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.     

Co-engaging CD47 and CD19 with a bispecific antibody abrogates B-cell receptor/CD19 association leading to impaired B-cell proliferation

CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.     

Using peptide mimetopes to elucidate anti-polysaccharide and anti-nucleic acid humoral responses.

Humoral responses against polysaccharide or nucleic acid antigens are often difficult to characterize and to induce. For example, the eliciting antigen for the development of anti-double-stranded(ds)DNA antibodies is unclear. dsDNA is a poor immunogen, yet antibodies to it bear the hallmark of a T cell dependent response. The microbial origin of polysaccharide antigens is, in general, readily known, but these antigens often do not elicit B cell memory responses, which are crucial for vaccine development. This review focuses on the use of peptide mimetopes to better understand humoral responses against non-protein antigens. First we describe a mimetope for dsDNA that was derived by probing a peptide phage library with an anti-dsDNA antibody. We discuss the usefulness of this mimetope in a search for candidate protein antigens and for examining the phenotype of antigen-specific B cells. Next, we discuss two mimetopes for phosphorylcholine (PC), a component of S. pneumoniae C polysaccharide. One was derived through mapping an anti-idiotype epitope and the other by probing a phagodisplay peptide library with an anti-PC antibody. Both of these peptide mimetopes for PC provide useful information regarding the requirements of a protective antibody response against pneumococcal infection, and define a critical role for adjuvant and carrier as well as mimetope.     

B cells

B cells are an important component of adaptive immunity. They produce and secrete millions of different antibody molecules, each of which recognizes a different (foreign) antigen. The fact that humans express a very large repertoire of antibodies is due to the complex mechanism of V(D)J recombination of immunoglobulin (Ig) genes as well as other processes including somatic hypermutation, gene conversion and class switching. The B cell receptor (BCR) is an integral membrane protein complex that is composed of two Ig heavy chains, two Ig light chains and two heterodimers of Igalpha and Igbeta. To eliminate foreign antigens, B cells cooperate with other cells of the immune system including macrophages, dendritic cells and T cells. B cell development is a tightly controlled process in which over 75% of the developing cells become apoptotic because of inappropriate immunoglobulin gene rearrangements or recognition of self antigens by Igs. Hence, the majority of B cell-associated disorders are caused by the incorrect function of genes/proteins involved in B cell development.     

Multiscale affinity maturation simulations to elicit broadly neutralizing antibodies against HIV

The design of vaccines against highly mutable pathogens, such as HIV and influenza, requires a detailed understanding of how the adaptive immune system responds to encountering multiple variant antigens (Ags). Here, we describe a multiscale model of B cell receptor (BCR) affinity maturation that employs actual BCR nucleotide sequences and treats BCR/Ag interactions in atomistic detail. We apply the model to simulate the maturation of a broadly neutralizing Ab (bnAb) against HIV. Starting from a germline precursor sequence of the VRC01 anti-HIV Ab, we simulate BCR evolution in response to different vaccination protocols and different Ags, which were previously designed by us. The simulation results provide qualitative guidelines for future vaccine design and reveal unique insights into bnAb evolution against the CD4 binding site of HIV. Our model makes possible direct comparisons of simulated BCR populations with results of deep sequencing data, which will be explored in future applications.     

Specific cross-protective antigonococcal immunity in the murine genital tract

Specific acquired immunity to gonococci was studied in systemically immunized mice, challenged with 10(7) gonococci by intrauterine inoculation. Protection after intraperitoneal immunization was monitored by vaginal cultures taken 24 h post-challenge, since events during the first 24 h postexposure to gonococci are crucial in determining the outcome of infection. Mice were protected against gonococcal challenge by two inoculations with either live or boiled gonococci given 4 weeks apart, whereas immunization with one inoculation did not protect against challenge 1 week later. Protection was correlated with high titers of IgG antibody in serum after two immunizations, but not with the high titers of serum IgM antibody found after the one immunization. IgG antibodies, but not IgM antibodies, were shown to pass into genital secretions. Protection could be passively transferred by serum with high titers of antibody. Of most practical importance was the finding that not only were heat-stable antigens protective, but also heterologous protection resulted after immunization with three strains differing in source (disseminated gonococcal infection versus gonorrhea), opacity-transparency characteristics, and serum sensitivity. The data indicate that IgG antibodies resulting from systemic immunization with heat-stable antigens may be able to provide cross-protection immunity against gonorrhea.     

