Regulation of BCR-mediated Ca2+ mobilization by MIZ1-TMBIM4 safeguards IgG1+ GC B cell-positive selection.

The transition from immunoglobulin M (IgM) to affinity-matured IgG antibodies is vital for effective humoral immunity. This is facilitated by germinal centers (GCs) through affinity maturation and preferential maintenance of IgG+ B cells over IgM+ B cells. However, it is not known whether the positive selection of the different Ig isotypes within GCs is dependent on specific transcriptional mechanisms. Here, we explored IgG1+ GC B cell transcription factor dependency using a CRISPR-Cas9 screen and conditional mouse genetics. We found that MIZ1 was specifically required for IgG1+ GC B cell survival during positive selection, whereas IgM+ GC B cells were largely independent. Mechanistically, MIZ1 induced TMBIM4, an ancestral anti-apoptotic protein that regulated inositol trisphosphate receptor (IP3R)-mediated calcium (Ca2+) mobilization downstream of B cell receptor (BCR) signaling in IgG1+ B cells. The MIZ1-TMBIM4 axis prevented mitochondrial dysfunction-induced IgG1+ GC cell death caused by excessive Ca2+ accumulation. This study uncovers a unique Ig isotype-specific dependency on a hitherto unidentified mechanism in GC-positive selection.

Long-term retention of antigens in germinal centers is controlled by the spatial organization of the follicular dendritic cell network.

Germinal centers (GCs) require sustained availability of antigens to promote antibody affinity maturation against pathogens and vaccines. A key source of antigens for GC B cells are immune complexes (ICs) displayed on follicular dendritic cells (FDCs). Here we show that FDC spatial organization regulates antigen dynamics in the GC. We identify heterogeneity within the FDC network. While the entire light zone (LZ) FDC network captures ICs initially, only the central cells of the network function as the antigen reservoir, where different antigens arriving from subsequent immunizations colocalize. Mechanistically, central LZ FDCs constitutively express subtly higher CR2 membrane densities than peripheral LZ FDCs, which strongly increases the IC retention half-life. Even though repeated immunizations gradually saturate central FDCs, B cell responses remain efficient because new antigens partially displace old ones. These results reveal the principles shaping antigen display on FDCs during the GC reaction.

Structural basis for Fc receptor recognition of immunoglobulin M.

Immunoglobulin Fc receptors are cell surface transmembrane proteins that bind to the Fc constant region of antibodies and play critical roles in regulating immune responses by activation of immune cells, clearance of immune complexes and regulation of antibody production. FcµR is the immunoglobulin M (IgM) antibody isotype-specific Fc receptor involved in the survival and activation of B cells. Here we reveal eight binding sites for the human FcµR immunoglobulin domain on the IgM pentamer by cryogenic electron microscopy. One of the sites overlaps with the binding site for the polymeric immunoglobulin receptor (pIgR), but a different mode of FcµR binding explains its antibody isotype specificity. Variation in FcµR binding sites and their occupancy reflects the asymmetry of the IgM pentameric core and the versatility of FcµR binding. The complex explains engagement with polymeric serum IgM and the monomeric IgM B-cell receptor (BCR).

Cryomicroscopy reveals the structural basis for a flexible hinge motion in the immunoglobulin M pentamer.

Immunoglobulin M (IgM) is the most ancient of the five isotypes of immunoglobulin (Ig) molecules and serves as the first line of defence against pathogens. Here, we use cryo-EM to image the structure of the human full-length IgM pentamer, revealing antigen binding domains flexibly attached to the asymmetric and rigid core formed by the Cµ4 and Cµ3 constant regions and the J-chain. A hinge is located at the Cµ3/Cµ2 domain interface, allowing Fabs and Cµ2 to pivot as a unit both in-plane and out-of-plane. This motion is different from that observed in IgG and IgA, where the two Fab arms are able to swing independently. A biased orientation of one pair of Fab arms results from asymmetry in the constant domain (Cµ3) at the IgM subunit interacting most extensively with the J-chain. This may influence the multi-valent binding to surface-associated antigens and complement pathway activation. By comparison, the structure of the Fc fragment in the IgM monomer is similar to that of the pentamer, but is more dynamic in the Cµ4 domain.

Unveiling the B cell receptor structure.

