Structural principles of B cell antigen receptor assembly.

The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition1-3 and a disulfide-linked Igα (also known as CD79A) and Igß (also known as CD79B) heterodimer (Igα/ß) that functions as the signalling entity through intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5. The organizing principle of the BCR remains unknown. Here we report cryo-electron microscopy structures of mouse full-length IgM BCR and its Fab-deleted form. At the ectodomain (ECD), the Igα/ß heterodimer mainly uses Igα to associate with Cµ3 and Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC') unbound. The transmembrane domain (TMD) helices of µHC and µHC' interact with those of the Igα/ß heterodimer to form a tight four-helix bundle. The asymmetry at the TMD prevents the recruitment of two Igα/ß heterodimers. Notably, the connecting peptide between the ECD and TMD of µHC intervenes in between those of Igα and Igß to guide TMD assembly through charge complementarity. Weaker but distinct density for the Igß ITAM nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B cell signalling and designing rational therapies against BCR-mediated diseases.

Isolation of Tailor-Made Antibody Fragments from Yeast-Displayed B-Cell Receptor Repertoires by Multiparameter Fluorescence-Activated Cell Sorting.

In the past decades, monoclonal antibodies have made an unprecedented transformation from research tools to a rapidly growing class of therapeutics. Advancements in the yeast surface display platform enable the selection of favorable mouse or human antibody variants from large B-cell receptor (BCR) gene repertoires that are derived from immunized normal or transgenic animals. Application of high-throughput fluorescence-activated cell sorting (FACS) screening along with well-chosen selection settings can be utilized to identify variants with desired affinities and predefined epitope binding properties. In the following chapter, we describe in detail a multiparameter screening protocol for the selection of antibody variants from yeast libraries generated from BCR gene repertoires from immunized transgenic rats. The procedure provides guidance for the selection of antigen-specific, high-affinity binding, and species cross-reactive human antibodies with a broad epitope coverage. Essentially, this can accelerate target-specific antibody characterization as multiple desirable antibody features can be easily integrated into the selection procedure. In addition, we provide information on how to validate binding behavior of selected candidates after expression as soluble, full-length IgG molecules.

Isolation of Anti-Hapten Antibodies by Fluorescence-Activated Cell Sorting of Yeast-Displayed B-Cell Receptor Gene Repertoires.

Anti-hapten antibodies are widely used as specific immunochemical detection tools in a variety of clinical and environmental analyses. The sensitivity, however, is limited due to the resulting antibody affinities to the haptens which, in turn, leads to a high demand for specific affinity reagents. A well-established path for the generation of high-affinity antibodies is the immunization of animals with the target antigen. However, the generation of anti-hapten antibodies via immunization remains challenging as small molecule haptens usually possess low immunogenicity and, therefore, must be coupled to an immunogenic and high molecular weight carrier to provoke an immune response.Consequently, antibodies are primarily raised against the carrier molecule or structural features of the hapten-linker fused to the carrier protein. This turns the generation of antibodies which bind exclusively to the hapten structure into a search for the needle in a haystack. In the following chapter, we describe how yeast surface display and high-throughput fluorescence-activated cell sorting can be used to isolate anti-hapten antibodies from a large, yeast-displayed B-cell receptor gene library derived from immunized animals. For this, we describe in detail the preparation of protein-hapten conjugates, the immunization procedure, and the subsequent screening process. Moreover, we provide a simple flow cytometry protocol that allows for a rapid analysis of the enriched clones toward free hapten binding.

Unravelling the suboptimal response of TP53-mutated chronic lymphocytic leukaemia to ibrutinib.

TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response to ibrutinib. We hereby report that ibrutinib-induced in vitro apoptosis and proliferation inhibition were significantly lower in TP53-mutated (TP53-M) CLL cells compared to TP53 wild-type cells. Contrariwise, venetoclax effectively killed TP53-M cells. Gene expression profile analysis of TP53-M cells revealed a downmodulation of B-cell receptor (BCR)-related genes and an upmodulation of genes with anti-apoptotic/pro-survival activity, suggesting that the survival and proliferation of TP53-M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53-M CLL patients.

