Showing papers on "B-cell receptor published in 2015"

Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma

Abstract: The two major subtypes of diffuse large B cell lymphoma (DLBCL)--activated B cell-like (ABC) and germinal center B cell-like (GCB)--arise by distinct mechanisms, with ABC selectively acquiring mutations that target the B cell receptor (BCR), fostering chronic active BCR signaling. The ABC subtype has a ∼40% cure rate with currently available therapies, which is worse than the rate for GCB DLBCL, and highlights the need for ABC subtype-specific treatment strategies. We hypothesized that ABC, but not GCB, DLBCL tumors would respond to ibrutinib, an inhibitor of BCR signaling. In a phase 1/2 clinical trial that involved 80 subjects with relapsed or refractory DLBCL, ibrutinib produced complete or partial responses in 37% (14/38) of those with ABC DLBCL, but in only 5% (1/20) of subjects with GCB DLBCL (P = 0.0106). ABC tumors with BCR mutations responded to ibrutinib frequently (5/9; 55.5%), especially those with concomitant myeloid differentiation primary response 88 (MYD88) mutations (4/5; 80%), a result that is consistent with in vitro cooperation between the BCR and MYD88 pathways. However, the highest number of responses occurred in ABC tumors that lacked BCR mutations (9/29; 31%), suggesting that oncogenic BCR signaling in ABC does not require BCR mutations and might be initiated by non-genetic mechanisms. These results support the selective development of ibrutinib for the treatment of ABC DLBCL.

Autoantibodies in systemic autoimmune diseases: specificity and pathogenicity

Abstract: In this Review we focus on the initiation of autoantibody production and autoantibody pathogenicity, with a special emphasis on the targeted antigens. Release of intracellular antigens due to excessive cell death or to ineffective clearance of apoptotic debris, modification of self-antigens during inflammatory responses, and molecular mimicry contribute to the initiation of autoantibody production. We hypothesize that those autoreactive B cells that survive and produce pathogenic autoantibodies have specificity for self-antigens that are TLR ligands. Such B cells experience both B cell receptor (BCR) activation and TLR engagement, leading to an escape from tolerance. Moreover, the autoantibodies they produce form immune complexes that can activate myeloid cells and thereby establish the proinflammatory milieu that further negates tolerance mechanisms of both B and T cells.

Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function

Abstract: Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity.

Checkpoints that control B cell development

Abstract: B cells differentiate from pluripotent hematopoietic stem cells (pHSCs) in a series of distinct stages. During early embryonic development, pHSCs migrate into the fetal liver, where they develop and mature to B cells in a transient wave, which preferentially populates epithelia and lung as well as gut-associated lymphoid tissues. This is followed by continuous B cell development throughout life in the bone marrow to immature B cells that migrate to secondary lymphoid tissues, where they mature. At early stages of development, before B cell maturation, the gene loci encoding the heavy and light chains of immunoglobulin that determine the B cell receptor composition undergo stepwise rearrangements of variable region-encoding gene segments. Throughout life, these gene rearrangements continuously generate B cell repertoires capable of recognizing a plethora of self-antigens and non-self-antigens. The microenvironment in which these B cell repertoires develop provide signaling molecules that play critical roles in promoting gene rearrangements, proliferation, survival, or apoptosis, and that help to distinguish self-reactive from non-self-reactive B cells at four distinct checkpoints. This refinement of the B cell repertoire directly contributes to immunity, and defects in the process contribute to autoimmune disease.

