The B-cell antigen receptor (BCR) plays a central role in the development and maintenance of B lymphocytes and the establishment of humoral immunity. In this review, we describe the basic structure of the BCR with a focus on the recently determined cryo-EM structure of the IgM-class BCR. Surprisingly, this structure shows that the ligand-binding part, the membrane-bound immunoglobulin (mIg) and the signalling part, the Igα/Igβ heterodimer, form an asymmetric 1:1 complex, the implications of which are discussed in this review. We then describe the nanoscale organisation of BCR receptor clusters on the B-cell surface and their localisation in the context of the three-dimensional topography of living B cells. We also discuss the different models for the regulation and activation of the BCR by a huge library of structurally different antigens. The second part deals with the lateral interactors and coreceptors of the BCR that either regulate (CD45), amplify (CD19) or terminate (CD22) BCR signalling. The final section provides a detailed and up-to-date overview of the many signalling proteins and pathways that are involved in B-cell activation starting with the kinases that phosphorylate and bind to the immunoreceptor tyrosine-based activation motif (ITAM) within the cytoplasmic tails of the Igα/Igβ signalling subunit. The many downstream substrates of these kinases connect the BCR to a multitude of signalling pathways that regulate the proliferation, survival and differentiation of B lymphocytes into antibody-producing plasma cells. These signalling pathways are intimately connected to the underlying cytoskeleton of the B cell as well as with the internalisation of antigen-bound BCRs. We hope that this review will provide the interested reader with an up-to-date overview of the current knowledge on the structure and signalling function of this important receptor of adaptive B-cell immunity.

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 B cell surface and/or bind with higher affinity to the viral entry receptor ACE2. However, we find a reduce expression of the chimeric RBD-CD8 carrying the point mutation N501Y and E484K characteristic for the alpha and beta variant, respectively. The comparison of the humoral immune response of 12 vaccinated probands with 12 COVID-19 patients shows that after the boost, the S-specific IgG class immune response in the vaccinated group is similar to that of the patient group. However, in comparison to WT the specific IgG serum antibodies bind less well to the alpha variant and only poorly to the beta variant S protein. This is in line with the notion that the beta variant is an immune escape variant of SARS-CoV-2. The IgA class immune response was more variable than the IgG response and higher in the COVID-19 patients than in the vaccinated group. In summary, we think that our BSFA represents a useful tool to evaluate the humoral immunity against emerging variants of SARS-CoV-2 and to analyze new vaccination protocols against these variants.

Emotions coordinate our behavior and physiological states during survival-salient events and pleasurable interactions. Even though we are often consciously aware of our current emotional state, such as anger or happiness, the mechanisms giving ...Emotions are often felt in the body, and somatosensory feedback has been proposed to trigger conscious emotional experiences. Here we reveal maps of bodily sensations associated with different emotions using a unique topographical self-report method. In ...

B-1 lymphocytes are neonatally-derived, self-reactive, innate-like B cells that produce natural IgM at steady state. B-1 cells also actively respond to infections with induced local IgM production. These two distinct functions of B-1 cells have been linked to the expression CD5, an inhibitor of BCR signaling, with CD5+ cells thought to contribute to steady state IgM, and CD5− cells to local IgM production during infection. The mechanisms underlying such “division of labor” are poorly understood. We aim to test our hypothesis that CD5+ B-1 cells are stimulated by innate signals via TLR, during infection, resulting in their activation and differentiation into CD5− IgM antibody-secreting cells. For that we first cultured CD5+ B-1 cells with TLR9 agonist, ODN-CpG. The cells proliferated, secreted IgM and showed increased Nur77 expression, Akt phosphorylation and interaction of Syk with CD79a, signs of BCR signaling. They lost expression of the BCR-inhibitor CD5 and upregulated the plasma cell marker CD138+. Following influenza infection of mice, CD5+ B-1 cells migrated to the mediastinal lymph nodes (medLN) where they lost CD5 and became IgM secreting cells. Both, global TLR−/− mice and chimeric mice lacking TLR only on B-1 cells had reduced numbers of B-1 derived plasma cells and antibody secreting cells in the medLN compared to controls. Our findings show that TLR-mediated activation of CD5+ B-1 cells results in the rapid reorganization of the BCR-complex, followed by loss of CD5 and that TLR stimulation of CD5+ B-1 is required for differentiation of B-1 cells to infection in vivo. Overall our data suggest that CD5+ and CD5− B-1 cells are not distinct subsets, but rather that CD5 expression indicates the activation state of B-1 cells.

