B Cell Receptor Internalization Research Articles

Role of Autoantigen Induced-B-Cell Receptor Internalization and Its Inhibition for the Biology and Survival of Diffuse Large B-Cell Lymphoma (DLBCL) Cells

In normal B-cells, antigen binding by the B cell receptor (BCR) triggers rapid BCR clustering, initialization of signaling, and subsequent endocytosis of the BCR-antigen complex. Numerous studies showed that BCR signaling is continued as the BCR is trafficked intracellularly. Similarly, the internalized BCR in complex with TLR9 in DLBCL cells elicits crucial pro-survival signaling for DLBCL cells. Despite these observations, the role of endocytosis in generating a pathological signal in DLBCL, and a potential of endocytosis blockade as a strategy to inhibit BCR signaling, have not been defined. To address these questions, we generated DLBCL cell models with modified, ovoalbumin (OVA)-specific hypervariable regions (HVR) in BCR using the knock-in CRISPR-Cas9 methodology. This model allows to study BCR receptor internalization and the signaling that accompanies this process in highly controlled conditions. The change of BCR specificity led to inhibition of spontaneous (autoantigen-initiated) BCR internalization, observed as an increased surface BCR level, and lower proliferative potential of autoautigen-dependent U2932 and HBL1 cells, but not in cells in which autoantigens were not detected (LY19). Using the kinome analysis platform (PamGene technology), we identified kinases activated by BCR-autoantigen binding, including proximal mediators of BCR signaling, cyclin-dependent kinases, protein kinase C and S6 ribosomal kinases. Simultaneous analysis of the OVA-induced BCR internalization kinetics and signaling activation in the above-described models indicated that the activation of certain signaling cascades may emanate from the inside of the cell. Using the Proximity Ligation Assay (PLA) method, we identified the presence of BCR-TLR9-IkB complexes (activators of the NF-kB transcription factor), in the endosomal compartment of autoantigen-dependent DLBCL cells. Cells with OVA-specific BCR showed a drastically decreased number of BCR-TLR9-IkB complexes, which was associated with lower expression of NF-kB-dependent genes. To confirm the crucial role of the autoantigen-induced BCR endocytosis in sustaining the signaling and promoting DLBCL cells survival, we reanalyzed available data from the CRISPR-Cas9-Screen studies. This approach revealed the key role of genes involved in endocytosis, including dynamin-2, for DLBCL cell survival. Based on these observations, we generated cell models characterized by inducible expression of the dominant-negative (DN) form of dynamin-2, which inhibits dynamin-2-dependent endocytosis. Experiments performed using these models showed that BCR internalization is dependent on dynamin-2, and the induction of the DN-dynamin-2 form blocks internalization of the BCR receptor, BCR-TLR9-IkB complexes assembly and activation of BCR-initiated signaling pathways, including the transcription factor NF-kB, which markedly inhibited the growth of DLBCL cells. In addition, clinically available Dynamin-2 inhibitors (e.g. certain phenothiazine derivatives), also inhibited BCR endocytosis and were toxic to DLBCL cells. In summary, our observations demonstrate that autoantigen-induced endocytosis of the BCR receptor is the key mechanism supporting the BCR signal and ensuring the survival of DLBCL cells with autoantigen-specific BCRs. Furthermore, we show that endocytosis is a rational therapeutic target in this group of lymphoid malignancies.

Targeting N-linked Glycosylation for the Therapy of Aggressive Lymphomas.

DLBCL depends on constitutive BCR activation and signaling. There are currently no therapeutics that target the BCR directly and attenuate its pathologic signaling. Here, we unraveled a therapeutically exploitable, OST-B-dependent glycosylation pathway that drives BCR organization and proximal BCR signaling. This article is highlighted in the In This Issue feature, p. 1749.