Expression and characterization of monomeric variable lymphocyte receptor B specific to the glycoprotein of viral hemorrhagic septicemia virus (VHSV).

Monomeric variable lymphocyte receptor B (VLRB) is one of the smallest binding scaffold (20-25 kDa) from jawless vertebrates, hagfish and lamprey. This relatively new class of binding scaffold has various advantages: i) it has a single peptide composition, amenable to molecular engineering for enhancing its stability and affinity; ii) it has a small size, contributing better tissue penetration and easier production using microorganism expression system. Monomeric arVLRB142, which can specifically bind to the glycoprotein of viral hemorrhagic septicemia virus (VHSV), was expressed in Pichia pastoris. High quantity recombinant monomeric arVLRB142 (rVLR142mono) was purified from 100 ml of culture with a resulting yield of 2.6 ±1.3 mg of target protein. Functional studies revealed that the purified rVLR142mono can specifically recognize low levels of the target antigen (recombinant glycoprotein) (i.e. as low as 0.1 nM), but also the native glycoprotein of VHSV. The expressed rVLR142mono exhibited high levels of stability and it retained it binding capacity over broad temperature (4 °C ~ 60 °C) and pH ranges (pH 1.5-12.5). We developed an effective expression system for mass production of monomeric VLRB based on P. pastoris. The recombinant protein that was obtained offers promising binding avidity and biophysical stability and its potential use in various biotechnological applications.

The immunomodulatory peptide bursopentin (BP5) enhances proliferation and induces sIgM expression in DT40 cells.

BACKGROUND: In the recent past, many studies have been focused on extracts of BF and multiple biologically active factors and their effects on humoral immune system in chickens and birds. However, the mechanism of those immunomodulatory peptides on the B lineage cells proliferation and antibody production in chicken is fairly unknown. DT40 cell line, an avian leucosis virus-induced chicken pre-B cell line, expresses immunoglobulin M (IgM) isotype B cell reporter in the plasma membrane. There are many evidences suggesting that DT40 cells are best characterized as a bursal stem cell line. Because of the unique characteristics of DT40 cell line, it has been widely used to observe biological processes of pre-B lymphocyte cell within living cells. METHODS: The chicken B cell line DT40 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and cytotoxicity was studied. Also, effect of BP5 on cell proliferation and cell cycle distribution of DT40 cells was studied. Also, the effect of BP5 on sIgM mRNA expression was studied by using real-time PCR. OBJECTIVES: To investigat the effects of Bursopentin (Cys-Lys-Arg-Val-Tyr, BP5) on a chicken promyelocyte cell line DT40, assays of cell proliferation, cell cycle distribution, detection of surface immunoglobulin G (sIgM) mRNA expression and gene microarray analysis were performed. RESULTS: The results showed that BP5 displayed concentration-dependent effects on the proliferation, cell cycle, and sIgM mRNA expression in DT40 cells. And the analysis of expression profiles identified a signature set of 3022 genes (1254 up regulated genes, 1762 down regulated genes), which clearly discriminated the BP5-treated DT40 cells from control with high certainty (P≤0.02). The results of microarray analysis were confirmed by quantitative reverse transcription-polymerase chain reaction for 12 of the differentially expressed genes. CONCLUSION: Theses findings showed the immuno-activity effect of BP5 on B lymphocyte and indicated that BP5 treatment regulated eight signaling pathways, in which Toll-like signaling pathway was the most significant enrichment pathway.

Lymphoblastic leukemia with surface light chain restriction: A diagnostic dilemma.

Surface light chain expression is a feature of mature B-cell neoplasms. Light chain restriction in precursor B acute lymphoblastic leukemia is infrequently seen. We report a case of a 28-year-old female with non-FAB L3 morphology blasts and immunophenotypic features showing overlap between a precursor and mature B-cell neoplasm.

B cell receptor induced Fc receptor-like 5 expression is mediated by multiple signaling pathways converging on NF-κB and NFAT.

