocking assay (SUBA) using immobilized recombinant human angiotensin‐converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS‐CoV‐2 spike protein. Spike protein‐expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell‐associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2‐ or 3‐approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell‐bound SARS‐CoV‐2 spike protein and can be rapidly adjusted to qui...
B cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteract... more B cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteracting phosphatases that currently are less well studied in their regulation of BCR signaling. Here, we used the B cell line Ramos to identify and quantify human B cell signaling components. Specifically, a protein tyrosine phosphatase profiling revealed a high expression of the protein tyrosine phosphatase 1B (PTP1B) in Ramos and human naïve B cells. The loss of PTP1B leads to increased B cell activation. Through substrate trapping in combination with quantitative mass spectrometry, we identified 22 putative substrates or interactors of PTP1B. We validated Igα, CD22, PLCγ1/2, CBL, BCAP, and APLP2 as specific substrates of PTP1B in Ramos B cells. The tyrosine kinase BTK and the two adaptor proteins GRB2 and VAV1 were identified as direct binding partners and potential substrates of PTP1B. We showed that PTP1B dephosphorylates the inhibitory receptor protein CD22 at phosphotyrosine 807. We c...
Membrane proteins are organized in nanoscale compartments. Their reorganization plays a crucial r... more Membrane proteins are organized in nanoscale compartments. Their reorganization plays a crucial role in receptor activation and cell signaling. To monitor the organization and reorganization of membrane proteins, we developed a new branched proximity hybridization assay (bPHA) allowing better quantification of the nanoscale protein-protein proximity. In this assay, oligo-coupled binding probes, such as aptamer, nanobody, and antibodies, are used to translate the proximity of target proteins to the proximity of oligos. The closely positioned oligos then serve as a template for a maximum of 400-fold branched DNA (bDNA) signal amplification. The amplified bPHA signal is recorded by flow cytometer, thus enabling proximity studies with high throughput, multiplexing, and single-cell resolution. To demonstrate the potential of the bPHA method, we measured the reorganization of the immunoglobulin M (IgM)-and immunoglobulin D (IgD)-class B cell antigen receptor (BCR) on the plasma membrane and the recruitment of spleen tyrosine kinase (Syk) to the BCR upon B lymphocyte activation.
During B cell development Vx gene rearrangement seems to occur only in 1t-positive pre-B cells. T... more During B cell development Vx gene rearrangement seems to occur only in 1t-positive pre-B cells. To study the role of the A chain in the activation of the Igx locus, we introduced expression vectors carrying different forms of the ,u gene into null pre-B cells. The activation of the Igx locus followed the expression of the membrane form (um) of the it chain. The expression of the secreted form (as) did not result in the activation of the Igx locus. We further show that both forms of the it chain differ in their intracellular transport in pre-B cells.
Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of ma... more Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are non- covalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-a) and 39 kd (Ig-,B), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L6m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD) expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the bm chain.
During the production process of this article, the phrase ''mean values ± SD'' was mistakenly add... more During the production process of this article, the phrase ''mean values ± SD'' was mistakenly added to the legends of Figures , and S1F. In addition, in the legend of Figure , the p value should be **p < 0.01, rather than **p < 0.001, as was indicated. These errors have now been corrected online. We apologize for any confusion this may have caused.
Germ-line transcripts of the immunoglobulin (Ig) and T cell receptor loci are thought to be invol... more Germ-line transcripts of the immunoglobulin (Ig) and T cell receptor loci are thought to be involved in the control of V gene rearrangement by rendering these loci accessible to the recombinases. We have analyzed the transcriptional activity of germ-line K alleles in two bone marrow-derived Abelsonmurine leukemia virus transformed pre-B cells: 300-19, a null cell line, and P8 a A-producing line. We found a novel germ-line JK transcript starting immediately upstream of JKI and spliced to CK. The potential role of this transcript in the opening of the Ig K locus as well as in the ordered usage of JK segments is discussed.