LAPTM5 mediates immature B cell apoptosis and B cell tolerance by regulating the WWP2-PTEN-AKT pathway

Elimination of autoreactive developing B cells is an important mechanism to prevent autoantibody production. However, how B cell receptor (BCR) signaling triggers apoptosis of immature B cells remains poorly understood. We show that BCR stimulation up-regulates the expression of the lysosomal-associated transmembrane protein 5 (LAPTM5), which in turn triggers apoptosis of immature B cells through two pathways. LAPTM5 causes BCR internalization, resulting in decreased phosphorylation of SYK and ERK. In addition, LAPTM5 targets the E3 ubiquitin ligase WWP2 for lysosomal degradation, resulting in the accumulation of its substrate PTEN. Elevated PTEN levels suppress AKT phosphorylation, leading to increased FOXO1 expression and up-regulation of the cell cycle inhibitor p27Kip1 and the proapoptotic molecule BIM. In vivo, LAPTM5 is involved in the elimination of autoreactive B cells and its deficiency exacerbates autoantibody production. Our results reveal a previously unidentified mechanism that contributes to immature B cell apoptosis and B cell tolerance.

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.

Cosmc deficiency causes spontaneous autoimmunity by breaking B cell tolerance.

Factors regulating the induction and development of B cell–mediated autoimmunity are not well understood. Here, we report that targeted deletion in murine B cells of X-linked Cosmc, encoding the chaperone required for expression of core 1 O-glycans, causes the spontaneous development of autoimmune pathologies due to a breakdown of B cell tolerance. BC-CosmcKO mice display multiple phenotypic abnormalities, including severe weight loss, ocular manifestations, lymphadenopathy, and increased female-associated mortality. Disruption of B cell tolerance in BC-CosmcKO mice is manifested as elevated self-reactive IgM and IgG autoantibodies. Cosmc-deficient B cells exhibit enhanced basal activation and responsiveness to stimuli. There is also an elevated frequency of spontaneous germinal center B cells in BC-CosmcKO mice. Mechanistically, loss of Cosmc confers enhanced B cell receptor (BCR) signaling through diminished BCR internalization. The results demonstrate that Cosmc, through control of core 1 O-glycans, is a previously unidentified immune checkpoint gene in maintaining B cell tolerance.

Examination of Antigen-Induced Endocytic Structure Formation in B Lymphocytes

B lymphocytes are an essential component of the adaptive immune response against pathogens. The ability of a B cell lineage to adapt to better recognize a given antigen depends critically upon the ability of each B cell to bind and internalize antigen through its B cell receptor (BCR). The mechanism of antigen internalization by B cells appears to require cooperation between BCR signaling, the actomyosin network and clathrin-mediated endocytic processes. However, it remains unclear how these actors are coordinated to achieve efficient and controlled uptake of structurally heterogeneous antigens. To better understand this process, we set out to directly observe antigen-induced endocytic structures in the human DG-75 B cell line using a combination of conventional and super-resolution microscopy techniques including confocal laser scanning microscopy (CLSM), total-internal reflected fluorescence microscopy (TIRF-M), and correlative super-resolution light and electron microscopy (CLEM). Cells expressing GFP-tagged BCR and red fluorescent protein-tagged endocytic proteins were stimulated with anti-human IgM Fab’2 to model BCR clustering and internalization. We observed recruitment of early adaptors of clathrin mediated endocytosis – FCHO1, Eps15, Epsin, CALM, and clathrin itself – to Fab’2-induced BCR clusters by TIRF-M and CLSM. CLEM revealed that BCR clusters are heterogeneous in size and frequently partition with clathrin on larger membrane invaginations. This work reveals novel structural features of antigen-induced endocytic structures and expands our understanding of the mechanism by which B cells may accommodate antigen of varying size during internalization.