Molecular biology of B cells

Abstract: ORGANIZATION, REARRANGEMENT AND TRANSCRIPTION OF IMMUNOGLOBULIN GENES Human Immunoglobulin heavy chains locus Immunoglobulin heavy chain genes of mouse Immunoglobulin ? genes of human and mouse Immunoglobulin lambdav (IGL) genes of human and mouse The Mechanism of V(D) J Recombination Transcription of Immunoglobulin genes B CELLS AND THEIR DEVELOPMENT Early B cell development to a mature , antigen-sensitive cell - cellular stages and molecular decisions Allelic exclusion, Isotypic exclusion and the Developmental Regulation of V(D)J Recombination The Development of Human B Lymphocytes Development and function of B cell subsets B CELL DIFFERENTIATION INDUCED BY Ag STIMULATION Structure and function of B cells antigen receptor complexes Regulation of antigen receptor signaling by the co-receptors, CD19 and CD22 The dynamic structure of antibody responses Dynamics of B cell migration to and within secondary lymphoid organs Characteristics of mucosal B cells with emphasis on the human secretory immune system The Cellular Basis of B Cell Memory Immunoglobulin assembly and secretion Fc And Complement Receptors Ig GENE ALTERATION IN PERIPHERY Regulation of Class Switch Recombination Molecular Mechanism of Class Switch Recombination Molecular Mechanism of Hypermutation Selection During Antigen-Driven B Cell Immune Responses: The Basis for High Affinity Antibody DISEASES OF B CELLS Chromosomal Translocations in B-cell Leukemias and Lymphomas Classification and Characteristics of Mouse B Cell-Lineage Lymphomas B cells producing pathogenic autoantibodies Immunodeficiencies Caused by B Cell Defects EVOLUTION OF Ig GENES Diverse forms of Immunoglobulin Genes in Lower Vertebrates Immunoglobulin Genes and Generation of Antibody Repertoires in Higher Vertebrates: A Key Role for GALT The zebrafish immune system The Origin of V(D)J Diversification FUNCTION AND APPLICATION OF IMMUNOGLOBULINS Antibody Structure and Recognition of Antigen Monoclonal antibodies from display libraries Humanization of Monoclonal Antibodies Human monoclonal antibodies from translocus mice

B-cell receptor signalling and its crosstalk with other pathways in normal and malignant cells.

Abstract: The physiology of B cells is intimately connected with the function of their B-cell receptor (BCR). B-cell lymphomas frequently (dys)regulate BCR signalling and thus take advantage of this pre-existing pathway for B-cell proliferation and survival. This has recently been underscored by clinical trials demonstrating that small molecules (fosfamatinib, ibrutinib, idelalisib) inhibiting BCR-associated kinases (SYK, BTK, PI3K) have an encouraging clinical effect. Here we describe the current knowledge of the specific aspects of BCR signalling in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukaemia (CLL) and normal B cells. Multiple factors can contribute to BCR pathway (dys)regulation in these malignancies and the activation of 'chronic' or 'tonic' BCR signalling. In lymphoma B cells, the balance of initiation, amplitude and duration of BCR activation can be influenced by a specific immunoglobulin structure, the expression and mutations of adaptor molecules (like GAB1, BLNK, GRB2, CARD11), the activity of kinases (like LYN, SYK, PI3K) or phosphatases (like SHIP-1, SHP-1 and PTEN) and levels of microRNAs. We also discuss the crosstalk of BCR with other signalling pathways (NF-κB, adhesion through integrins, migration and chemokine signalling) to emphasise that the 'BCR inhibitors' target multiple pathways interconnected with BCR, which might explain some of their clinical activity.

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

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

Apoptosis and antigen affinity limit effector cell differentiation of a single naïve B cell

Abstract: When exposed to antigens, naive B cells differentiate into different types of effector cells: antibody-producing plasma cells, germinal center cells, or memory cells. Whether an individual naive B cell can produce all of these different cell fates remains unclear. Using a limiting dilution approach, we found that many individual naive B cells produced only one type of effector cell subset, whereas others produced all subsets. The capacity to differentiate into multiple subsets was a characteristic of clonal populations that divided many times and resisted apoptosis, but was independent of isotype switching. Antigen receptor affinity also influenced effector cell differentiation. These findings suggest that diverse effector cell types arise in the primary immune response as a result of heterogeneity in responses by individual naive B cells.

