Discovering immunoreceptor coupling and organization motifs.

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 important receptor types and viral envelope proteins. This discovery suggests that antigen receptors do not function as isolated entities but rather as part of an ICOM-based interactome that controls their nanoscale organization on resting cells and their dynamic remodeling on activated lymphocytes.

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters.

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 used lattice light-sheet microscopy in combination with tailored custom-built 4D image analysis to study the cell-surface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM-BCR. Ramos B-cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membrane-localized IgM-BCR was found in clusters, which were mainly 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 dynamic topographical features of the cell surface govern the localization and transport of IgM-BCR clusters to facilitate antigen screening by B cells.

Structural principles of B cell antigen receptor assembly.

The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition1-3 and a disulfide-linked Igα (also known as CD79A) and Igß (also known as CD79B) heterodimer (Igα/ß) that functions as the signalling entity through intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5. The organizing principle of the BCR remains unknown. Here we report cryo-electron microscopy structures of mouse full-length IgM BCR and its Fab-deleted form. At the ectodomain (ECD), the Igα/ß heterodimer mainly uses Igα to associate with Cµ3 and Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC') unbound. The transmembrane domain (TMD) helices of µHC and µHC' interact with those of the Igα/ß heterodimer to form a tight four-helix bundle. The asymmetry at the TMD prevents the recruitment of two Igα/ß heterodimers. Notably, the connecting peptide between the ECD and TMD of µHC intervenes in between those of Igα and Igß to guide TMD assembly through charge complementarity. Weaker but distinct density for the Igß ITAM nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B cell signalling and designing rational therapies against BCR-mediated diseases.

Human CD38 regulates B cell antigen receptor dynamic organization in normal and malignant B cells.

CD38 is a multifunctional protein expressed on the surface of B cells in healthy individuals but also in B cell malignancies. Previous studies have suggested a connection between CD38 and components of the IgM class B cell antigen receptor (IgM-BCR) and its coreceptor complex. Here, we provide evidence that CD38 is closely associated with CD19 in resting B cells and with the IgM-BCR upon engagement. We show that targeting CD38 with an antibody, or removing this molecule with CRISPR/Cas9, inhibits the association of CD19 with the IgM-BCR, impairing BCR signaling in normal and malignant B cells. Together, our data suggest that CD38 is a new member of the BCR coreceptor complex, where it exerts a modulatory effect on B cell activation upon antigen recognition by regulating CD19. Our study also reveals a new mechanism where α-CD38 antibodies could be a valuable option in therapeutic approaches to B cell malignancies driven by aberrant BCR signaling.

B cells expressing IgM B cell receptors of HIV-1 neutralizing antibodies discriminate antigen affinities by sensing binding association rates.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

Surface Ig variable domain glycosylation affects autoantigen binding and acts as threshold for human autoreactive B cell activation.

The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

The IL-3, IL-5, and GM-CSF common receptor beta chain mediates oncogenic activity of FLT3-ITD-positive AML.

FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML.

A Barcoded Flow Cytometric Assay to Explore the Antibody Responses Against SARS-CoV-2 Spike and Its Variants.

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.

Quantitative proteomics identifies PTP1B as modulator of B cell antigen receptor signaling.

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 conclude that PTP1B negatively modulates BCR signaling by dephosphorylating distinct phosphotyrosines in B cell-specific receptor proteins and various downstream signaling components.

A surrogate cell-based SARS-CoV-2 spike blocking assay.

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 quickly pre-screen already approved therapeutic antibodies or sera from vaccinated individuals for their ACE2 blocking activities against any emerging SARS-CoV-2 variants.

Designing a SARS-CoV-2 T-Cell-Inducing Vaccine for High-Risk Patient Groups.

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 weakened lymphocyte performance. Meanwhile, an according vaccine, incorporating T cell epitopes predominant in convalescents, is undergoing clinical trial testing.

CD20 as a gatekeeper of the resting state of human B cells.