Notch receptors play various roles for cell fate decisions in developing organs, although their f... more Notch receptors play various roles for cell fate decisions in developing organs, although their functions at the cell level are poorly understood. Recently, we found that Notch1 and its ligand are each expressed in juxtaposed cell compartments in the follicles of the bursa of Fabricius, the central organ for chicken B cell development. To examine the function of Notch1 in B cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line, DT40, by a Cre/loxPmediated inducible expression system. Remarkably, the active Notch1 caused growth suppression of the cells, accompanied by a cell cycle inhibition at the G 1 phase and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling, also induced the apoptosis, but no cell cycle inhibition. Thus, Notch1 signaling induces apoptosis of the B cells through Hairy1, and the G 1 cell cycle arrest through other pathways. This novel function of Notch1 may account for the recent observations indicating the selective inhibition of early B cell development in mice by Notch1.
During the preparation of figures for the above article, we inadvertently rotated the astrin blot... more During the preparation of figures for the above article, we inadvertently rotated the astrin blot in Figure by 180 , and we inserted incorrect images in Figures and. The correct and incorrect images were derived from experiments conducted in parallel and are quite similar in appearance. We mislabeled them when we retrieved them for figure preparation. In Figure , we inserted an incorrect image for astrin. In Figure , we inserted an incorrect image for raptor-astrin PLA under arsenite treatment in the right panel. Furthermore, we used DAPI for nuclear staining in PLA but instead indicated Hoechst in the figure legends. These errors do not affect the results or interpretation of any of these experiments. The corrected images have been inserted into the figures below, and the figures and figure legends of PLA panels have been corrected online. We regret these errors and apologize for the inconvenience that they may have caused.
Signals processed through the B cell antigen receptor (BCR) control both the proliferation and di... more Signals processed through the B cell antigen receptor (BCR) control both the proliferation and differentiation of B lymphocytes. How these different signaling modes are established at the BCR is poorly understood. We show that a conserved arginine in the tail sequence of the Igα subunit of the BCR is methylated by the protein arginine methyltransferase 1. This modification negatively regulates the calcium and PI-3 kinase pathways of the BCR while promoting signals leading to B cell differentiation. Thus, Igα arginine methylation can play an important role in specifying the outcome of BCR signaling.
Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD ... more Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD immunoglobulins. The structural and functional differences between the two receptor classes are poorly understood. Recently two proteins of 29 and 31 kDa (BAP29 and BAP31) have been described that are preferentially associated with membrane IgD but only weakly with membrane IgM. We describe here the cloning of fulllength murine and human BAP31 cDNAs encoding proteins of 245 and 246 amino acids respectively. The two BAP31 proteins are 95% identical. The BAP31 gene is ubiquitously expressed in murine tissues and is located on the X chromosome in both mouse and man. The murine BAP31 protein has 43% sequence identity to murine BAP29. Both proteins have a hydrophobic N-terminus and an a-helical C-terminus which ends with a KKXX motif implicated in vesicular transport. By a mutational analysis we have identified amino acids in the transmembrane sequence of the 6m chain that are critical for binding to BAP31/BAP29. A structural model of the BAPs and their potential functions are discussed.
B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling streng... more B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR)1 or hyperactivation above maximum (for example, self-reactive BCR)2,3 thresholds of signalling strength causes negative selection. In 25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), whichmimics constitutively active pre-BCR signalling4,5. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival6. We tested the hypothesis that targeted hyperactivation—above a maximum threshold—will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR–ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patientderived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1)9, we demonstrated that pharmacological hyperactivation of SYKand engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
The recently determined cryo-EM structures of the T cell antigen receptor (TCR) and B cell antige... more The recently determined cryo-EM structures of the T cell antigen receptor (TCR) and B cell antigen receptor (BCR) show in molecular details the interactions of the ligand-binding part with the signaling subunits but they do not reveal the signaling mechanism of these antigen receptors. Without knowing the molecular basis of antigen sensing by these receptors, a rational design of optimal vaccines is not possible. The existence of conserved amino acids (AAs) that are not involved in the subunit interaction suggests that antigen receptors form higher complexes and/or have lateral interactors that control their activity. Here, I describe evolutionary conserved leucine zipper (LZ) motifs within the transmembrane domains (TMD) of antigen and coreceptor components that are likely to be involved in the oligomerization and lateral interaction of antigen receptor complexes on T and B cells. These immunoreceptor coupling and organization motifs (ICOMs) are also found within the TMDs of other ...