Identification of Antigen-Specific B Cell Receptor Sequences Using Public Repertoire Analysis

Abstract: High-throughput sequencing allows detailed study of the BCR repertoire postimmunization, but it remains unclear to what extent the de novo identification of Ag-specific sequences from the total BCR repertoire is possible. A conjugate vaccine containing Haemophilus influenzae type b (Hib) and group C meningococcal polysaccharides, as well as tetanus toxoid (TT), was used to investigate the BCR repertoire of adult humans following immunization and to test the hypothesis that public or convergent repertoire analysis could identify Ag-specific sequences. A number of Ag-specific BCR sequences have been reported for Hib and TT, which made a vaccine containing these two Ags an ideal immunological stimulus. Analysis of identical CDR3 amino acid sequences that were shared by individuals in the postvaccine repertoire identified a number of known Hib-specific sequences but only one previously described TT sequence. The extension of this analysis to nonidentical, but highly similar, CDR3 amino acid sequences revealed a number of other TT-related sequences. The anti-Hib avidity index postvaccination strongly correlated with the relative frequency of Hib-specific sequences, indicating that the postvaccination public BCR repertoire may be related to more conventional measures of immunogenicity correlating with disease protection. Analysis of public BCR repertoire provided evidence of convergent BCR evolution in individuals exposed to the same Ags. If this finding is confirmed, the public repertoire could be used for rapid and direct identification of protective Ag-specific BCR sequences from peripheral blood.

B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation.

Abstract: The B cell antigen receptors (BCRs) play an important role in the clonal selection of B cells and their differentiation into antibody-secreting plasma cells. Mature B cells have both immunoglobulin M (IgM) and IgD types of BCRs, which have identical antigen-binding sites and are both associated with the signaling subunits Igα and Igβ, but differ in their membrane-bound heavy chain isoforms. By two-color direct stochastic optical reconstruction microscopy (dSTORM), we showed that IgM-BCRs and IgD-BCRs reside in the plasma membrane in different protein islands with average sizes of 150 and 240 nm, respectively. Upon B cell activation, the BCR protein islands became smaller and more dispersed such that the IgM-BCRs and IgD-BCRs were found in close proximity to each other. Moreover, specific stimulation of one class of BCR had minimal effects on the organization of the other. These conclusions were supported by the findings from two-marker transmission electron microscopy and proximity ligation assays. Together, these data provide evidence for a preformed multimeric organization of BCRs on the plasma membrane that is remodeled after B cell activation.

Peanut oral immunotherapy transiently expands circulating Ara h 2-specific B cells with a homologous repertoire in unrelated subjects.

Abstract: Background Peanut oral immunotherapy (PNOIT) induces persistent tolerance to peanut in a subset of patients and induces specific antibodies that might play a role in clinical protection. However, the contribution of induced antibody clones to clinical tolerance in PNOIT is unknown. Objective We hypothesized that PNOIT induces a clonal, allergen-specific B-cell response that could serve as a surrogate for clinical outcomes. Methods We used a fluorescent Ara h 2 multimer for affinity selection of Ara h 2–specific B cells and subsequent single-cell immunoglobulin amplification. The diversity of related clones was evaluated by means of next-generation sequencing of immunoglobulin heavy chains from circulating memory B cells with 2x250 paired-end sequencing on the Illumina MiSeq platform. Results Expression of class-switched antibodies from Ara h 2–positive cells confirms enrichment for Ara h 2 specificity. PNOIT induces an early and transient expansion of circulating Ara h 2–specific memory B cells that peaks at week 7. Ara h 2–specific sequences from memory cells have rates of nonsilent mutations consistent with affinity maturation. The repertoire of Ara h 2–specific antibodies is oligoclonal. Next-generation sequencing–based repertoire analysis of circulating memory B cells reveals evidence for convergent selection of related sequences in 3 unrelated subjects, suggesting the presence of similar Ara h 2–specific B-cell clones. Conclusions Using a novel affinity selection approach to identify antigen-specific B cells, we demonstrate that the early PNOIT-induced Ara h 2–specific B-cell receptor repertoire is oligoclonal and somatically hypermutated and shares similar clonal groups among unrelated subjects consistent with convergent selection.

Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia

Abstract: NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBe, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBe protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBe loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

A high density of tertiary lymphoid structure B cells in lung tumors is associated with increased CD4(+) T cell receptor repertoire clonality.

Abstract: T and B cell receptor (TCR and BCR, respectively) Vβ or immunoglobulin heavy chain complementarity-determining region 3 sequencing allows monitoring of repertoire changes through recognition, clonal expansion, affinity maturation, and T or B cell activation in response to antigen. TCR and BCR repertoire analysis can advance understanding of antitumor immune responses in the tumor microenvironment. TCR and BCR repertoires of sorted CD4+, CD8+ or CD19+ cells in tumor, non-tumoral distant tissue (NT), and peripheral compartments (blood/draining lymph node [P]) from 47 non-small cell lung cancer (NSCLC) patients (agemedian = 68 y) were sequenced. The clonotype spectra were assessed among different tissues and correlated with clinical and immunological parameters. In all tissues, CD4+ and CD8+ TCR repertoires had greater clonality relative to CD19+ BCR. CD4+ T cells exhibited greater clonality in NT compared to tumor (p = 0.002) and P (p 68). Younger patients ...

Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor.

Abstract: Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

In-Depth Assessment of Within-Individual and Inter-Individual Variation in the B Cell Receptor Repertoire.

Abstract: High-throughput sequencing of the B cell receptor (BCR) repertoire can provide rapid characterization of the B cell response in a wide variety of applications in health, after vaccination and in infectious, inflammatory and immune-driven disease, and is starting to yield clinical applications. However, the interpretation of repertoire data is compromised by a lack of studies to assess the intra and inter-individual variation in the BCR repertoire over time in healthy individuals. We applied a standardized isotype-specific BCR repertoire deep sequencing protocol to a single highly sampled participant, and then evaluated the method in 9 further participants to comprehensively describe such variation. We assessed total repertoire metrics of mutation, diversity, VJ gene usage and isotype subclass usage as well as tracking specific BCR sequence clusters. There was good assay reproducibility (both in PCR amplification and biological replicates), but we detected striking fluctuations in the repertoire over time that we hypothesize may be due to subclinical immune activation. Repertoire properties were unique for each individual, which could partly be explained by a decrease in IgG2 with age, and genetic differences at the immunoglobulin locus. There was a small repertoire of public clusters (0.5, 0.3, and 1.4% of total IgA, IgG, and IgM clusters, respectively), which was enriched for expanded clusters containing sequences with suspected specificity toward antigens that should have been historically encountered by all participants through prior immunization or infection. We thus provide baseline BCR repertoire information that can be used to inform future study design, and aid in interpretation of results from these studies. Furthermore, our results indicate that BCR repertoire studies could be used to track changes in the public repertoire in and between populations that might relate to population immunity against infectious diseases, and identify the characteristics of inflammatory and immunological diseases.

Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome

Abstract: Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder frequently associated with systemic autoimmunity, including autoantibody-mediated cytopenias WAS protein (WASp)-deficient B cells have increased B cell receptor (BCR) and Toll-like receptor (TLR) signaling, suggesting that these pathways might impact establishment of the mature, naive BCR repertoire To directly investigate this possibility, we evaluated naive B cell specificity and composition in WASp-deficient mice and WAS subjects (n = 12) High-throughput sequencing and single-cell cloning analysis of the BCR repertoire revealed altered heavy chain usage and enrichment for low-affinity self-reactive specificities in murine marginal zone and human naive B cells Although negative selection mechanisms including deletion, anergy, and receptor editing were relatively unperturbed, WASp-deficient transitional B cells showed enhanced proliferation in vivo mediated by antigen- and Myd88-dependent signals Finally, using both BCR sequencing and cell surface analysis with a monoclonal antibody recognizing an intrinsically autoreactive heavy chain, we show enrichment in self-reactive cells specifically at the transitional to naive mature B cell stage in WAS subjects Our combined data support a model wherein modest alterations in B cell-intrinsic, BCR, and TLR signals in WAS, and likely other autoimmune disorders, are sufficient to alter B cell tolerance via positive selection of self-reactive transitional B cells