CD20 is a B cell-specific membrane protein and represents an attractive target for therapeutic antibodies. Despite widespread usage of anti-CD20 antibodies for B cell depletion therapies, the biological function of their target remains unclear. Here, we demonstrate that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. CRISPR/Cas9-mediated ablation of CD20 in resting B cells resulted in relocalization and interaction of the IgM-class B cell antigen receptor with the coreceptor CD19. This receptor rearrangement led to a transient activation of B cells, accompanied by the internalization of many B cell surface marker proteins. Reexpression of CD20 restored the expression of the B cell surface proteins and the resting state of Ramos B cells. Similarly, treatment of Ramos or naive human B cells with the anti-CD20 antibody rituximab induced nanoscale receptor rearrangements and transient B cell activation in vitro and in vivo. A departure from the resting B cell state followed by the loss of B cell identity of CD20-deficient Ramos B cells was accompanied by a PAX5 to BLIMP-1 transcriptional switch, metabolic reprogramming toward oxidative phosphorylation, and a shift toward plasma cell development. Thus, anti-CD20 engagement or the loss of CD20 disrupts membrane organization, profoundly altering the fate of human B cells.

Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes.

The major function of B lymphocytes is to sense antigens and to produce protective antibodies after activation. This function requires the expression of a B-cell antigen receptor (BCR), and evolutionary conserved mechanisms seem to exist that ensure that B cells without a BCR do not develop nor survive in the periphery. Here, we show that the loss of BCR expression on Burkitt lymphoma cells leads to decreased mitochondrial function and impaired metabolic flexibility. Strikingly, this phenotype does not result from the absence of a classical Syk-dependent BCR signal but rather from compromised ER expansion. We show that the reexpression of immunoglobulins (Ig) in the absence of the BCR signaling subunits Igα and Igß rescues the observed metabolic defects. We demonstrate that immunoglobulin expression is needed to maintain ER homeostasis not only in lymphoma cells but also in resting B cells. Our study provides evidence that the expression of BCR components, which is sensed in the ER and shapes mitochondrial function, represents a novel mechanism of metabolic control in B cells.

A new branched proximity hybridization assay for the quantification of nanoscale protein-protein proximity.

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.

TLR induces reorganization of the IgM-BCR complex regulating murine B-1 cell responses to infections.

In mice, neonatally-developing, self-reactive B-1 cells generate steady levels of natural antibodies throughout life. B-1 cells can, however, also rapidly respond to infections with increased local antibody production. The mechanisms regulating these two seemingly very distinct functions are poorly understood, but have been linked to expression of CD5, an inhibitor of BCR-signaling. Here we demonstrate that TLR-mediated activation of CD5+ B-1 cells induced the rapid reorganization of the IgM-BCR complex, leading to the eventual loss of CD5 expression, and a concomitant increase in BCR-downstream signaling, both in vitro and in vivo after infections of mice with influenza virus and Salmonella typhimurium. Both, initial CD5 expression and TLR-mediated stimulation, were required for the differentiation of B-1 cells to IgM-producing plasmablasts after infections. Thus, TLR-mediated signals support participation of B-1 cells in immune defense via BCR-complex reorganization.

A symmetric geometry of transmembrane domains inside the B cell antigen receptor complex.

B lymphocytes have the ability to sense thousands of structurally different antigens and produce cognate antibodies against these molecules. For this they carry on their surface multiple copies of the B cell antigen receptor (BCR) comprising the membrane-bound Ig (mIg) molecule and the Igα/Igß heterodimer functioning as antigen binding and signal transducing components, respectively. The mIg is a symmetric complex of 2 identical membrane-bound heavy chains (mHC) and 2 identical light chains. How the symmetric mIg molecule is asymmetrically associated with only one Igα/Igß heterodimer has been a puzzle. Here we describe that Igα and Igß both carry on one side of their α-helical transmembrane domain a conserved amino acid motif. By a mutational analysis in combination with a BCR rebuilding approach, we show that this motif is required for the retention of unassembled Igα or Igß molecules inside the endoplasmic reticulum and the binding of the Igα/Igß heterodimer to the mIg molecule. We suggest that the BCR forms within the lipid bilayer of the membrane a symmetric Igα-mHC:mHC-Igß complex that is stabilized by an aromatic proline-tyrosine interaction. Outside the membrane this symmetry is broken by the disulfide-bridged dimerization of the extracellular Ig domains of Igα and Igß. However, symmetry of the receptor can be regained by a dimerization of 2 BCR complexes as suggested by the dissociation activation model.