Uncoupling of BCR Internalization and Signaling in Treatment Naive and Btki-Treated Patients with Chronic Lymphocytic Leukemia

Ligation of the B-cell receptor (BCR) results in activation of intracellular signaling as well as internalization and processing of ligand/receptor complexes. BCR responsiveness has been shown to vary markedly between patients with chronic lymphocytic leukaemia (CLL) and is linked to prognosis. Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, relatively little is known about the capacity of CLL B-cells to internalize ligands that bind to the BCR.In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR following stimulation. First, we assessed to what extent this varies between various prognostic subgroups. Second, given that BCR signaling is thought to be more pronounced within lymphoid tissue, we investigated whether internalization varies between different anatomic sites of the same individual. Finally, we examined the effect of agents that inhibit BCR function by comparing BCR expression and internalization in a cohort of patient before and during therapy with Bruton's tyrosine kinase inhibitors (BTKi).BCR internalization was assessed in two ways. First, we used a pH sensitive dye linked to agonistic anti-IgM (pHrodo-αIgM) to detect the uptake and retention of ligand/receptor complexes in acidified endosomes. Second, BCR internalization was assessed directly by measuring the rate of disappearance of surface IgM following ligation by agonistic anti-IgM. An increase in the percentage of cells showing pHrodo fluorescence above control was detected in all CLL cases studied (mean percentage pHrodo-αIgM uptake = 30.2±2.5%, p<0.0001, n=40) although there was considerable inter-patient variation. We observed a significant correlation between pHrodo-αIgM uptake and high surface IgM (sIgM) expression (r2=0.529, p=0.0001, n=40), and an association with markers of adverse prognosis including, CD38 positivity (p=0.001, n=40), and the capacity to mobilize calcium ions following BCR ligation (p=0.001, n=26). Cases of CLL with unmutated IGHV genes showed higher uptake of pHrodo-αIgM than mutated cases however this did not reach statistical significance (p=0.053, n=40).Similarly, when BCR function within individual CLL patients was examined, we also found that pHrodo-αIgM uptake varied substantially and was maximal in lymph node (LN) derived CLL cells (p=0.03, n=6) and those in the peripheral blood (PB) that express the highest levels of CD5 (p=0.0001, n=26), a marker that is upregulated following BCR activation. In addition, we found that LN CLL cells expressed higher levels of sIgM than those derived from the PB (p=0.03, n=6). This was a surprising finding, as BCR stimulation is thought to occur within LNs, which might be expected to result in down regulated BCR expression.When the level of BCR internalization and accumulation in the endosomes was adjusted for the number of sIgM molecules, we found that BCR internalization and retention was actually more efficient in anergic cases of CLL (defined by lack of ability to mobilize Ca2+ following stimulation with anti-IgM; p=0.0002, n=26). This observation was supported by direct measurement of the rate of BCR endocytosis, which showed a more rapid internalization in anergic B-cells compared to signaling competent and normal B-cells. Similar findings have previously been reported in a murine model of B-cell anergy.Finally, data from BTKi treated CLL patients showed that, 12 months after commencing treatment, CLL B-cells exhibit lower levels of sIgM expression (p=0.02, n=7) and have more efficient BCR internalization than at the outset (p=0.05, n=7). Since low sIgM expression and more efficient BCR internalization is a feature of B-cell anergy, these results suggest that therapy with BTK inhibitors selectively depletes B-cells that are capable of signaling through the BCR and enriches for those displaying features of anergy.Overall our data show that BCR internalization is uncoupled from intracellular signaling in CLL and is most efficient in cells that demonstrate poor downstream BCR signaling or show features of recent BCR stimulation. These observations are similar to those previously reported in anergic B-cells and provide further evidence for ongoing BCR activation and anergy in CLL. DisclosuresPatten:Gilead: Research Funding. Devereux:Gilead: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Consultancy, Other: Travel, Accommodations, Expenses ; GSK: Consultancy.

Lipid raft-dependent plasma membrane repair interferes with the activation of B lymphocytes.

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca(2+)-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca(2+)-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR-lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte-mediated immune responses.

HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis.

