Acceleration of intracellular targeting of antigen by the B-cell antigen receptor: importance depends on the nature of the antigen-antibody interaction.

The B-cell antigen receptor (BCR) internalizes bound antigen such that antigen-derived peptides become associated with emigrating major histocompatibility complex (MHC) class II molecules for presentation to T cells. Experiments with B-cell transfectants reveal that BCR confers a specificity of intracellular targeting since chimeric antigen receptors which internalize antigen by virtue of a heterologous cytoplasmic domain do not necessarily give rise to presentation. In contrast, however, previous studies have shown that antigen binding to irrelevant cell surface molecules (e.g. transferrin receptor, MHC class I) can ultimately lead to presentation. The solution to this paradox appears to be that the intracellular targeting by BCR actually reflects an acceleration of antigen delivery. Depending on the nature of the BCR-antigen interaction, this accelerated targeting can be essential in determining whether or not internalization leads to significant presentation. Physiologically, the accelerated delivery of antigen by BCR could prove of particular importance early in the immune response when antigen-BCR interaction is likely to be poor.     

The Syk-binding ubiquitin ligase c-Cbl mediates signaling-dependent B cell receptor ubiquitination and B cell receptor-mediated antigen processing and presentation

B cell receptor (BCR)-mediated antigen (Ag) processing and presentation lead to B cell-T cell interactions, which support affinity maturation and immunoglobulin class switching. These interactions are supported by generation of peptide-MHC class II complexes in multivesicular body-like MIIC compartments of B cells. Previous studies have shown that trafficking of Ag·BCR complexes to MVB-like MIIC occurs via an ubiquitin-dependent pathway and that ubiquitination of Ag·BCR complexes occurs by an Src family kinase signaling-dependent mechanism that is restricted to lipid raft-resident Ag·BCR complexes. This study establishes that downstream Syk-dependent BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and presentation. Knockdown studies reveal that of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central role in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and presentation. These results establish the novel role for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and presentation of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is modulated to impact the initiation and maturation of the humoral immune response.     

Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules

Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.     

A Highlights from MBoC Selection: Structurally distinct endocytic pathways for B cell receptors in B lymphocytes

B lymphocytes play a critical role in adaptive immunity. On antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative superresolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab’)2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when cluster size increases beyond the size of a single clathrin-coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.     

Plasticity of B cell receptor internalization upon conditional depletion of clathrin

B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized by specifically blocking each potential mechanism of internalization. BCR uptake was reduced by approximately 70% in B cells conditionally deficient in clathrin heavy chain expression. Actin or raft antagonists were both able to block the residual, clathrin-independent BCR internalization. These agents also affected clathrin-dependent internalization, indicating that clathrin-coated pits, in concert with mechanisms dependent on rafts and actin, mediate the majority of BCR internalization. Clustering G(M1) gangliosides enhanced clathrin-independent BCR internalization, and this required actin. Thus, although rafts or actin independently did not mediate BCR internalization, they apparently cooperate to promote some internalization even in the absence of clathrin. Simultaneous inhibition of all BCR uptake pathways resulted in sustained tyrosine phosphorylation and activation of the extracellular signal-regulated kinase (ERK), strongly suggesting that downstream BCR signaling can occur without receptor translocation to endosomes and that internalization leads to signal attenuation.     

Antibody-independent functions of B cells during viral infections

The humoral immune response and antibody-mediated functions of B cells during viral infections are well described. However, we have limited understanding of antibody-independent B cell functions, such as cytokine production and antigen presentation, in acute and chronic viral infections and their role in protection and/or immunopathogenesis. Here, we summarize the current literature on these antibody-independent B cell functions and identify remaining knowledge gaps. B cell subsets produce anti- and pro-inflammatory cytokines, which can have both beneficial and detrimental effects during viral clearance. As professional antigen presenting cells, B cells also play an important role in immune regulation/shaping of the developing adaptive immune responses. Since B cells primarily express TLR7 and TLR9, we specifically discuss the role of Toll-like receptor (TLR)-mediated B cell responses to viral infections and their role in augmenting adaptive immunity through enhanced cytokine production and antigen presentation. However, viruses have evolved strategies to subvert TLR signaling and additional stimulation via B cell receptor (BCR) may be required to overcome the defective TLR response in B cells. To conclude, antibody-independent B cell functions seem to have an important role in regulating both acute and chronic viral infections and may form the basis for novel therapeutic approaches in treatment of viral infections in the future.     