B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen r... more B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have studied the cell surface features of Ramos B cells and the spatiotemporal organization of the IgM-BCR using lattice light sheet microscopy in combination with tailored custom-built 4D image analysis. Ramos B cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A proportion of membrane-localized IgM-BCR was found in clusters, which were associated with the ridges and the microvilli. The dynamic ridge network organization and the IgM-BCR cluster mobility were linked and both were controlled by Arp2/3 complex activity. Our results suggest that topographical features of the cell surface govern the distribution and dynamic localization of IgM-BCR clusters to facilitate ...
The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M... more The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M, D, G, A or E for antigen recognition and a disulfide-linked heterodimer between Igα and Igβ (Igα/β, also known as CD79A and CD79B) that functions as the signalling entity. The organizing principle of BCR assembly remains elusive. Here we report the cryo-electron microscopy structures of the intact IgM class BCR at 8.2 Å resolution and its Fab-deleted form (IgM BCRΔFab) at 3.6 Å resolution. At the ectodomain (ECD), Igα and Igβ position their respective Ig folds roughly in parallel with an approximate 2-fold symmetry, which is distinct from structures of Igβ/β homodimers. Unlike previous predictions, the BCR structure displays an asymmetric arrangement, in which the Igα/β ECD heterodimer mainly uses Igα to associate with Cµ3-Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC’) empty. The transmembrane domain (TMD) helices of the two µHCs also deviate from the 2...
We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-C... more We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-CoV-2-derived peptides performed in March and April 2020, respectively. The first set of peptides contained eight peptides predicted to bind to the individual’s HLA molecules. The second set consisted of ten peptides predicted to bind promiscuously to several HLA-DR allotypes. The vaccine formulation contained the new TLR 1/2 agonist XS15 and was administered as an emulsion in Montanide as a single subcutaneous injection. Peripheral blood mononuclear cells isolated from blood drawn before and after vaccinations were assessed using Interferon-γ ELISpot assays and intracellular cytokine staining. We detected vaccine-induced CD4 T cell responses against six out of 11 peptides predicted to bind to HLA-DR after 19 days, following vaccination, for one peptide already at day 12. We used these results to support the design of a T-cell-inducing vaccine for application in high-risk patients, with w...
The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneum... more The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneumonia and other severe disease manifestations known as COVID-19. This health crisis has resulted in a significant effort to stop the spread of this new coronavirus. However, while propagating itself in the human population, the virus accumulates mutations and generates new variants with increased fitness and the ability to escape the human immune response. Here we describe a color-based barcoded spike flow cytometric assay (BSFA) that is particularly useful to evaluate and directly compare the humoral immune response directed against either wild type (WT) or mutant spike (S) proteins or the receptor-binding domains (RBD) of SARS-CoV-2. This assay employs the human B lymphoma cell line Ramos, transfected for stable expression of WT or mutant S proteins or a chimeric RBD-CD8 fusion protein. We find that the alpha and beta mutants are more stably expressed than the WT S protein on the Ramos ...
To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and succ... more To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and successful vaccination against coronavirus disease 2019 (COVID‐19), the kinetics of neutralizing or blocking anti‐SARS‐CoV‐2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS‐CoV‐2 blocking assay (SUBA) using immobilized recombinant human angiotensin‐converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS‐CoV‐2 spike protein. Spike protein‐expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell‐associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2‐ or 3‐approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell‐bound SARS‐CoV‐2 spike protein and can be rapidly adjusted to qui...
B cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteract... more B cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteracting phosphatases that currently are less well studied in their regulation of BCR signaling. Here, we used the B cell line Ramos to identify and quantify human B cell signaling components. Specifically, a protein tyrosine phosphatase profiling revealed a high expression of the protein tyrosine phosphatase 1B (PTP1B) in Ramos and human naïve B cells. The loss of PTP1B leads to increased B cell activation. Through substrate trapping in combination with quantitative mass spectrometry, we identified 22 putative substrates or interactors of PTP1B. We validated Igα, CD22, PLCγ1/2, CBL, BCAP, and APLP2 as specific substrates of PTP1B in Ramos B cells. The tyrosine kinase BTK and the two adaptor proteins GRB2 and VAV1 were identified as direct binding partners and potential substrates of PTP1B. We showed that PTP1B dephosphorylates the inhibitory receptor protein CD22 at phosphotyrosine 807. We c...
Membrane proteins are organized in nanoscale compartments. Their reorganization plays a crucial r... more Membrane proteins are organized in nanoscale compartments. Their reorganization plays a crucial role in receptor activation and cell signaling. To monitor the organization and reorganization of membrane proteins, we developed a new branched proximity hybridization assay (bPHA) allowing better quantification of the nanoscale protein-protein proximity. In this assay, oligo-coupled binding probes, such as aptamer, nanobody, and antibodies, are used to translate the proximity of target proteins to the proximity of oligos. The closely positioned oligos then serve as a template for a maximum of 400-fold branched DNA (bDNA) signal amplification. The amplified bPHA signal is recorded by flow cytometer, thus enabling proximity studies with high throughput, multiplexing, and single-cell resolution. To demonstrate the potential of the bPHA method, we measured the reorganization of the immunoglobulin M (IgM)-and immunoglobulin D (IgD)-class B cell antigen receptor (BCR) on the plasma membrane and the recruitment of spleen tyrosine kinase (Syk) to the BCR upon B lymphocyte activation.
During B cell development Vx gene rearrangement seems to occur only in 1t-positive pre-B cells. T... more During B cell development Vx gene rearrangement seems to occur only in 1t-positive pre-B cells. To study the role of the A chain in the activation of the Igx locus, we introduced expression vectors carrying different forms of the ,u gene into null pre-B cells. The activation of the Igx locus followed the expression of the membrane form (um) of the it chain. The expression of the secreted form (as) did not result in the activation of the Igx locus. We further show that both forms of the it chain differ in their intracellular transport in pre-B cells.
Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of ma... more Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are non- covalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-a) and 39 kd (Ig-,B), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L6m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD) expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the bm chain.
During the production process of this article, the phrase ''mean values ± SD'' was mistakenly add... more During the production process of this article, the phrase ''mean values ± SD'' was mistakenly added to the legends of Figures , and S1F. In addition, in the legend of Figure , the p value should be **p < 0.01, rather than **p < 0.001, as was indicated. These errors have now been corrected online. We apologize for any confusion this may have caused.
Germ-line transcripts of the immunoglobulin (Ig) and T cell receptor loci are thought to be invol... more Germ-line transcripts of the immunoglobulin (Ig) and T cell receptor loci are thought to be involved in the control of V gene rearrangement by rendering these loci accessible to the recombinases. We have analyzed the transcriptional activity of germ-line K alleles in two bone marrow-derived Abelsonmurine leukemia virus transformed pre-B cells: 300-19, a null cell line, and P8 a A-producing line. We found a novel germ-line JK transcript starting immediately upstream of JKI and spliced to CK. The potential role of this transcript in the opening of the Ig K locus as well as in the ordered usage of JK segments is discussed.