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.

B-cell development and function depend on stage-specific signaling through the B-cell antigen receptor (BCR). Signaling and intracellular trafficking of the BCR are connected, but the molecular mechanisms of this link are incompletely understood. Here, we investigated the role of the endosomal adaptor protein and member of the LAMTOR/Ragulator complex LAMTOR2 (p14) in B-cell development. Efficient conditional deletion of LAMTOR2 at the pre-B1 stage using mb1-Cre mice resulted in complete developmental arrest. Deletion of LAMTOR2 using Cd19-Cre mice permitted analysis of residual B cells at later developmental stages, revealing that LAMTOR2 was critical for the generation and activation of mature B lymphocytes. Loss of LAMTOR2 resulted in aberrant BCR signaling due to delayed receptor internalization and endosomal trafficking. In conclusion, we identify LAMTOR2 as critical regulator of BCR trafficking and signaling that is essential for early B-cell development in mice.

The B-cell antigen receptor (BCR) is one of the most abundant receptors on the surface of B cells with roughly 100,000-200,000 copies per cell. Signaling through the BCR is crucial for the activation and differentiation of B cells. Unlike other receptors, the BCR can be activated by a large set of structurally different ligands, but the molecular mechanism of BCR activation is still a matter of controversy. Although dominant for a long time, the cross-link model (CLM) of BCR activation is not supported by recent studies of the nanoscale organization of the BCR on the surface of resting B cells. In contrast to the prediction of CLM, the numerous BCR complexes on these cells are not randomly distributed monomers but rather form oligomers which reside within membrane confinements. This finding is more in line with the dissociation activation model (DAM), wherein B-cell activation is accompanied by an opening of the auto-inhibited BCR oligomers instead of a cross-linking of the BCR monomers. In this review, we discuss in detail the new findings and their implications for BCR signaling.

B-cell chronic lymphocytic leukemia (CLL) is one of the most prevalent B cell malignancies in adults and characterized by expansion of monoclonal mature B cells. Survival and proliferation of CLL cells depends on microenvironmental contact in lymphoid organs. The transmembrane glycoprotein CD38 acts as an important mediator of survival, proliferation and migration signals for CLL cells, and its expression is associated with poor prognosis. Spleen tyrosine kinase (SYK) is a central element of the B-cell receptor signal transduction pathway and has additionally been shown to be involved in cytokine and integrine signaling. In this study we demonstrate direct involvement of SYK in the CD38 signaling pathway in primary CLL samples.

A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis , on the surface of the same B cell or in trans , on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis- ligand binding of CD22 is crucial for the regulation of B-cell Ca 2+ signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca 2+ signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca 2+ responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.

Upon B-cell antigen receptor (BCR) activation, the protein tyrosine kinase Syk phosphorylates the adaptor protein SH2 domain-containing leukocyte protein of 65 kDa (SLP-65), thus coupling the BCR to diverse signalling pathways. Here, we report that SLP-65 is not only a downstream target and substrate of Syk but also a direct binding-partner and activator of this kinase. This positive feedback is mediated by the binding of the SH2 domain of SLP-65 to an autophosphorylated tyrosine of Syk. The mutant B cells that cannot form the Syk/SLP-65 complex are defective in BCR-induced extracellular signal-regulated kinase, nuclear factor kappa B and nuclear factor of activated T cells, but not Akt activation, and are blocked in B-cell development. Furthermore, we show that formation of the Syk/SLP-65 complex is required for sustained Ca(2+) responses in activated B cells. We suggest that after activation and internalization of the BCR, Syk remains active as part of a membrane-bound Syk/SLP-65 complex controlling sustained signalling and calcium influx.

