B Cell Receptor Microclusters Research Articles

Spatial organization and early signaling of the B-cell receptor in CLL.

Most chronic lymphocytic leukemia (CLL) clones express B-cell receptors (BcR) of both IgM/IgD isotypes; however, 5%–10% of CLL cases express isotype-switched immunoglobulin G (IgG). The early signaling and spatial patterning of the various BcRs at steady state and after activation are still fully unresolved. Herein, we show higher expression of the BcR signalosome elements and a more robust constitutive cell-intrinsic proximal BcR signaling in CLL with unmutated IGHV expressing IgM isotype (IgM U-CLL), compared with IGHV-mutated CLL (M-CLL) expressing either IgM or IgG isotypes. IgM in U-CLL is frequently located in the membrane plane in polarized patches, occasionally in caps, and sometimes inside the cells. Among M-CLL, IgM is scattered laterally in the membrane plane in a similar pattern as seen in normal B cells, whereas IgG is dispersed around the cell membrane in smaller clusters than in IgM U-CLL. Upon BcR engagement, both IgG and IgM expressing M-CLL showed attenuated signaling and only slight spatial reorganization dynamics of BcR microclusters and internalization, compared with the extensive reorganization and internalization of the BcR in IgM expressing U-CLL. The global gene signature of IgG M-CLL was closely related to that of IgM M-CLL rather than IgM U-CLL. Overall, we report fundamental differences in the basal composition, biochemical status, and spatial organization of the BcR in the three examined immunogenetic CLL subtypes that correlate with their clinical behavior. On the basis of our findings, IgG class-switched M-CLL likely represents the same disease as IgM M-CLL rather than a different biological and/or clinical entity.

A B-cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation.

B-cell activation and immune synapse (IS) formation with membrane-bound antigens are actin-dependent processes that scale positively with the strength of antigen-induced signals. Importantly, ligating the B-cell integrin, LFA-1, with ICAM-1 promotes IS formation when antigen is limiting. Whether the actin cytoskeleton plays a specific role in integrin-dependent IS formation is unknown. Here, we show using super-resolution imaging of mouse primary B cells that LFA-1:ICAM-1 interactions promote the formation of an actomyosin network that dominates the B-cell IS. This network is created by the formin mDia1, organized into concentric, contractile arcs by myosin 2A, and flows inward at the same rate as B-cell receptor (BCR):antigen clusters. Consistently, individual BCR microclusters are swept inward by individual actomyosin arcs. Under conditions where integrin is required for synapse formation, inhibiting myosin impairs synapse formation, as evidenced by reduced antigen centralization, diminished BCR signaling, and defective signaling protein distribution at the synapse. Together, these results argue that a contractile actomyosin arc network plays a key role in the mechanism by which LFA-1 co-stimulation promotes B-cell activation and IS formation.

A PI(4,5)P2-derived “gasoline engine model” for the sustained B cell receptor activation

Abstract To efficiently initiate activation responses against rare ligands in the microenvironment, lymphocytes employ sophisticated mechanisms involving signaling amplification. Recently, a signaling amplification mechanism initiated from phosphatidylinositol (PI) 4, 5-biphosphate [PI(4,5)P2] hydrolysis and synthesis for sustained B cell activation has been reported. Antigen and B cell receptor (BCR) recognition triggered the prompt reduction of PI(4,5)P2 density within the BCR microclusters, which led to the positive feedback for the synthesis of PI(4,5)P2 outside of the BCR microclusters. At single molecule level, the diffusion of PI(4,5)P2 was slow, allowing for the maintenance of a PI(4,5)P2 density gradient between the inside and outside of the BCR microclusters and the persistent supply of PI(4,5)P2 from outside to inside of the BCR microclusters. Here, we review studies that have contributed to uncovering the molecular mechanisms of PI(4,5)P2-derived signaling amplification model. Based on these studies, our recent work supported a “gasoline engine model” in which the activation of B cell signaling inside the microclusters is similar to the working principle of burning gasoline within the engine chamber of a gasoline engine.

A PI(4,5)P2-derived "gasoline engine model" for the sustained B cell receptor activation.