Notch receptors play various roles for cell fate decisions in developing organs, although their f... more Notch receptors play various roles for cell fate decisions in developing organs, although their functions at the cell level are poorly understood. Recently, we found that Notch1 and its ligand are each expressed in juxtaposed cell compartments in the follicles of the bursa of Fabricius, the central organ for chicken B cell development. To examine the function of Notch1 in B cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line, DT40, by a Cre/loxPmediated inducible expression system. Remarkably, the active Notch1 caused growth suppression of the cells, accompanied by a cell cycle inhibition at the G 1 phase and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling, also induced the apoptosis, but no cell cycle inhibition. Thus, Notch1 signaling induces apoptosis of the B cells through Hairy1, and the G 1 cell cycle arrest through other pathways. This novel function of Notch1 may account for the recent observations indicating the selective inhibition of early B cell development in mice by Notch1.
During the preparation of figures for the above article, we inadvertently rotated the astrin blot... more During the preparation of figures for the above article, we inadvertently rotated the astrin blot in Figure by 180 , and we inserted incorrect images in Figures and. The correct and incorrect images were derived from experiments conducted in parallel and are quite similar in appearance. We mislabeled them when we retrieved them for figure preparation. In Figure , we inserted an incorrect image for astrin. In Figure , we inserted an incorrect image for raptor-astrin PLA under arsenite treatment in the right panel. Furthermore, we used DAPI for nuclear staining in PLA but instead indicated Hoechst in the figure legends. These errors do not affect the results or interpretation of any of these experiments. The corrected images have been inserted into the figures below, and the figures and figure legends of PLA panels have been corrected online. We regret these errors and apologize for the inconvenience that they may have caused.
Signals processed through the B cell antigen receptor (BCR) control both the proliferation and di... more Signals processed through the B cell antigen receptor (BCR) control both the proliferation and differentiation of B lymphocytes. How these different signaling modes are established at the BCR is poorly understood. We show that a conserved arginine in the tail sequence of the Igα subunit of the BCR is methylated by the protein arginine methyltransferase 1. This modification negatively regulates the calcium and PI-3 kinase pathways of the BCR while promoting signals leading to B cell differentiation. Thus, Igα arginine methylation can play an important role in specifying the outcome of BCR signaling.
Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD ... more Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD immunoglobulins. The structural and functional differences between the two receptor classes are poorly understood. Recently two proteins of 29 and 31 kDa (BAP29 and BAP31) have been described that are preferentially associated with membrane IgD but only weakly with membrane IgM. We describe here the cloning of fulllength murine and human BAP31 cDNAs encoding proteins of 245 and 246 amino acids respectively. The two BAP31 proteins are 95% identical. The BAP31 gene is ubiquitously expressed in murine tissues and is located on the X chromosome in both mouse and man. The murine BAP31 protein has 43% sequence identity to murine BAP29. Both proteins have a hydrophobic N-terminus and an a-helical C-terminus which ends with a KKXX motif implicated in vesicular transport. By a mutational analysis we have identified amino acids in the transmembrane sequence of the 6m chain that are critical for binding to BAP31/BAP29. A structural model of the BAPs and their potential functions are discussed.
The IgM and IgD classes of antigen receptor can perform different functions on B cells. However, ... more The IgM and IgD classes of antigen receptor can perform different functions on B cells. However, so far no class-specific components communicating with the cytoplasm have been found in the two antigen receptors. We have employed a new biotinylation protocol to search for intracellular membrane Ig-associated proteins. Here we describe two proteins of 29 and 31 kDa that are associated with membrane IgD and to some extent with membrane IgM. The membrane IgM molecule is associated specifically with three proteins of 32, 37 and 41 kDa. The purification and sequencing of the two mIgD-associated proteins revealed that they are novel proteins which are related to each other. These proteins may be the missing link between the antigen receptor and the cytoskeleton and may contribute to functional differences between membrane IgM and membrane IgD.
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Papers cited selleck products by Michael Reth
(ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1)9, we demonstrated that pharmacological hyperactivation of SYKand engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
Papers by Michael Reth
(ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1)9, we demonstrated that pharmacological hyperactivation of SYKand engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.