Deletion of HAX1 in mice causes a severe reduction in the numbers of lymphocytes in the bone marrow and in the spleen. Additionally, B220+ B progenitor cells in the bone marrow are reduced, suggesting an important function of HAX1 in B cell development. HAX1 is thought to play a protective role in apoptotic processes; therefore, we investigated the role of HAX1 in bone marrow B progenitor cells and splenic B cells. We did not observe an effect on the survival of Hax1−/− bone marrow cells but detected enhanced survival of splenic Hax1−/− B cells upon in vitro starvation/growth-factor withdrawal. To explain this apparent inconsistency with previous reports of HAX1 function, we also studied the B cell receptor (BCR)-induced apoptosis of IgM-stimulated splenic naïve B cells and found that apoptosis decreased in these cells. We further found impaired internalization of the BCR from Hax1−/− splenic B cells after IgM crosslinking; this impaired internalization may result in decreased BCR signaling and, consequently, decreased BCR-mediated apoptosis. We measured HAX1 binding to the cytoplasmic domains of different Ig subtypes and identified KVKWI(V)F as the putative binding motif for HAX1 within the cytoplasmic domains. Because this motif can be found in almost all Ig subtypes, it is likely that HAX1 plays a general role in BCR-mediated internalization events and BCR-mediated apoptosis.

Molecular Mechanisms of B Cell Antigen Gathering and Endocytosis.

Generation of high-affinity, protective antibodies requires B cell receptor (BCR) signaling, as well as antigen internalization and presentation to helper T cells. B cell antigen internalization is initiated by antigen capture, either from solution or from immune synapses formed on the surface of antigen-presenting cells, and proceeds via clathrin-dependent endocytosis and intracellular routing to late endosomes. Although the components of this pathway are still being discovered, it has become clear that antigen internalization is actively regulated by BCR signaling at multiple steps and, vice versa, that localization of the BCR along the endocytic pathway modulates signaling. Accordingly, defects in BCR internalization or trafficking contribute to enhanced B cell activation in models of autoimmune diseases and in B cell lymphomas. In this review, we discuss how BCR signaling complexes regulate each of the steps of this endocytic process and why defects along this pathway manifest as hyperactive B cell responses in vivo.

Abstract 3641: Antibody targeting the IgM B-cell receptor (mBCR) induces growth inhibition and apoptosis

Abstract Background: The B-cell Receptor (BCR) initiates a driver pathway in B-cell lymphoma-leukemia. Inhibition of downstream pathway tyrosine kinases is therapeutic. Both vertical and horizontal membrane B-cell Receptor Complex (BCRC) interactions render this pathway complex. As a consequence of the BCR's sequence homology to serum IgM (sIgM), specific B-cell membrane IgM (mIgM) targeting in vivo was thought not to be feasible. A unique sequence identified in the membrane-bound BCR, designated proximal domain (PD), is not expressed in sIgM. Here we identify an additional conformational epitope discovered in the extracellular domains of mIgM, not detected in sIgM. Methods: We have generated high affinity anti-PD monoclonal antibodies (mAbs) by proprietary immunization techniques. These mAbs are shown by ELIZA, Westerns blots and Scanning Immuno-Electron Microscopy (SEM) to bind to mIgM protein and mIgM+ expressing cell lines (CA 46, SU-DHL-5, CRL-1596, CRL-1432). Using these high affinity anti-PD mAbs, mIgM was immune-affinity purified. Second generation antibodies detecting conformational epitopes on mBCR and not reacting with sIgM in ELIZA/Western/SEM assays were collected. Growth inhibition was assessed by MTT and clonogenic assays. Four mAbs were selected for further studies. Results: Cell surface binding assays demonstrated the specificity of these mAbs by testing against a panel of mIgM + vs mIgM- (mIgG+) cells. Normal or Waldenstrom's Macroglobulinemia sera failed to block or reduce mAb binding to mIgM+ cells and results were confirmed by SEM. At 37°C anti-PD mAbs (mAb1-1, mAb2-2b, mAb3-2b) internalize mIgM by 30 mins, but they do not modulate cell growth inhibition. Second generation mAb4-2b mediates BCR internalization and in low density cultures, cell growth inhibition, anti-clonogenic activity and apoptosis is observed. Apoptosis is seen in a variety of malignant B-cell lines including high and low mIgM/Igα/β expressers. Conclusions: These data support the contention that BCR internalization is insufficient to interrupt the BCRC signaling cascade despite the lack of detectable residual mIgM on the cell surface. Upon mAb4-2b binding to a non-ligand binding site on mIgM, it induces both BCR internalization and in another distinct event, apoptosis. By competitive mAb studies the apoptosis mediating conformational epitope appears to reside outside of the linear PD sequence. These mAbs are candidates for clinical development as drug/radioisotope targeting vehicles or as a mediator of inhibition of the BCR signaling pathway (mAb 4-2b). As these agents have a high level of specificity because they do not bind to non-mIgM B-cells, normal lymphocytes and non-lymphatic tissues may be spared toxicity. Citation Format: Rachel S. Welt, Yamuna D. Gangadharan, Jonathan A. Welt, Virginia Raymond, David Kostyal, Sydney Welt. Antibody targeting the IgM B-cell receptor (mBCR) induces growth inhibition and apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3641. doi:10.1158/1538-7445.AM2014-3641