The cover image was specifically designed based on an illustration provided by Minguet et al., the authors of Enhanced B-cell activation mediated by TLR4 and BCR crosstalk (pp. 2475–2487). The authors demonstrate that while simultaneous stimulation of TLR4 and BCR activates B cells in an additive manner, antigen-coupled LPS activates B cells synergistically. The authors proposed this image based on the idea that “science is multidimensional and will never be finished or completely understood”. The illustration was further enhanced with images taken from various studies published in this issue of the European Journal of Immunology.

Nature 433, 647–653 (2005) It has been drawn to Nature's attention that K.R.C., S.E., C.-L.C., A.M. and K.-L.L. filed a patent application relevant to this work (patent number WO 2004/070013) in 2004, which should therefore have been declared as a competing financial interest.

Human T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia and induces autoimmune disease. Previous analyses of tax transgenic mice suggested that protection of peripheral T-cells from Fas-mediated apoptosis by virus-encoded oncoprotein Tax was relevant to the onset of HTLV-I-induced diseases. Here, we show the high level expression of cellular FLICE/caspase-8-inhibitory protein (c-FLIP) in Tax-expressing HTLV-I-infected T-cells. The silencing of c-FLIP expression by a lentivirus-based RNA interference system rendered Tax-positive HTLV-I-infected T-cells sensitive to Fas-mediated apoptosis. Exogenously expressed Tax by using a conditional Cre-loxP-mediated inducible system also inhibited Fas-mediated apoptosis by up-regulating c-FLIP expression in HTLV-I-negative T-cells. Tax mutant d3 which cannot activate CREB/ATF1, while another M22 mutant which cannot activate NF-kappa B did not, suppressed Fas-mediated apoptosis by inducing c-FLIP expression. Furthermore, expression of the dominant negative mutant of either NF-kappa B or I kappa B alpha canceled not only c-FLIP expression but also inhibitory activity against Fas-mediated apoptosis by Tax. Inactivation of NFAT, however, did not decrease the expression of c-FLIP in HTLV-I-infected T-cells. Taken together, Tax inhibits Fas-mediated apoptosis by up-regulating c-FLIP expression in HTLV-I-infected cells, and NF-kappa B activity plays an essential role in the up-regulation of c-FLIP.

The proliferation and differentiation of lymphocytes are regulated by receptors localized on the cell surface. Engagement of these receptors induces the activation of intracellular signaling proteins that transmit the receptor signals to distinct targets and control the cellular responses. The first signaling proteins to be discovered in higher organisms were the products of oncogenes. For example, the kinases Src and Abelson (Abl) were originally identified as oncogenes and were later characterized as important proteins for signal transduction in various cell types, including lymphocytes. Now, as many cellular signaling molecules have been discovered and ordered into certain pathways, we can better understand why particular signaling proteins are associated with tumorigenesis. In this review, we discuss recent progress in unraveling the molecular mechanisms of signaling pathways that control the proliferation and differentiation of early B cells. We point out the concepts of auto-inhibition and subcellular localization as crucial aspects in the regulation of B cell signaling.

The chicken c- mil / raf -1 gene (formerly also known as c- mht ) was originally identified in the search for the cellular counterpart to the v- mil oncogene of the Mill Hill 2 retrovirus and was among the first cellular proto-oncogenes discovered. Although the c- mil / raf -1 promotor, as well as the exons transduced into v- mil , were characterized in detail, an entire map of this locus has never been published. Here, we now report the location of five previously unmapped exons. In addition, we have noticed inconsistent numbering of the c- mil / raf -1 exons in the literature and the GenBank database. Thus, we provide here a complete map of the c- mil / raf -1 gene and a revision of the exon numbers. Comparison of the chicken c- mil / raf -1 gene with those of other vertebrates suggests that the numbers and lengths of the translated exons of the raf -1 locus were established early in the vertebrate lineage and have been conserved during the divergent evolution of teleosts and tetrapods.