Fc receptor-like (FCRL) proteins are novel regulators of the B cell response to antigen. Human FCRL5 binds intact IgG and modifies the strength of antigen receptor (BCR) signaling. Altering FCRL5 expression could therefore regulate the B cell response to antigen. In this study, we found that FCRL5 expression is induced specifically upon BCR stimulation and dissected the molecular mechanism. FCRL5 mRNA and cell surface protein expression required prolonged BCR stimulation and de novo protein synthesis. Using chemical inhibitors and activators, we identified roles for several signaling pathways, indicating a complex mechanism. Specifically, the PI3K/AKT, JNK, PKC and IKK2-dependent classical NF-κB pathways were involved in induced FCRL5 expression. Furthermore, induced FCRL5 expression required elevation of intracellular Ca(++) and was partially blocked by cyclosporine A, a calcineurin inhibitor. The importance of the transcription factors NF-κB, NFAT and CREB-binding protein was revealed based on sensitivity to inhibitors. Using reporter gene assays, we showed that the core FCRL5 promoter was sufficient to drive induced gene expression. Mutations of two predicted NF-κB sites or an NFAT site in the core promoter abrogated induced gene expression, suggesting direct regulation of the FCRL5 gene by NF-κB and NFAT. In support, we detected binding of NF-κB and NFAT family proteins to oligonucleotides corresponding to the predicted sites. We propose that the identified intricate mechanism serves to ensure that FCRL5 is expressed on B cells at a precise time following antigen encounter, with potential implications regarding regulation of the B cell response.

Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation.

B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association with effector proteins and with endo-lysosomal compartments. In addition, we show that BCL10 is modified by LUBAC-mediated linear ubiquitylation, and demonstrate an important function of LUBAC in BCR-induced NF-κB signaling. Our results offer a global and integrated view of BCR signaling, and the provided datasets can serve as a valuable resource for further understanding BCR signaling networks.

Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells.

Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, µ heavy (µH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, µH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

The class-specific BCR tonic signal modulates lymphomagenesis in a c-myc deregulation transgenic model.

Deregulation of c-myc by translocation onto immunoglobulin (Ig) loci can promote B cell malignant proliferations with phenotypes as diverse as acute lymphoid leukemia, Burkitt lymphoma, diffuse large B cell lymphoma, myeloma... The B cell receptor (BCR) normally providing tonic signals for cell survival and mitogenic responses to antigens, can also contribute to lymphomagenesis upon sustained ligand binding or activating mutations. BCR signaling varies among cell compartments and BCR classes. For unknown reasons, some malignancies associate with expression of either IgM or class-switched Ig. We explored whether an IgA BCR, with strong tonic signaling, would affect lymphomagenesis in c-myc IgH 3'RR transgenic mice prone to lymphoproliferations. Breeding c-myc transgenics in a background where IgM expression was replaced with IgA delayed lymphomagenesis. By comparison to single c-myc transgenics, lymphomas from double mutant animals were more differentiated and less aggressive, with an altered transcriptional program. Larger tumor cells more often expressed CD43 and CD138, which culminated in a plasma cell phenotype in 10% of cases. BCR class-specific signals thus appear to modulate lymphomagenesis and may partly explain the observed association of specific Ig classes with human B cell malignancies of differential phenotype, progression and prognosis.

The Eµ enhancer region influences H chain expression and B cell fate without impacting IgVH repertoire and immune response in vivo.

The IgH intronic enhancer region Eµ is a combination of both a 220-bp core enhancer element and two 310-350-bp flanking scaffold/matrix attachment regions named MARsEµ. In the mouse, deletion of the core-enhancer Eµ element mainly affects VDJ recombination with minor effects on class switch recombination. We carried out endogenous deletion of the full-length Eµ region (core plus MARsEµ) in the mouse genome to study VH gene repertoire and IgH expression in developing B-lineage cells. Despite a severe defect in VDJ recombination with partial blockade at the pro-B cell stage, Eµ deletion (core or full length) did not affect VH gene usage. Deletion of this regulatory region induced both a decrease of pre-B cell and newly formed B cell compartments and a strong orientation toward the marginal zone B cell subset. Because Igµ H chain expression was decreased in Eµ-deficient pre-B cells, we propose that modification of B cell homeostasis in deficient animals was caused by "weak" pre-B cell and BCR expression. Besides imbalances in B cell compartments, Ag-specific Ab responses were not impaired in animals carrying the Eµ deletion. In addition to its role in VDJ recombination, our study points out that the full-length Eµ region does not influence VH segment usage but ensures efficient Igµ-chain expression required for strong signaling through pre-B cells and newly formed BCRs and thus participates in B cell inflow and fate.