The tipping points in the initiation of B cell signalling: how small changes make big differences

B cells are selected by the binding of antigen to clonally distributed B cell receptors (BCRs), triggering signalling cascades that result in B cell activation. With the recent application of high-resolution live-cell imaging, we are gaining an understanding of the events that initiate BCR signalling within seconds of its engagement with antigen. These observations are providing a molecular explanation for fundamental aspects of B cell responses, including antigen affinity discrimination and the value of class switching, as well as insights into the underlying causes of B cell tumorigenesis. Advances in our understanding of the earliest molecular events that follow antigen binding to the BCR may provide a general framework for the initiation of signalling in the adaptive immune system.     

Distinct sequences in the cytoplasmic domain of complement receptor 2 are involved in antigen internalization and presentation

B cells express randomly rearranged surface Ig that forms part of a multiprotein complex known as the B cell receptor (BCR). Recognition of Ag via this receptor results in its capture, internalization, proteolysis and presentation to CD4+ T cells. The recognition of Ag by CD4+ T cells is critical for the selection of individual B cells, leading to the eventual secretion of a high affinity version of the BCR as an effective circulating Ab. B cells also express other receptors that recognize Ags associated with components of innate immunity. One of these receptors, CR2, binds Ags coated with activated complement components. Studies have shown that cross-linking CR2 and the BCR with complement-tagged Ags leads to enhanced Ag presentation by B cells. In addition, Ags targeted to B cell CR2 in the absence of BCR coligation are also efficiently presented to T cells. In this report, we identify several distinct sequences within the cytoplasmic domain of mouse CR2 (mCR2) that are essential for mCR2-mediated Ag presentation in both the presence and the absence of BCR cross-linking. The finding that distinct sequences in the cytoplasmic domain of mCR2 are essential for BCR-independent Ag presentation leads us to propose a novel role for CR2.     

Role of B cell antigen processing and presentation in the humoral immune response

In the 25 years since it was first indicated that lymphocyte subpopulations must interact during the generation of a humoral immune response, there has been an explosion of data on the molecular mechanism of this interaction. It has been demonstrated that T cells recognize a processed antigen fragment presented by a major histocompatibility complex molecule on the surface of an antigen-presenting cell. The minimal peptides required for T cell recognition of several proteins have been determined, the molecular genetics of many of the cell surface molecules involved have been defined, and the three-dimensional structure of the T cell receptor and the major histocompatibility antigens have been deduced. Several cell types have been found to act as antigen-presenting cells, although the roles of these populations in vivo remain unclear. However, it is clear that there must be a physical interaction between a B cell and a T cell before the B cell can respond to a T-dependent antigen. This interaction requires processing and presentation of the antigen by the B cell. Therefore this review focuses on antigen processing and presentation by resting B cells, one of the key steps in initiation of a humoral immune response.     

Mannosylation of Virus-Like Particles Enhances Internalization by Antigen Presenting Cells

Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.     

Membrane heterogeneities in the formation of B cell receptor-Lyn kinase microclusters and the immune synapse

Antigen binding to the B cell receptors (BCRs) induces BCR clustering, phosphorylation of BCRs by the Src family kinase Lyn, initiation of signaling, and formation of an immune synapse. We investigated B cells as they first encountered antigen on a membrane using live cell high resolution total internal reflection fluorescence microscopy in conjunction with fluorescence resonance energy transfer. Newly formed BCR microclusters perturb the local membrane microenvironment, leading to association with a lipid raft probe. This early event is BCR intrinsic and independent of BCR signaling. Association of BCR microclusters with membrane-tethered Lyn depends on Lyn activity and persists as microclusters accumulate and form an immune synapse. Membrane perturbation and BCR-Lyn association correlate both temporally and spatially with the transition of microclustered BCRs from a "closed" to an "open" active signaling conformation. Visualization and analysis of the earliest events in BCR signaling highlight the importance of the membrane microenvironment for formation of BCR-Lyn complexes and the B cell immune synapse.     

B cells as antigen presenting cells

Several characteristics confer on B cells the ability to present antigen efficiently: (1) they can find T cells in secondary lymphoid organs shortly after antigen entrance, (2) BCR-mediated endocytosis allows them to concentrate small amounts of specific antigen, and (3) BCR signaling and HLA-DO expression direct their antigen processing machinery to favor presentation of antigens internalized through the BCR. When presenting antigen in a resting state, B cells can induce T cell tolerance. On the other hand, activation by antigen and T cell help converts them into APC capable of promoting immune responses. Presentation of self antigens by B cells is important in the development of autoimmune diseases, while presentation of tumor antigens is being used in vaccine strategies to generate immunity. Thus, detailed understanding of the antigen presenting function of B cells can lead to their use for the generation or inhibition of immune responses.     

