B‐cell intrinsic and extrinsic signals that regulate central tolerance of mouse and human B cells*

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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CVID-Associated B Cell Activating Factor Receptor Variants Change Receptor Oligomerization, Ligand Binding and Signaling Responses

10.21203/rs.3.rs-1068596/v1 ◽

2021 ◽

Author(s):

Violeta Block ◽

Eirini Sevdali ◽

Mike Recher ◽

Hassan Abolhassani ◽

Lennart Hammarstrom ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Receptor ◽

Ectodomain Shedding ◽

Cellular Functions ◽

Single Nucleotide Variants ◽

B Cell Activating Factor ◽

Human B Cells ◽

Receptor Component ◽

Primary Amino

Abstract Purpose B cell activating factor (BAFF) binding to BAFF-receptor(BAFFR) activates essential cellular functions required forthe survival of mature, human B cells. Thus,deletion ofthe BAFFR gene blocks the development of B cells at the transition from immature to mature B cells resulting in B lymphopenia and hypogammaglobulinemia. In addition to complete BAFFR deficiency, single nucleotide variants changing the primary amino acid sequence of BAFFR gene exist. Some of these variants were foundin patients suffering from immunodeficiency, autoimmunity, or B cell lymphomas. However, it remains unclearto which extent such variants disturb the activity of BAFFR. Methods Since individual differences and genetic/environmental modifiers change the expression and activity of BAFFR, we developed a cellular system that allows the unbiased analysis of BAFFR variants P21R, A52T, G64V, Dup92-95, P146S, and H159Y regarding oligomerization, signaling, and ectodomain shedding.Results Here we show that several of these variants impair BAFFR oligomerization, direct interactions between BAFFR and the B cell receptor component CD79B, BAFFR ectodomain shedding and the activation of AKT and ERK1/2. Conclusion All of these variants are pathogenic and have the potential to contribute to the development of primary antibody deficiencies, autoimmunity and lymphoma, but they most likely do not cause B lymphopenia and agammaglobulinemia like complete BAFFR deficiency.

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Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection

Blood ◽

10.1182/blood-2010-01-263533 ◽

2010 ◽

Vol 115 (25) ◽

pp. 5191-5201 ◽

Cited By ~ 209

Author(s):

Stephen A. Beers ◽

Ruth R. French ◽

H. T. Claude Chan ◽

Sean H. Lim ◽

Timothy C. Jarrett ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Type I ◽

Type Ii ◽

Antigenic Modulation ◽

Lymphatic Leukemia ◽

Human B Cells ◽

Anti Cd20

Abstract Rituximab, a monoclonal antibody that targets CD20 on B cells, is now central to the treatment of a variety of malignant and autoimmune disorders. Despite this success, a substantial proportion of B-cell lymphomas are unresponsive or develop resistance, hence more potent anti-CD20 monoclonal antibodies (mAbs) are continuously being sought. Here we demonstrate that type II (tositumomab-like) anti-CD20 mAbs are 5 times more potent than type I (rituximab-like) reagents in depleting human CD20 Tg B cells, despite both operating exclusively via activatory Fcγ receptor–expressing macrophages. Much of this disparity in performance is attributable to type I mAb-mediated internalization of CD20 by B cells, leading to reduced macrophage recruitment and the degradation of CD20/mAb complexes, shortening mAb half-life. Importantly, human B cells from healthy donors and most cases of chronic lymphatic leukemia and mantle cell lymphoma, showed rapid CD20 internalization that paralleled that seen in the Tg mouse B cells, whereas most follicular lymphoma and diffuse large B-cell lymphoma cells were far more resistant to CD20 loss. We postulate that differences in CD20 modulation may play a central role in determining the relative efficacy of rituximab in treating these diseases and strengthen the case for focusing on type II anti-CD20 mAb in the clinic.