Copresentation of antigen and ligands of Siglec-G induces B cell tolerance independent of CD22.

Differentiation of self from nonself is indispensable for maintaining B cell tolerance in peripheral tissues. CD22 and Siglec-G (sialic acid-binding Ig-like lectin G) are two inhibitory coreceptors of the BCR that are implicated in maintenance of tolerance to self Ags. Enforced ligation of CD22 and the BCR by a nanoparticle displaying both Ag and CD22 ligands induces a tolerogenic circuit resulting in apoptosis of the Ag-reactive B cell. Whether Siglec-G also has this property has not been investigated in large part owing to the lack of a selective Siglec-G ligand. In this article, we report the development of a selective high-affinity ligand for Siglec-G and its application as a chemical tool to investigate the tolerogenic potential of Siglec-G. We find that liposomal nanoparticles decorated with Ag and Siglec-G ligand inhibit BCR signaling in both B1 and B2 B cells compared with liposomes displaying Ag alone. Not only is inhibition of B cell activation observed by ligating the BCR with Siglec-G, but robust tolerance toward T-independent and T-dependent Ags is also induced in mice. The ability of Siglec-G to inhibit B cell activation equally in both B1 and B2 subsets is consistent with our observation that Siglec-G is expressed at a relatively constant level throughout numerous B cell subsets. These results suggest that Siglec-G may contribute to maintenance of B cell tolerance toward self Ags in various B cell compartments.

Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.

Tolerant anti-insulin B cells are effective APCs.

Autoreactive B lymphocytes that are not culled by central tolerance in the bone marrow frequently enter the peripheral repertoire in a state of functional impairment, termed anergy. These cells are recognized as a liability for autoimmunity, but their contribution to disease is not well understood. Insulin-specific 125Tg B cells support T cell-mediated type 1 diabetes in NOD mice, despite being anergic to B cell mitogens and T cell-dependent immunization. Using this model, the potential of anergic, autoreactive B cells to present Ag and activate T cells was investigated. The data show that 1) insulin is captured and rapidly internalized by 125Tg BCRs, 2) these Ag-exposed B cells are competent to activate both experienced and naive CD4(+) T cells, 3) anergic 125Tg B cells are more efficient than naive B cells at activating T cells when Ag is limiting, and 4) 125Tg B cells are competent to generate low-affinity insulin B chain epitopes necessary for activation of diabetogenic anti-insulin BDC12-4.1 T cells, indicating the pathological relevance of anergic B cells in type 1 diabetes. Thus, phenotypically tolerant B cells that are retained in the repertoire may promote autoimmunity by driving activation and expansion of autoaggressive T cells via Ag presentation.

TLR2 may influence the behavior of the malignant clone in B-CLL.

B-cell receptor (BCR) and Toll-like receptor (TLR) stimulation and integration with signals from the pathogen or immune cells and their products determine the B-cell antibody response. Low expression of BCR is the hallmark of B lymphocytes in CLL, however little is known about the expression and function of TLR in B-CLL. We studied TLR2, TLR4, IL-6 and mIL-6Rα expression on mRNA and protein level in CD19(+) subpopulation of normal lymphocytes and the CD19(+)CD5(+) subpopulation from B-CLL. Experiments were performed on unstimulated and stimulated lymphocytes [Staphylococcus aureus Cowan I (SAC) and lipopolysaccharide (LPS) from Escherichia coli - TLR2- and TLR4-specific agonists, respectively]. We showed undetectable or low IL-6 expression, which seems to be specific for B-CLL lymphocytes. Induction of TLR4 mRNA upon LPS stimulation affected the expression of IL-6, but not of mIL-6Rα. Increased expression of TLR2 (MFI) after LPS and SAC stimulation did not correlate with mIL-6Rα receptor expression. B-CLL CD19(+)CD5(+) lymphocytes showed a significant increase in TLR2 expression at the protein level after stimulation with SAC and LPS compared to normal CD19(+) lymphocytes. TLR2 may influence the behaviour of the malignant clone in B-CLL.