Cutting Edge: An In Vivo Reporter Reveals Active B Cell Receptor Signaling in the Germinal Center

Abstract: Long-lasting Ab responses rely on the germinal center (GC), where B cells bearing high-affinity Ag receptors are selected from a randomly mutated pool to populate the memory and plasma cell compartments. Signaling downstream of the BCR is dampened in GC B cells, raising the possibility that Ag presentation and competition for T cell help, rather than Ag-dependent signaling per se, drive these critical selection events. In this study we use an in vivo reporter of BCR signaling, Nur77-eGFP, to demonstrate that although BCR signaling is reduced among GC B cells, a small population of cells exhibiting GC light zone phenotype (site of Ag and follicular helper T cell encounter) express much higher levels of GFP. We show that these cells exhibit somatic hypermutation, gene expression characteristic of signaling and selection, and undergo BCR signaling in vivo.

Core fucosylation of IgG B cell receptor is required for antigen recognition and antibody production

Abstract: Ag recognition and Ab production in B cells are major components of the humoral immune response. In the current study, we found that the core fucosylation catalyzed by α1,6-fucosyltransferase (Fut8) was required for the Ag recognition of BCR and the subsequent signal transduction. Moreover, compared with the 3-83 B cells, the coalescing of lipid rafts and Ag-BCR endocytosis were substantially reduced in Fut8-knockdown (3-83-KD) cells with p31 stimulation and then completely restored by reintroduction of the Fut8 gene to the 3-83-KD cells. Indeed, Fut8-null (Fut8(-/-)) mice evoked a low immune response following OVA immunization. Also, the frequency of IgG-producing cells was significantly reduced in the Fut8(-/-) spleen following OVA immunization. Our results clearly suggest an unexpected mode of BCR function, in which the core fucosylation of IgG-BCR mediates Ag recognition and, concomitantly, cell signal transduction via BCR and Ab production.

Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity

Abstract: The small Rho GTPase Cdc42, known to interact with Wiskott–Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell–intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.

CD19 and BAFF-R can signal to promote B-cell survival in the absence of Syk.

Abstract: The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B-cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B-cell-specific deletion of the Syk gene and found that a considerable fraction of mature Syk-negative B cells can survive in the periphery for an extended time. Syk-negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL-4, anti-CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk-deficient B cells require BAFF receptor and CD19/PI3K signaling for their long-term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B-cell pool.

Targeting neoplastic B cells and harnessing microenvironment: the “double face” of ibrutinib and idelalisib

Abstract: Tyrosine kinase inhibitors (TKIs) targeting signaling molecules downstream B cell receptor (BCR) are powerfully spreading in the therapeutic landscape of B cell lymphoproliferative disease, due to a manageable toxicity profile and encouraging clinical effectiveness. In particular, ibrutinib, previously called PCI-32765, is a potent inhibitor of Bruton tyrosine kinase (Btk), recently approved for the treatment of relapsed mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Moreover, idelalisib (formerly GS-1101 and CAL-101) is a selective reversible inhibitor of the p110δ isoform of phosphoinositol 3 kinase (PI3K) approved for the treatment of patients with relapsed follicular lymphoma (FL) and CLL. These agents directly affect the neoplastic clone, disrupting the supportive platform provided by BCR signaling cascade and by other microenvironmental mutualistic interactions, and also interfering with chemokine gradients and adhesive properties of neoplastic B cells. In the present review, we describe the clinical efficacy of ibrutinib and idelalisib in CLL and B cell non-Hodgkin lymphoma (B-NHL), then focusing on the mode of action (MOA) of these TKIs towards the neoplastic B cell compartment. At last, the review would further expand the view on potential additional targets of ibrutinib and idelalisib belonging to other microenvironmental cellular elements.