Molecular structures provide a road map for understanding and controlling B cell receptor activation.

Digital holography-based 3D particle localization for single-molecule tweezer techniques.

We present a three-dimensional (3D) imaging technique for the fast tracking of microscopic objects in a fluid environment. Our technique couples digital holographic microscopy with three-dimensional localization via parabolic masking. Compared with existing approaches, our method reconstructs 3D volumes from single-plane images, which greatly simplifies image acquisition, reduces the demand on microscope hardware, and facilitates tracking higher densities of microscopic particles while maintaining similar levels of precision. We demonstrate utility of this method in magnetic tweezer experiments, opening their use to multiplexed single-molecule force spectroscopy assays, which were previously limited by particle crowding and fast dissociation times. We propose that our technique will also be useful in other applications that involve the tracking of microscopic objects in three dimensions, such as studies of microorganism motility and 3D flow characterization of microfluidic devices.

Chronic calcium signaling in IgE+ B cells limits plasma cell differentiation and survival.

In contrast to other antibody isotypes, B cells switched to IgE respond transiently and do not give rise to long-lived plasma cells (PCs) or memory B cells. To better understand IgE-BCR-mediated control of IgE responses, we developed whole-genome CRISPR screening that enabled comparison of IgE+ and IgG1+ B cell requirements for proliferation, survival, and differentiation into PCs. IgE+ PCs exhibited dependency on the PI3K-mTOR axis that increased protein amounts of the transcription factor IRF4. In contrast, loss of components of the calcium-calcineurin-NFAT pathway promoted IgE+ PC differentiation. Mice bearing a B cell-specific deletion of calcineurin B1 exhibited increased production of IgE+ PCs. Mechanistically, sustained elevation of intracellular calcium in IgE+ PCs downstream of the IgE-BCR promoted BCL2L11-dependent apoptosis. Thus, chronic calcium signaling downstream of the IgE-BCR controls the self-limiting character of IgE responses and may be relevant to the accumulation of IgE-producing cells in allergic disease.

Endophilin A2 regulates B-cell endocytosis and is required for germinal center and humoral responses.

Antigen-specific B-cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B-cell endosomal trafficking pathways and their specific roles in B-cell responses have not been systematically investigated. Here, we report high-throughput identification of genes regulating B-cell receptor (BCR)-mediated antigen internalization using genome-wide functional screens. We show that antigen internalization depends both on constitutive, clathrin-mediated endocytosis and on antigen-induced, clathrin-independent endocytosis mediated by endophilin A2. Although endophilin A2-mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B-cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2-deficient mice show defects in GC B-cell responses and production of high-affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin-independent intracellular trafficking in GC B-cell clonal expansion and antibody responses.

Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation.

Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are debilitating diseases that share causal mutations in ACVR1, a TGF-ß family type I receptor. ACVR1R206H is a frequent mutation in both diseases. Pathogenic signaling via the SMAD1/5 pathway is mediated by Activin A, but how the mutation triggers aberrant signaling is not known. We show that ACVR1 is essential for Activin A-mediated SMAD1/5 phosphorylation and is activated by two distinct mechanisms. Wild-type ACVR1 is activated by the Activin type I receptors, ACVR1B/C. In contrast, ACVR1R206H activation does not require upstream kinases, but is predominantly activated via Activin A-dependent receptor clustering, which induces its auto-activation. We use optogenetics and live-imaging approaches to demonstrate Activin A-induced receptor clustering and show it requires the type II receptors ACVR2A/B. Our data provide molecular mechanistic insight into the pathogenesis of FOP and DIPG by linking the causal activating genetic mutation to disrupted signaling.

B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments.

Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in synapses of primary mouse B cells and human B cell lines forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction.

MHC class II cell-autonomously regulates self-renewal and differentiation of normal and malignant B cells.

Best known for presenting antigenic peptides to CD4+ T cells, major histocompatibility complex class II (MHC II) also transmits or may modify intracellular signals. Here, we show that MHC II cell-autonomously regulates the balance between self-renewal and differentiation in B-cell precursors, as well as in malignant B cells. Initiation of MHC II expression early during bone marrow B-cell development limited the occupancy of cycling compartments by promoting differentiation, thus regulating the numerical output of B cells. MHC II deficiency preserved stem cell characteristics in developing pro-B cells in vivo, and ectopic MHC II expression accelerated hematopoietic stem cell differentiation in vitro. Moreover, MHC II expression restrained growth of murine B-cell leukemia cell lines in vitro and in vivo, independently of CD4+ T-cell surveillance. Our results highlight an important cell-intrinsic contribution of MHC II expression to establishing the differentiated B-cell phenotype.