Augmented B Lymphocyte Response to Antigen in the Absence of Antigen-Induced B Lymphocyte Signaling in an IgG-Transgenic Mouse Line

IgG-containing B cell antigen receptor (IgG-BCR), the BCR mostly expressed on memory B cells, contains a distinct signaling function from IgM-BCR or IgD-BCR expressed on naïve B cells. Because naïve B cells transgenic for IgG exhibit augmented response to antigens similar to memory B cells, the distinct signaling function of IgG-BCR appears to play a role in augmented antibody responses of memory B cells. However, how IgG-BCR signaling augments B cell responses is not yet well understood. Here we demonstrate that B cells from IgG-transgenic mice are anergic with defect in generation of BCR signaling upon BCR ligation. However, these IgG-transgenic B cells generate markedly augmented antibody response to a T cell-dependent antigen, probably due to hyper-responsiveness to a T cell-derived signal through CD40. Both BCR signaling defect and augmented response to CD40 ligation are partially restored in xid IgG-transgenic mice in which BCR signaling is down-modulated due to a loss-of-function mutation in the tyrosine kinase Btk crucial for BCR signaling. Thus, IgG-BCR induces augmented B cell responses in the absence of antigen-induced BCR signaling probably through high ligand-independent BCR signaling that may “idle” B cells to make them ready to respond to T cell help. This finding strongly suggests a crucial role of ligand-independent signaling in receptor function.     

Bruton’s Tyrosine Kinase Mediates the Synergistic Signalling between TLR9 and the B Cell Receptor by Regulating Calcium and Calmodulin

B cells signal through both the B cell receptor (BCR) which binds antigens and Toll-like receptors (TLRs) including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton’s tyrosine kinase (BTK) is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.     

It��s All About Change: The Antigen-driven Initiation of B-Cell Receptor Signaling

B-cell responses are initiated by the binding of foreign antigens to the clonally distributed B-cell receptors (BCRs) resulting in the triggering of signaling cascades that activate a variety of genes associated with B-cell activation. Although we now understand the molecular nature of the signaling pathways in considerable detail what remains only poorly understood are the mechanisms by which the information that antigen has bound to the BCR ectodomain is transduced across the B-cell membrane to the BCR cytoplasmic domains to trigger signaling. To a large part this gap in knowledge is because of the paucity of techniques to temporally and spatially resolve changes in the behavior of the BCR that occur within several seconds of antigen binding. With the advent of new live-cell imaging technologies we are gaining our first clear views of the events that lead up to the triggering of BCR signaling cascades. These events may provide potential new targets for therapeutic intervention in disease involving hyper or chronic activation of B cells.Specific, high-affinity antibody responses are the result of processes based on clonal selection (reviewed in Rajewsky 1996). In the absence of antigen, individuals generate a B-cell repertoire in which each B cell expresses a single heavy and light chain gene, the product of somatic recombination of variable and constant region gene segments. Self-reactive B cells are removed from the repertoire and when antigen enters the immune system it selects those B cells expressing BCR’s with highest affinity for the antigen. Under the influence of both T cell and innate immune system regulation the antigen-selected B cells are induced to differentiate into short-lived antibody producing cells or enter germinal centers where they undergo the molecularly linked processes of somatic hypermutation and isotype switching. Antigen selection within the germinal centers results in high-affinity memory B cells expressing isotype switched BCRs. These memory B cells account, in large part, for the high titered, high affinity IgG antibody responses observed upon re-exposure to antigen. Thus, we presume that B cells are capable of initiating responses to the universe of foreign antigens to which individuals are exposed and do so through mechanisms that are sensitive to the affinity of the BCR for antigen and by which isotype switched BCRs are more effective. Until recently, the events by which the binding of antigen to the BCRs triggered signaling remained largely unknown due in a large part to the paucity of experimental approaches that were able to provide the spatial and temporal resolution necessary to capture the earliest events that follow the binding of antigens to BCRs that result in triggering the B cell’s signaling cascades. The conventional biochemical techniques that were used so successfully to describe the components of the BCR signaling cascades were too slow to study early events and could not provide spatial information. The application of new live-cell imaging technologies that allow resolution of single molecules over a timeframe of several seconds to the study of antigen-induced B-cell responses is providing the first views of these processes. Here we review progress in understanding the initiation of the BCR signaling using live-cell imaging technologies and how this new knowledge may explain in part the mechanisms that underlie hyper or chronic activation of B cells in autoimmunity and in B-cell cancers.     

Inducing specific reactivity against B cells in mice by immunizing with an Fc fusion protein containing self-Igβ

A recombinant chimeric fusion protein, muIgbeta-hugamma4.Fc, composed of the extracellular domain of mouse Igbeta (CD79b) and the CH2-CH3 domains of human IgGgamma4.Fc (hugamma4.Fc), linked via an immunologically inert flexible peptide, was prepared. The fusion protein was evaluated for its ability to induce specific auto-reactive immune response against Igbeta and to modulate B cell activity in Balb/c mice. Upon immunization with muIgbeta-hugamma4.Fc, mice developed immunoglobulin (IgG) against self-Igbeta, which could bind to the cells of a mouse B cell line expressing Igbeta on the cell surface. The proportion of B cells in mononuclear cells in the peripheral blood (PBMC) of treated mice decreased as compared to that of mice immunized with hugamma4.Fc without the Igbeta component. Furthermore, mice immunized against muIgbeta-hugamma4.Fc displayed a reduced antibody response against an irrelevant antigen. The implications of employing the present approach in developing a therapeutic strategy for regulating B cell activity has been discussed.     

BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment

Marginal-zone (MZ) B cells represent a first line of defense against particulate blood-borne antigens. Together with the B1 cells, they are responsible for the early response against type II T-independent antigens. The molecular pathways controlling the development of MZ B cells are only poorly understood. We found that these cells are virtually absent in mice deficient in the BOB.1/OBF.1 coactivator. Loss of these B cells was demonstrated by the lack of cells showing the appropriate cell surface phenotype but also by histological analyses and tri-nitro-phenol-Ficoll capturing. The lack of these cells is a B-cell-intrinsic defect, as shown by bone marrow complementation experiments. We also show that the expression of BOB.1/OBF.1 in peripheral B cells is required for the development of MZ B lymphocytes. Our analysis of BOB.1/OBF.1-deficient splenic B cells reveals alterations in cell motility, tumor necrosis factor receptor expression, and B-cell receptor (BCR) signaling. These changes could contribute to the loss of MZ B lymphocytes by altering the maturation of the cells. Interestingly, development of and BCR signaling in B1 B cells are completely normal in BOB.1/OBF.1 mutant mice.     

The pre-B cell receptor checkpoint

B lymphocytes are essential antibody-producing cells of the immune system. During the development of progenitor B cells to mature B cells that express a membrane-bound antibody, the B cell receptor (BCR), the cells undergo selection at several checkpoints, which ensures that a diverse antibody repertoire is generated and that the BCRs recognise foreign-, but not self-, antigens. In this review, we consider the pre-BCR checkpoint. Mutations or alterations that affect this checkpoint underpin the development of pre-B cell leukemias, primary immunodeficiency, and possibly, systemic autoimmunity.     

Functional heterogeneity of memory B lymphocytes: in vivo analysis of TD-primed B cells responsive to secondary stimulation with TD and TI antigens

The functional heterogeneity of memory B cells induced by a single determinant, consisting of a decapeptide representing amino acid residues 103-112 of tobacco mosaic virus protein (TMVP), was analyzed. Decapeptide specific antibodies were elicited in mice adoptively transferred with TMVP-immune spleen cells when challenged with TMVP, decapeptide conjugated to succinylated human gamma-globulin (SHGG), or decapeptide conjugated to Brucella abortus (BA). Whereas secondary stimulation by either TMVP or decapeptide-SHGG was dependent on appropriately primed T cells, stimulation by decapeptide-BA was independent of conventional T cell help. Furthermore, memory B cells responsive to TMVP (TD), decapeptide-SHGG (TD), or decapeptide-BA (TI. 1 prototype) were shown to consist of overlapping populations because adoptive recipients of TMVP-primed cells challenged simultaneously with TD and TI decapeptide antigens did not result in a higher antibody response than that elicited by one of the TD antigens injected alone. However, decapeptide-BA consistently induced a smaller antidecapeptide response than either TMVP or decapeptide-SHGG. This suggested that only a fraction of the memory B cell population which was activated by the original priming antigen (thymus-dependent) was also responsive to secondary in vivo stimulation by the priming hapten conjugated to Brucella abortus. Detailed analyses of the antibodies induced in the recipients of TMVP-immune spleen cells after secondary challenge with either TMVP, decapeptide-SHGG, or decapeptide-BA failed to distinguish between the responsive memory B cells; the antidecapeptide antibodies induced by all three immunogens shared the same fine specificities and immunoglobulin isotype composition. These data are viewed as further evidence that subsets of TD-primed B cells, which may display differential sensitivity to cross-stimulation with TD and TI forms of the antigen, represent distinct stages of memory B cell maturation within a common B cell lineage. In support of this conclusion, we establish a developmental relationship between TI and/or TD responsive decapeptide memory B cell in the following communication.     

B cells from common variable immunodeficiency patients fail to differentiate to antibody secreting cells in response to TLR9 ligand (CpG-ODN) or anti-CD40+IL21

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.     

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.     

Multiplexed measurement of serum antibodies using an array biosensor

The array biosensor provides the capability for simultaneously measuring titers of antibody against multiple antigens. Human antibodies against four different targets, tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B (SEB) and hepatitis B, were measured simultaneously in sera from eight different donors in a single assay and titers were determined. The assays could measure amounts of bound antibody as low as approximately 100 fg. Each individual serum exhibited a different pattern of reactivity against the four target antigens. Applications of this biosensor capability include monitoring for exposure to pathogens and for efficacy of vaccination.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

The in vitro antibody response to cell surface antigens. II. Monoclonal antibodies to human leukemia cells.

A method has been developed for the production of monoclonal mouse antibody responses in vitro against human cell surface antigens. Limiting numbers of immune spleen cells were transferred to syngeneic, irradiated recipients whose spleen fragments were then cultured in vitro and stimulated to produce antibody. The majority of the antibody from any one fragment culture was likely to be the product of a single donor B cell and thus monoclonal. Evidence for this included a linear relationship between donor cell transferred and spleen fragments producing antibody, extremely restricted isoelectric focusing patterns of the individual antibody products, and unique reactivity patterns of these antibodies against a panel of human lymphoid cells. Different human B leukemia cells were seen as immunogenically distinct by the mouse. By using the monoclonal mouse antibodies as probes, a fine analysis of cell surface antigens is jow possible.