Intravenous immunoglobulin induces a functional silencing program similar to anergy in human B cells

Chronic inflammatory and autoimmune diseases are largely due to inappropriate response of hyperactive or autoreactive B cells. These autoreactive B cells can evade central tolerance checkpoints and migrate to the periphery, where they would be silenced by anergy. Such anergic cells are characterized by B-cell receptor (BCR) desensitization and altered downstream signaling. We sought to determine whether intravenous immunoglobulin (IVIg) induces a nonresponsive state of B cells and to address the similarities of this mechanism to those described in anergy. Human B cells were stimulated with anti-IgM antibody, and effects of IVIg on several parameters, such as calcium release, tyrosine phosphorylation, BCR aggregation, BCR internalization, or transcriptional activity, were studied by using flow cytometry, confocal microscopy, Western blotting, and a quantitative PCR array. IVIg-treated B cells show defects in activating coreceptor expression, calcium signaling, and BCR aggregation on engagement by antigen. IVIg also induces suppression of phosphoinositide 3-kinase signaling, which plays a central role in determining B-cell fate. All these events ultimately lead to profound modifications in gene expression, resulting in long-term functional but reversible silencing of IVIg-treated B cells. Our findings provide insights into the effectiveness of IVIg in treating autoimmune or inflammatory pathologies associated with the loss of B-cell tolerance. Furthermore, these data provide a model to explore the complexity of positive versus negative selection in B cells.

Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

CIN85 is required for Cbl-mediated regulation of antigen receptor signaling in human B cells

AbstractThe aberrant regulation of B-cell receptor (BCR) signaling allows unwanted B cells to persist, thereby potentially leading to autoimmunity and B-cell malignancies. Casitas B-lineage lymphoma (Cbl) proteins suppress BCR signaling; however, the molecular mechanisms that control Cbl function in human B cells remain unclear. Here, we demonstrate that CIN85 (c-Cbl interacting protein of 85 kDa) is constitutively associated with c-Cbl, Cbl-b, and B-cell linker in B cells. Experiments using CIN85-overexpressing and CIN85-knockdown B-cell lines revealed that CIN85 increased c-Cbl phosphorylation and inhibited BCR-induced calcium flux and phosphorylation of Syk and PLCγ2, whereas it did not affect BCR internalization. The Syk phosphorylation in CIN85-overexpressing and CIN85-knockdown cells was inversely correlated with the ubiquitination and degradation of Syk. Moreover, CIN85 knockdown in primary B cells enhanced BCR-induced survival and growth, and increased the expression of BcLxL, A1, cyclin D2, and myc. Following the stimulation of BCR and Toll-like receptor 9, B-cell differentiation- associated molecules were up-regulated in CIN85-knockdown cells. Together, these results suggest that CIN85 is required for Cbl-mediated regulation of BCR signaling and for downstream events such as survival, growth, and differentiation of human B cells.