LAMTOR2 (p14) Controls B Cell Differentiation by Orchestrating Endosomal BCR Trafficking.

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.

Pten controls B-cell responsiveness and germinal center reaction by regulating the expression of IgD BCR.

In contrast to other B-cell antigen receptor (BCR) classes, the function of IgD BCR on mature B cells remains largely elusive as mature B cells co-express IgM, which is sufficient for development, survival, and activation of B cells. Here, we show that IgD expression is regulated by the forkhead box transcription factor FoxO1, thereby shifting the responsiveness of mature B cells towards recognition of multivalent antigen. FoxO1 is repressed by phosphoinositide 3-kinase (PI3K) signaling and requires the lipid phosphatase Pten for its activation. Consequently, Pten-deficient B cells expressing knock-ins for BCR heavy and light chain genes are unable to upregulate IgD. Furthermore, in the presence of autoantigen, Pten-deficient B cells cannot eliminate the autoreactive BCR specificity by secondary light chain gene recombination. Instead, Pten-deficient B cells downregulate BCR expression and become unresponsive to further BCR-mediated stimulation. Notably, we observed a delayed germinal center (GC) reaction by IgD-deficient B cells after immunization with trinitrophenyl-ovalbumin (TNP-Ova), a commonly used antigen for T-cell-dependent antibody responses. Together, our data suggest that the activation of IgD expression by Pten/FoxO1 results in mature B cells that are selectively responsive to multivalent antigen and are capable of initiating rapid GC reactions and T-cell-dependent antibody responses.

Antigen Receptor Function in the Context of the Nanoscale Organization of the B Cell Membrane.

The B cell antigen receptor (BCR) plays a central role in the self/nonself selection of B lymphocytes and in their activation by cognate antigen during the clonal selection process. It was long thought that most cell surface receptors, including the BCR, were freely diffusing and randomly distributed. Since the advent of superresolution techniques, it has become clear that the plasma membrane is compartmentalized and highly organized at the nanometer scale. Hence, a complete understanding of the precise conformation and activation mechanism of the BCR must take into account the organization of the B cell plasma membrane. We review here the recent literature on the nanoscale organization of the lymphocyte membrane and discuss how this new information influences our view of the conformational changes that the BCR undergoes during activation.

Carbohydrate-dependent B cell activation by fucose-binding bacterial lectins.

Bacterial lectins are typically multivalent and bind noncovalently to specific carbohydrates on host tissues to facilitate bacterial adhesion. Here, we analyzed the effects of two fucose-binding lectins, BambL from Burkholderia ambifaria and LecB from Pseudomonas aeruginosa, on specific signaling pathways in B cells. We found that these bacterial lectins induced B cell activation, which, in vitro, was dependent on the cell surface expression of the B cell antigen receptor (BCR) and its co-receptor CD19, as well as on spleen tyrosine kinase (Syk) activity. The resulting release of intracellular Ca2+ was followed by an increase in the cell surface abundance of the activation marker CD86, augmented cytokine secretion, and subsequent cell death, replicating all of the events that are observed in vitro upon canonical and antigen-mediated B cell activation. Moreover, injection of BambL in mice resulted in a substantial, BCR-independent loss of B cells in the bone marrow with simultaneous, transient enlargement of the spleen (splenomegaly), as well as an increase in the numbers of splenic B cells and myeloid cells. Together, these data suggest that bacterial lectins can initiate polyclonal activation of B cells through their sole capacity to bind to fucose.