Internalization of rituximab and the efficiency of B Cell depletion in rheumatoid arthritis and systemic lupus erythematosus.

Abstract: Objective: Rituximab, a type I anti-CD20 monoclonal antibody (mAb), induces incomplete B cell depletion in some patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), thus contributing to a poor clinical response. The mechanisms of this resistance remain elusive. The purpose of this study was to determine whether type II mAb are more efficient than type I mAb at depleting B cells from RA and SLE patients, whether internalization influences the efficiency of depletion, and whether Fc? receptor type IIb (Fc?RIIb) and the B cell receptor regulate this internalization process. Methods: We used an in vitro whole blood B cell–depletion assay to assess the efficiency of depletion, flow cytometry to study cell surface protein expression, and surface fluorescence–quenching assays to assess rituximab internalization, in samples from patients with RA and patients with SLE. Paired t-test or Mann-Whitney U test was used to compare groups, and Spearman's rank correlation test was used to assess correlation. Results: We found that type II mAb internalized significantly less rituximab than type I mAb and depleted B cells from patients with RA and SLE at least 2-fold more efficiently than type I mAb. Internalization of rituximab was highly variable between patients, was regulated by Fc?RIIb, and inversely correlated with cytotoxicity in whole blood B cell–depletion assays. The lowest levels of internalization were seen in IgD– B cells, including postswitched (IgD–CD27+) memory cells. Internalization of type I anti-CD20 mAb was also partially inhibited by anti-IgM stimulation. Conclusion: Variability in internalization of rituximab was observed and was correlated with impaired B cell depletion. Therefore, slower-internalizing type II mAb should be considered as alternative B cell–depleting agents for the treatment of RA and SLE.

Role of Calcium Signaling in B Cell Activation and Biology.

Abstract: Increase in intracellular levels of calcium ions (Ca2+) is one of the key triggering signals for the development of B cell response to the antigen. The diverse Ca2+ signals finely controlled by multiple factors participate in the regulation of gene expression, B cell development, and effector functions. B cell receptor (BCR)-initiated Ca2+ mobilization is sourced from two pathways: one is the release of Ca2+ from the intracellular stores, endoplasmic reticulum (ER), and other is the prolonged influx of extracellular Ca2+ induced by depleting the stores via store-operated calcium entry (SOCE) and calcium release-activated calcium (CRAC) channels. The identification of stromal interaction molecule 1(STIM1), the ER Ca2+ sensor, and Orai1, a key subunit of the CRAC channel pore, has now provided the tools to understand the mode of Ca2+ influx regulation and physiological relevance. Herein, we discuss our current understanding of the molecular mechanisms underlying BCR-triggered Ca2+ signaling as well as its contribution to the B cell biological processes and diseases.

Inhibitors of BCR signalling interrupt the survival signal mediated by the micro-environment in mantle cell lymphoma.

Abstract: Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B-cell receptor (BCR)-mediated signalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through therapies targeting BCR-associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high amount of IL-1β, IL-6, IL-8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased autocrine secretion of IL-1β, IL-6, IL-8, CCL5, IL-10, TNFα and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamatinib (R406) reversed these protective effects and decreased both basal and BCR-induced autocrine cytokine secretions associated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR-induced MCL cell adhesion to human bone marrow stromal cells (HMSCs) in short- and long-term co-culture. We demonstrated that BCR-induced survival relies on autocrine secretion of IL-1β, TNFα and CCL5 that might facilitate adhesion of MCL cells to HMSC. Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis.

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: Modulation by BCR and CD40, and relevance to T-independent antibody responses

Abstract: Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS-mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing effective T-independent antibody responses to microbial pathogens, allergens and vaccines.