To efficiently initiate activation responses against rare ligands in the microenvironment, lymphocytes employ sophisticated mechanisms involving signaling amplification. Recently, a signaling amplification mechanism initiated from phosphatidylinositol (PI) 4, 5-biphosphate [PI(4,5)P2] hydrolysis and synthesis for sustained B cell activation has been reported. Antigen and B cell receptor (BCR) recognition triggered the prompt reduction of PI(4,5)P2 density within the BCR microclusters, which led to the positive feedback for the synthesis of PI(4,5)P2 outside of the BCR microclusters. At single molecule level, the diffusion of PI(4,5)P2 was slow, allowing for the maintenance of a PI(4,5)P2 density gradient between the inside and outside of the BCR microclusters and the persistent supply of PI(4,5)P2 from outside to inside of the BCR microclusters. Here, we review studies that have contributed to uncovering the molecular mechanisms of PI(4,5)P2-derived signaling amplification model. Based on these studies, we proposed a "gasoline engine model" in which the activation of B cell signaling inside the microclusters is similar to the working principle of burning gasoline within the engine chamber of a gasoline engine. We also discuss the evidences showing the potential universality of this model and future prospects.

Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.

The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production

A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab′)2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2–Rac axis in PC differentiation and IgG antibody responses.

PI3K- and PTEN-Governed PIP<sub>2</sub> and PIP<sub>3</sub> Equilibrium Regulates the Activation and Function of B Cells Through Dock2 Controlled Growth of Antigen Receptor Microclusters

The growth of B cell receptor (BCR) microclusters upon antigen stimulation is one of the most important events in B cell activation, however the underlying molecular mechanism unknown. Here, we systematically addressed this question through a combination of molecular imaging, genetic ablation, pharmacological inhibitor and mice model approaches. We showed that the earliest BCR signaling molecules, Lyn, Syk and PLCγ2 are required for B cells to initiate the growth of BCR microclusters. PI3K-mediated PIP3 production but not Akt and PDK1 activation was required for the sustained growth of the BCR microclusters. The PI3K counterpart signaling molecule, PTEN, potently inhibited the growth of the BCR microclusters, which was dependent on its membrane binding activities and on the lipid phosphatase but not protein phosphatase activity. Mechanistically, we showed that PIP3-dependent recruitment and activation of a unique guanine nucleotide exchange factor, Dock2, was required for the sustained growth of the BCR microclusters through the remodeling of the F-actin cytoskeleton. As a consequence, the formation of the antigen-induced B cell immunological synapse (IS) in Dock2 deficient B cells was drastically impaired. Moreover, Dock2 deficient B cells exhibited decreased Ca2 influx. Physiologically, we found that mice with reduced expression of PTEN in B cells showed enhanced antibody responses and a spontaneous lupus-like autoimmune phenotype. Clinically, we showed that primary B cells from lupus patients exhibited more prominent BCR and PI3K microclusters than B cells from healthy controls. Thus, these results demonstrated the importance of PI3K- and PTEN-governed PIP2 and PIP3 equilibrium in regulating the dynamic growth of BCR microclusters during the initiation of B cell activation.

Growth of B Cell Receptor Microclusters Is Regulated by PIP2 and PIP3 Equilibrium and Dock2 Recruitment and Activation

The growth of B cell receptor (BCR) microclusters upon antigen stimulation drives B cell activation. Here, we show that PI3K-mediated PIP3 production is required for the growth of BCR microclusters. This growth is likely inhibited by PTEN and dependent on its plasma membrane binding and lipid phosphatase activities. Mechanistically, we find that PIP3-dependent recruitment and activation of a guanine nucleotide exchange factor, Dock2, is required for the sustained growth of BCR microclusters through remodeling of the F-actin cytoskeleton. As a consequence, Dock2 deficiency significantly disrupts the structure of the B cell immunological synapse. Finally, we find that primary B cells from systemic lupus erythematosus (SLE) patients exhibit more prominent BCR and PI3K microclusters than B cells from healthy controls. These results demonstrate the importance of a PI3K- and PTEN-governed PIP2 and PIP3 equilibrium in regulating the activation of B cells through Dock2-controlled growth of BCR microclusters.

Human tonsil naive B cells and germinal center B cells show distinct behaviors upon contact of antigen in lipid bilayers

Abstract During antibody responses to T-dependent antigens B cells undergo clonal expansion and somatic hyper-mutation in the dark zones of germinal centers (GCs) and subsequently undergo isotype switching and affinity based selection in the GC light zones. Only a small portion of B cells survive from GC reactions and differentiate into either plasma cells or memory B cells. However, the mechanism underlying selection within the GC is not fully understood. We compared the behavior of human tonsil naïve (IgD+, CD10−) and GC (IgD−, CD10+) B cells as they engaged antigen (using Light chain specific antibodies as a surrogate antigen) incorporated into planar lipid bilayers using a combination of TIRF and confocal microscopy. We observed marked differences in the cellular dynamics of naïve and GC B cells. Upon touching the antigen-containing bilayer naïve B cells rapidly form B cell receptor (BCR) micro-clusters that grow rapidly with time and signal for Ca++ fluxes. Within five minutes naïve B cells formed stable areas of contact with the bilayers. In contrast, GC B cells only slowly form BCR micro-clusters that grow slowly with time and signal for delayed Ca++ fluxes. Notably, the behavior of GC B cells over the same time period is far more dynamic with only transient interactions with the bilayer and active ruffling in the cells periphery suggesting that the GC B cells in contrast to naïve B cells continue to sample antigen for longer periods of time. Experiments in progress are aimed at relating the observed behaviors to the affinity of naïve and GC B cells for antigen.

Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.

Survival of human lymphoma cells requires B-cell receptor engagement by self-antigens

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on chronic active B-cell receptor (BCR) signaling. BCR pathway inhibitors induce remissions in a subset of ABC DLBCL patients. BCR microclusters on the surface of ABC cells resemble those generated following antigen engagement of normal B cells. We speculated that binding of lymphoma BCRs to self-antigens initiates and maintains chronic active BCR signaling in ABC DLBCL. To assess whether antigenic engagement of the BCR is required for the ongoing survival of ABC cells, we developed isogenic ABC cells that differed solely with respect to the IgH V region of their BCRs. In competitive assays with wild-type cells, substitution of a heterologous V region impaired the survival of three ABC lines. The viability of one VH4-34(+) ABC line and the ability of its BCR to bind to its own cell surface depended on V region residues that mediate the intrinsic autoreactivity of VH4-34 to self-glycoproteins. The BCR of another ABC line reacted with self-antigens in apoptotic debris, and the survival of a third ABC line was sustained by reactivity of its BCR to an idiotypic epitope in its own V region. Hence, a diverse set of self-antigens is responsible for maintaining the malignant survival of ABC DLBCL cells. IgH V regions used by the BCRs of ABC DLBCL biopsy samples varied in their ability to sustain survival of these ABC lines, suggesting a screening procedure to identify patients who might benefit from BCR pathway inhibition.

FcγRIIB-mediated dissociation of BCR-CD19 microclusters and hyperreactivity of lupus B cells (HUM7P.303)

Abstract B cell activation is regulated through the interplay between the B cell receptor (BCR) and the inhibitory co-receptor FcγRIIB or activating co-receptor CD19. Recent studies suggest that the BCR microcluster is converted to a signaling active state upon its colocalization with CD19 microclusters to activate B cells. Here using TIRF microscopy-based live cell imaging techniques, we show that FcγRIIB can inhibit B cell activation by a previously uncharacterized mechanism of blocking the spatial-temporal colocalization of BCR and CD19 microclusters within the immunological synapse (IS). This inhibitory function of FcγRIIB mainly relies on its transmembrane domain (TM). Indeed, human primary B cells from SLE patients homozygous for the gene encoding FcγRIIB-I232T lose the ability to block the synaptic colocalization of BCR with CD19, leading to the dis-regulated recruitment of pCD19, pSyk and pPI3K to membrane proximal signalosome. Furthermore, FcγRIIB-I232T homozygous SLE patients are more likely to develop severe clinical symptoms characterized by high anti-ds DNA antibody titers and the involvement of kidney, central nervous system, gastrointestinal tract, lung and heart compared to patients carrying either one or both WT copies of FcγRIIB. These observations may explain the hyper-reactive features of SLE patient B cells and provide a potential target through FcγRIIB for the prophylactic diagnosis, evaluation of disease progression and potential therapies.

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell's encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.

The Effect of Lipid Mediated Attraction and Antigen Affinity on B-Cell Receptor Microcluster Formation

We present findings from our energy-based Monte Carlo simulation involving antigen (Ag) bound, B-cell receptor (BCR) interaction with crucial cellular components, namely the lipid membrane, src family tyrosine kinase and Syk kinase activity. After encountering antigen presenting cells and binding Ags, one of the earliest events of signalsome assembly is the formation of BCR microclusters. BCR recognition of antigen results in a monotonically increasing graded signaling response with increasing BCR-Ag affinity. Recent experiments indicate an important role of very early (within seconds) signaling events in this affinity discrimination process. Our current studies improve upon our most recent biophysical models for BCR-Ag microcluster and macrocluster formation by incorporating lipid-mediated BCR attraction factors. We examine the effect of varying attractive forces and interactions between lipids, BCR bound state and kinase activity. Our model can show that strong coupling between BCR/BCR and BCR/lipid (sphingolipids) molecules generates affinity-dependent microclustering similar to what has been observed in experiments. Our results indicate that microcluster size and number increase with increasing affinity while the formation times of such early BCR-Ag clusters decrease. These features can have important implications for the B cell affinity discrimination problem.