Defective calcium signaling and disrupted CD20–B-cell receptor dissociation in patients with common variable immunodeficiency disorders

B cells of patients with common variable immunodeficiency (CVID) disorders display impairment in production of immunoglobulin class-switched antibodies, which is possibly contributed to by defects in early B-cell activation. On resting B cells, B-cell receptors (BCRs) are organized in oligomers that are signaling inactive. Their triggering by cognate antigen causes the lateral reorganization of BCRs and associated proteins into signalosomes, resulting in BCR-activated calcium entry. In resting cells the B-cell surface antigen CD20 is associated with the BCR but dissociates on signalosome formation. We sought to determine whether CD20 dissociation from the BCR during early B-cell activation might contribute to the development of CVID disorders. We evaluated BCR signalosome formation, internalization, and signaling in primary B cells of pediatric patients with CVID disorders and healthy control subjects. In many pediatric patients with CVID disorders, Bcells exhibit significant deficits in BCR triggering-mediated calcium entry in the cytosol, which correlates with impaired plasmablast differentiation invitro. These alterations did not originate from upregulation of CD22 or defects in calcium channels and did not involve gene mutations in phospholipase Cγ2 or Bruton tyrosine kinase. Instead, B cells from patients with CVID disorders exhibited reduced BCR dissociation from CD20. BCR or CD20 cross-linking induced less BCR internalization, and antibody-mediated CD20 triggering elicited less BCR downstream signaling, as measured based on secondary fluxes. We propose that CD20 dissociation from the BCR signalosome is pivotal to BCR-mediated calcium mobilization in the cytosol. Defects in CD20/BCR signalosome conformation might predispose to the spectrum of CVID disorders.

CD40 Co-stimulation Inhibits Sustained BCR-induced Ca2+Signaling in Response to Long-term Antigenic Stimulation of Immature B Cells

Regulation of B cell receptor (BCR)-induced Ca(2+) signaling by CD40 co-stimulation was compared in long-term BCR-stimulated immature (WEHI-231) and mature (Bal-17) B cells. In response to long-term pre-stimulation of immature WEHI-231 cells to α-IgM antibody (0.5~48 hr), the initial transient decrease in BCR-induced [Ca(2+)](i) was followed by spontaneous recovery to control level within 24 hr. The recovery of Ca(2+) signaling in WEHI-231 cells was not due to restoration of internalized receptor but instead to an increase in the levels of PLCγ2 and IP(3)R-3. CD40 co-stimulation of WEHI-231 cells prevented BCR-induced cell cycle arrest and apoptosis, and it strongly inhibited the recovery of BCR-induced Ca(2+) signaling. CD40 co-stimulation also enhanced BCR internalization and reduced expression of PLCγ2 and IP(3)R-3. Pre-treatment of WEHI-231 cells with the antioxidant N-acetyl-L-cysteine (NAC) strongly inhibited CD40-mediated prevention of the recovery of Ca(2+) signaling. In contrast to immature WEHI-231 cells, identical long-term α-IgM pre-stimulation of mature Bal-17 cells abolished the increase in BCR-induced [Ca(2+)](i), regardless of CD40 co-stimulation. These results suggest that CD40-mediated signaling prevents antigen-induced cell cycle arrest and apoptosis of immature B cells through inhibition of sustained BCR-induced Ca(2+) signaling.

B Cell Activation by Outer Membrane Vesicles—A Novel Virulence Mechanism

Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca2+ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19+ IgD+ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