The actin cytoskeleton is required for the trafficking of the B cell antigen receptor to the late endosomes

The B cell antigen receptor (BCR) plays two central roles in B cell activation: to internalize antigens for processing and presentation, and to initiate signal transduction cascades that both promote B cells to enter the cell cycle and facilitate antigen processing by accelerating antigen transport. An early event in B cell activation is the association of BCR with the actin cytoskeleton, and an increase in cellular F-actin. Current evidence indicates that the organization of actin filaments changes in response to BCR-signaling, making actin filaments good candidates for regulation of BCR-antigen targeting. Here, we have analyzed the role of actin filaments in BCR-mediated antigen transport, using actin filament-disrupting reagents, cytochalasin D and latrunculin B, and an actin filament-stabilizing reagent, jasplakinolide. Perturbing actin filaments, either by disrupting or stabilizing them, blocked the movement of BCR from the plasma membrane to late endosomes/lysosomes. Cytochalasin D-treatment dramatically reduced the rate of internalization of BCR, and blocked the movement of the BCR from early endosomes to late endosomes/lysosomes, without affecting BCR-signaling. Thus, BCR-trafficking requires functional actin filaments for both internalization and movement to late endosomes/lysosomes, defining critical control points in BCR-antigen targeting.     

Phosphoinositide 3-kinase activation by Igbeta controls de novo formation of an antigen-processing compartment

Antigens that bind B cell antigen receptor (BCR) are preferentially and rapidly processed for antigen presentation. The BCR is a multimeric complex containing a signaling module composed of Igalpha and Igbeta. Signaling pathways implicated in antigen presentation through the BCR are ill defined. Here we demonstrate that phosphoinositide 3-kinase (PI3K) inhibitors preclude antigen presentation induced by BCR or Igbeta but not Igalpha. Unraveling the mechanisms responsible for this inhibition, we show that PI3K inhibitors block neither antigen internalization nor degradation. Rather PI3K inhibitors block de novo formation of a multivesicular antigen processing compartment, which is induced by triggering of the BCR or Igbeta. Strikingly, we found using fluorescent probes binding specifically to PI3K products that BCR and Igbeta but not Igalpha induce PI3K activation in endocytic compartments wherein antigen is transported. Altogether, these results strongly suggest that Igbeta couples the BCR to PI3K activation that is instrumental for de novo formation of the antigen processing compartment and efficient antigen presentation.     

The NeuARt II system: a viewing tool for neuroanatomical data based on published neuroanatomical atlases

Background

Activation of naïve B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cluster Assignment for Biological Inference), which identifies significant co-clustering of genes with similar Gene Ontology? annotation.

Results

CLASSIFI analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the BCR-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. Proton pump components that were not included in the initial microarray data set were also upregulated in response to BCR stimulation in follow up experiments. MHC Class II expression was found to be maintained specifically in response to BCR stimulation. Furthermore, ligand-specific internalization of the BCR, a first step in B cell antigen processing and presentation, was demonstrated.

Conclusion

These observations provide experimental validation of the computational approach implemented in CLASSIFI, demonstrating that CLASSIFI-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. Furthermore, this analysis has identified at least thirty-eight candidate components of the B cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to B cell antigen processing and presentation.     

Molecular Control of B Cell Activation and Immunological Synapse Formation

B cells form an essential part of the adaptive immune system by producing specific antibodies that can neutralize toxins and target infected or malignant cells for destruction. During B cell activation, a fundamental role is played by a specialized intercellular structure called the immunological synapse (IS). The IS serves as a platform for B cell recognition of foreign, often pathogenic, antigens on the surface of antigen‐presenting cells (APC). This recognition is elicited by highly specific B cell receptors (BCR) that subsequently trigger carefully orchestrated intracellular signaling cascades that lead to cell activation. Furthermore, antigen internalization, essential for full B cell activation and differentiation into antibody producing effector cells or memory cells, occurs in the IS. Recent developments especially in various imaging‐based methods have considerably advanced our understanding of the molecular control of B cell activation. Interestingly, the cellular cytoskeleton is emerging as a key player at several stages of B cell activation, including the initiation of receptor signaling. Here, we discuss the functions and molecular mechanisms of the IS and highlight the multifaceted role of the actin cytoskeleton in several aspects of B cell activation.      

Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.     

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.