B Cell Receptor-Mediated Antigen Gathering Requires Ubiquitin Ligase Cbl and Adaptors Grb2 and Dok-3 to Recruit Dynein to the Signaling Microcluster

The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to Tcells. Although the amount of antigen presentation to Tcells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein. Furthermore, we visualize the localization and movement of these microclusters on the underlying microtubule network. Importantly, disruption of this network or diminished dynein recruitment in Grb2-, Dok-3-, or Cbl-deficient B cells, does not influence microcluster formation or actin-dependent spreading, but abrogates directed movement of microclusters and antigen accumulation. Thus we identify a surprising but pivotal role for dynein and the microtubule network alongside Grb2, Dok-3, and Cbl in antigen gathering during B cell activation.

Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.

Expression of a functional B cell receptor with the specificity of VRC01, a broadly neutralizing antibody against HIV (159.8)

Abstract VRC01, a CD4-binding site antibody that neutralizes over 90% of HIV-1 isolates has recently been identified. One of its unusual features is that the variable domain goes through extensive somatic hypermutation (30%) from its germline precursors, which leads to greatly enhanced binding affinity and neutralization capacity. To understand the somatic hypermutation process of VRC01 during B cell development and to identify immunogens that drive the affinity maturation of VRC01, the B cell receptor (BCR) form of VRC01 and its germline precursor 02*02 was expressed and examined for antigen-binding, BCR microclustering and BCR signaling. Co-expression of surface IgM and kappa chain transgenes was detected on 293T and Ramos cells by either transfection or lentivirus-mediated transduction. The transgenic VRC01 BCR bound to different gp120 variants as determined by flow cytometry. The VRC01 BCR also exhibited higher binding affinity to the gp120 Resurfaced Stabilized Cores (RSC3) presented on lipid bilayers than to the mutant form ΔRSC3 as indicated by more BCR microclusters upon binding to RSC3. Stimulation of VRC01-BCR-expressing B cells with RSC3 or anti-IgM induced phosphorylation of the B cell signaling tyrosine kinases Fyn and Syk. In conclusion, a functional VRC01-BCR was successfully expressed on 293T and B cells. This transgenic system allows us to test the feasibility of guiding the maturation of VRC01 with appropriate antigens.

The cytoskeleton coordinates the early events of B-cell activation.

B cells contribute to protective adaptive immune responses through generation of antibodies and long-lived memory cells, following engagement of the B-cell receptor (BCR) with specific antigen. Recent imaging investigations have offered novel insights into the ensuing molecular and cellular events underlying B-cell activation. Following engagement with antigen, BCR microclusters form and act as sites of active signaling through the recruitment of intracellular signaling molecules and adaptors. Signaling through these "microsignalosomes" is propagated and enhanced through B-cell spreading in a CD19-dependent manner. Subsequently, the mature immunological synapse is formed, and functions as a platform for antigen internalization, enabling the antigen presentation to helper T cells required for maximal B-cell activation. In this review, we discuss the emerging and critical role for the cytoskeleton in the coordination and regulation of these molecular events during B-cell activation.

Intrinsic Properties of immunoglobulin IgG1 Isotype-Switched B Cell Receptors Promote Microclustering and the Initiation of Signaling

Memory B cells express high-affinity, immunoglobulin GB cell receptors (IgG BCRs) that enhance B cell responses, giving rise to the rapid production of high-affinity, IgG antibodies. Despite the central role of IgG BCRs in memory responses, the mechanisms by which the IgG BCRs function to enhance B cell responses are not fully understood. Using high-resolution live-cell imaging, we showed that IgG1 BCRs dramatically enhanced the earliest BCR-intrinsic events that followed within seconds of B cells' encounter with membrane bound antigen, including BCR oligomerization and BCR microcluster growth, leading to Syk kinase recruitment and calcium responses. The enhancement of these early events was dependent on a membrane proximal region of the IgG1 cytoplasmic tail not previously appreciated to play a role in IgG1 BCR signaling. Thus, intrinsic properties of the IgG1 BCR enhance early antigen-driven events that ultimately translate into heightened signaling.

Early Events in B Cell Activation

B cell activation is initiated by the ligation of the B cell receptor (BCR) with antigen and ultimately results in the production of protective antibodies against potentially pathogenic invaders. Here we review recent literature concerned with the spatiotemporal dynamic characterization of the early molecular events of B cell activation, including the initiation of BCR triggering, the formation of BCR microclusters, and the dynamic regulation of BCR signaling. Because these events involve the considerable reorganization of molecules within the membrane, an important role for the cytoskeleton is emerging in the regulation of B cell activation. At each stage we highlight the role of the cytoskeleton, establishing its pivotal position during the initiation and regulation of B cell activation.