Receptor Rendezvous

B cell systems that protect organisms from viral infection walk a fine line because the molecules they detect--such as unmethylated DNA--can also be derived from host cells, and an overzealous defense results in autoimmune disease. Chaturvedi et al . describe new insight into how components of the adaptive and innate immune systems--B cell receptors (BCRs) and Toll-like receptor 9 (TLR9), respectively--act together to sense DNA-containing antigens. The answer was not obvious because the BCR is thought to act primarily on the cell surface, whereas TLR9 is normally present on endocytic vesicles. The authors used confocal fluorescence microscopy and immunoelectron microscopy to show that in mouse B cells in which the BCR was activated, TLR9 was relocated into autophagosomes. When cells were stimulated just through TLR9 with unmethylated DNA, phosphorylated (thus, activated) p38 mitogen-activated protein kinase (MAPK) was detected only in endosomes. However, if the BCR was activated with an antibody to immunoglobulin M (IgM) (alone or with DNA to activate TLR9), p38 was detected in large autophagosome-like structures. The recruitment of TLR9 to this compartment appeared to depend on physical internalization of the BCR because treatment of cells with an inhibitor of BCR internalization that still allowed early signaling events prevented relocalization of TLR9. Pharmacological inhibition of phospholipase D (PLD) revealed that activation of the lipase in response to the BCR was necessary for the recruitment of TLR9 to the autophagosomes. In commentary, Monroe and Keir discuss how the cooperation of BCR and TLR appears to be spatially restricted to vesicles where TLRs can interact with unmethylated DNA in the antigen cargo carried by the BCR. They emphasize that the paper opens new areas for further exploration, including the spatial organization of signaling by immune receptors and the continued signaling of internalized BCR after its movement away from the plasma membrane. A. Chaturvedi, D. Dorward, S. K. Pierce, The B cell receptor governs the subcellular location of Toll-like receptor 9 leading to hyperresponses to DNA-containing antigens. Immunity 28 , 799-809 (2008). [PubMed] J. G. Monroe, M. E. Keir, Bridging Toll-like- and B cell-receptor signaling: Meet me at the autophagosome. Immunity 28 , 729-731 (2008). [PubMed]

Establishing the molecular mechanisms of B cell receptor internalization

The B cell receptor (BCR) is comprised of ligand binding (IgH/L) and signaling (CD79a/b) subunits and mediates the processing/presentation of bound antigen. Internalization of the BCR had been reported to occur both through clathrin‐coated pits (CCPs) and lipid rafts (LRs), although it is unclear which pathway predominates. Use of a previously described model, consisting of a CD32‐CD79b fusion protein, demonstrates that the cytoplasmic tail of CD79b contains one or more functional endocytosis motifs. Internalization is crosslinking‐dependent, recapitulating the behavior of the native BCR when ligated with an antibody against the variable region of the IgH/L subunit. CD79b contains an ITAM with two Yxx? motifs that may bind the clathrin adaptor AP‐2, suggesting CCPs may be involved in internalization. However, there is also an increased association between the BCR and LRs upon receptor crosslinking, implying that under certain circumstances, LR localization of the BCR may lead to internalization subsequent to signaling. Current work focuses on mutating the conserved ITAM residues of CD79b to disrupt these potential AP‐2 binding sites. Additionally, by visualizing the BCR in morphologically identifiable CCPs by electron microscopy or in LRs by sucrose fractionation, the primary mechanism of BCR internalization will be further established. Supported by NIH grant AI‐065773 to JRD.

Dual role of Cbl links critical events in BCR endocytosis

Receptor endocytosis down-regulates ligand-induced signaling in a timely manner and depends on cytoskeletal remodeling. In B lymphocytes, internalization of B cell receptors (BCRs) is also critical to antigen presentation. However, the mechanisms underlying BCR endocytosis are not fully understood. Similarly, the molecular mechanisms linking endocytosis to cytoskeletal remodeling remain poorly defined. We used flow cytometry, pull-down assays, immunochemistry and fluorescence microscopy to investigate BCR internalization in the DT40 B cell line. We demonstrate that ablation of Cbl impacts BCR endocytosis and the underlying cytoskeletal dynamics. Specifically, we demonstrate that ligand-induced endocytosis is impaired in Cbl-/- cells and that the ubiquitin ligase activity is required for Cbl to promote endocytosis. We also show that phosphorylation of CrkII, activation of Rac downstream of CrkII and BCR capping require Cbl. Furthermore, we show that the association of Cbl and CrkII requires phosphorylation of Cbl, but not its ubiquitin ligase activity. Our data indicate that Cbl promotes BCR endocytosis and attenuates ligand-induced signaling by virtue of its ability to orchestrate receptor ubiquitylation and cytoskeletal dynamics.