Direct reduction of antigen receptor expression in polyclonal B cell populations developing in vivo results in light chain receptor editing.

Secondary Ab V region gene segment rearrangement, termed receptor editing, is a major mechanism contributing to B lymphocyte self-tolerance. However, the parameters that determine whether a B cell undergoes editing are a current subject of debate. We tested the role that the level of BCR expression plays in the regulation of receptor editing in a polyclonal population of B cells differentiating in vivo. Expression of a short hairpin RNA for κ L chain RNA in B cells resulted in reduction in levels of this RNA and surface BCRs. Strikingly, fully mature and functional B cells that developed in vivo and efficiently expressed the short hairpin RNA predominantly expressed BCRs containing λ light chains. This shift in L chain repertoire was accompanied by inhibition of development, increased Rag gene expression, and increased λ V gene segment-cleavage events at the immature B cell stage. These data demonstrated that reducing the translation of BCRs that are members of the natural repertoire at the immature B cell stage is sufficient to promote editing.

Fc receptor-like 5 promotes B cell proliferation and drives the development of cells displaying switched isotypes.

The biological roles of B cell membrane proteins in the FCRL family are enigmatic. FCRL proteins, including FCRL5, were shown to modulate early BCR signaling, although the subsequent, functional consequences of receptor engagement are poorly understood. We found that FCRL5 surface protein itself was induced temporarily upon BCR stimulation of human, naive B cells, indicating precise control over timing of FCRL5 engagement. Cross-linking of FCRL5 on cells induced to express FCRL5 enhanced B cell proliferation significantly. This enhancement required costimulation of the BCR and TLR9, two signals required for optimal proliferation of naive B cells, whereas T cell help in the form of anti-CD40 and IL-2 was dispensable. In addition, we found that FCRL5 stimulation generated a high proportion of cells displaying surface IgG and IgA. Optimal development of cells expressing switched isotypes required T cell help, in addition to stimuli found necessary for enhanced proliferation. Surprisingly, cells that developed upon FCRL5 stimulation simultaneously displayed surface IgM, IgG, and IgA. Cells expressing multiple Ig isotypes were described in hairy cell leukemia, a disease in which FCRL5 is overexpressed. Enhanced proliferation and downstream isotype expression upon FCRL5 stimulation could reflect a physiological role for FCRL5 in the expansion and development of antigen-primed B cells. In addition, FCRL5 may promote growth of malignant cells in hairy cell leukemia and other FCRL5-expressing tumors.

Expansion of functionally anergic CD21-/low marginal zone-like B cell clones in hepatitis C virus infection-related autoimmunity.

Homeostasis of peripheral B cell subsets is disturbed during chronic hepatitis C virus (HCV) infection, leading to the occurrence of autoimmunity and B cell lymphoproliferation. However, mechanisms by which HCV causes lymphoproliferation remain controversial. We report in this article on the elevated number of clonal CD21(-/low)IgM(+)CD27(+) marginal zone (MZ)-like B cells, which correlates with autoimmunity and lymphoproliferation in HCV patients. We found an increase in autoreactive BCRs using V(H)1-69 and V(H)4-34 genes in CD21(-/low) MZ B cells. CD21(-/low) MZ B cells showed impaired calcium-mediated signaling, did not upregulate activation markers, and did not proliferate in response to BCR triggering. CD21(-/low) MZ B cells also were prone to dying faster than their CD21(+) counterparts, suggesting that these B cells were anergic. CD21(-/low) MZ B cells, in contrast, remained responsive to TLR9 stimulation. Gene array analyses revealed the critical role of Early growth response 2 and Cbl-b in the induction of anergy. Therefore, HCV patients who display high frequencies of unresponsive CD21(-/low) MZ B cells are more susceptible to developing autoimmunity and/or lymphoproliferation. These cells remain in peripheral blood controlled by functional anergy instead of being eliminated, and chronic antigenic stimulation through TLR stimulation may create a favorable environment for breaking tolerance and activating these cells.