Intrinsic properties of human germinal center B cells set antigen affinity thresholds.

Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind, gather, and present antigen to T follicular helper (Tfh) cells. Here, we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens, whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.

Myosin II Synergizes with F-Actin to Promote DNGR-1-Dependent Cross-Presentation of Dead Cell-Associated Antigens.

Conventional type 1 DCs (cDC1s) excel at cross-presentation of dead cell-associated antigens partly because they express DNGR-1, a receptor that recognizes exposed actin filaments on dead cells. In vitro polymerized F-actin can be used as a synthetic ligand for DNGR-1. However, cellular F-actin is decorated with actin-binding proteins, which could affect DNGR-1 recognition. Here, we demonstrate that myosin II, an F-actin-associated motor protein, greatly potentiates the binding of DNGR-1 to F-actin. Latex beads coated with F-actin and myosin II are taken up by DNGR-1+ cDC1s, and antigen associated with those beads is efficiently cross-presented to CD8+ T cells. Myosin II-deficient necrotic cells are impaired in their ability to stimulate DNGR-1 or to serve as substrates for cDC1 cross-presentation to CD8+ T cells. These results provide insights into the nature of the DNGR-1 ligand and have implications for understanding immune responses to cell-associated antigens and for vaccine design.

Mechanics of antigen extraction in the B cell synapse.

B cell encounter with antigen displayed on antigen-presenting cells leads to B cell immune synapse formation, internalisation of the antigen, and stimulation of antibody responses. The sensitivity with which B cells detect antigen, and the quality and quantity of antigen that B cells acquire, depend upon mechanical properties of the immune synapse including interfacial tension, the strength of intermolecular bonds, and the compliance of the molecules and membranes that participate in antigen presentation. In this review, we discuss our current understanding of how these various physical parameters influence B cell antigen extraction in the immune synapse and how a more comprehensive understanding of B cell mechanics may promote the development of new approaches to stimulate the production of desired antibodies.

Myosin IIa Promotes Antibody Responses by Regulating B Cell Activation, Acquisition of Antigen, and Proliferation.

B cell responses are regulated by antigen acquisition, processing, and presentation to helper T cells. These functions are thought to depend on contractile activity of non-muscle myosin IIa. Here, we show that B cell-specific deletion of the myosin IIa heavy chain reduced the numbers of bone marrow B cell precursors and splenic marginal zone, peritoneal B1b, and germinal center B cells. In addition, myosin IIa-deficient follicular B cells acquired an activated phenotype and were less efficient in chemokinesis and extraction of membrane-presented antigens. Moreover, myosin IIa was indispensable for cytokinesis. Consequently, mice with myosin IIa-deficient B cells harbored reduced serum immunoglobulin levels and did not mount robust antibody responses when immunized. Altogether, these data indicate that myosin IIa is a negative regulator of B cell activation but a positive regulator of antigen acquisition from antigen-presenting cells and that myosin IIa is essential for B cell development, proliferation, and antibody responses.

DNA-Based Probes for Measuring Mechanical Forces in Cell-Cell Contacts: Application to B Cell Antigen Extraction from Immune Synapses.

The production of antibodies requires the expansion and selection of high-affinity B cell clones. This process is initiated by antigen uptake through the B cell receptor (BCR), which recognizes and binds antigen displayed on the surface of an antigen-presenting cell (APC). To acquire the antigen, B cells use myosin contractility to physically pull BCR-antigen clusters from the APC membrane. These mechanical forces influence association and dissociation rates of BCR-antigen bonds, resulting in affinity-dependent acquisition of antigen by B cells. Mechanical regulation of B cell antigen acquisition from APCs remains poorly understood, although the recent development of DNA-based force sensors has enabled the measurement of mechanical forces generated in B cell-APC contacts. In this chapter, we describe a protocol to design, synthesize, and purify DNA-based force sensors to measure B cell antigen extraction forces using fluorescence microscopy.