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Production of HIV-1 by Human B Cells Infectedin Vitro:Characterization of an EBV Genome-Negative B Cell Line Chronically Synthetizing a Low Level of HIV-1 after Infection

Virology ◽

10.1006/viro.1998.9120 ◽

1998 ◽

Vol 244 (2) ◽

pp. 542-551 ◽

Cited By ~ 14

Author(s):

Laurent Fritsch ◽

Vincent Marechal ◽

Véronique Schneider ◽

Corinne Barthet ◽

Willy Rozenbaum ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Line ◽

Low Level ◽

Human B Cells ◽

Hiv 1

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Rewiring of B cell receptor signaling by Epstein–Barr virus LMP2A

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007946117 ◽

2020 ◽

Vol 117 (42) ◽

pp. 26318-26327

Author(s):

Kamonwan Fish ◽

Federico Comoglio ◽

Arthur L. Shaffer ◽

Yanlong Ji ◽

Kuan-Ting Pan ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Cell Receptor ◽

B Cell Receptor ◽

Barr Virus ◽

Human B Cells ◽

Bcr Signaling ◽

Epstein Barr

Epstein–Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

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Antigen Extraction and B Cell Activation Enable Identification of Rare Membrane Antigen Specific Human B Cells

Frontiers in Immunology ◽

10.3389/fimmu.2019.00829 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 3

Author(s):

Maria Zimmermann ◽

Natalie Rose ◽

John M. Lindner ◽

Hyein Kim ◽

Ana Rita Gonçalves ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Activation ◽

B Cell Activation ◽

Human B Cells

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Active PI3K abrogates central tolerance in high-avidity autoreactive B cells

Journal of Experimental Medicine ◽

10.1084/jem.20181652 ◽

2019 ◽

Vol 216 (5) ◽

pp. 1135-1153 ◽

Cited By ~ 5

Author(s):

Sarah A. Greaves ◽

Jacob N. Peterson ◽

Pamela Strauch ◽

Raul M. Torres ◽

Roberta Pelanda

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Peripheral Tolerance ◽

High Avidity ◽

Cell Tolerance ◽

B Cell Tolerance ◽

Central Tolerance ◽

Autoreactive B Cells ◽

Self Antigen

Autoreactive B cells that bind self-antigen with high avidity in the bone marrow undergo mechanisms of central tolerance that prevent their entry into the peripheral B cell population. These mechanisms are breached in many autoimmune patients, increasing their risk of B cell–mediated autoimmune diseases. Resolving the molecular pathways that can break central B cell tolerance could therefore provide avenues to diminish autoimmunity. Here, we show that B cell–intrinsic expression of a constitutively active form of PI3K-P110α by high-avidity autoreactive B cells of mice completely abrogates central B cell tolerance and further promotes these cells to escape from the bone marrow, differentiate in peripheral tissue, and undergo activation in response to self-antigen. Upon stimulation with T cell help factors, these B cells secrete antibodies in vitro but remain unable to secrete autoantibodies in vivo. Overall, our data demonstrate that activation of the PI3K pathway leads high-avidity autoreactive B cells to breach central, but not late, stages of peripheral tolerance.

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Glucocorticoid-induced leucine zipper (GILZ) inhibits B cell activation in systemic lupus erythematosus

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2015-207744 ◽

2015 ◽

Vol 75 (4) ◽

pp. 739-747 ◽

Cited By ~ 21

Author(s):

Sarah A Jones ◽

Andrew E J Toh ◽

Dragana Odobasic ◽

Marie-Anne Virginie Oudin ◽

Qiang Cheng ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

Leucine Zipper ◽

Germinal Centre ◽

Cell Phenotype ◽

Systemic Lupus ◽

Human B Cells

ObjectivesSystemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids.MethodsWe conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice.ResultsReduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli.ConclusionsOur findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

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Production and characterization of a bispecific IgG capable of inducing T-cell-mediated lysis of malignant B cells

Blood ◽

10.1182/blood.v81.12.3343.3343 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3343-3349 ◽

Cited By ~ 10

Author(s):

BK Link ◽

GJ Weiner

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Hybridoma Cells ◽

Activated T Cells ◽

Human B Cells ◽

B Cell Malignancy ◽

Cell Malignancy

Abstract Bispecific monoclonal antibodies (bsabs) recognizing both CD3 and a tumor antigen can redirect T-cell-mediated cytotoxicity toward cells bearing that antigen. Such bsabs have been shown to be more effective than monospecific monoclonal antibodies (MoAbs) at preventing tumor growth in animal models of B-cell malignancy. The current studies describe the production and preliminary evaluation of a bsab designed to induce the lysis of malignant human B cells by human T cells. The bsab was obtained from a hybrid-hybridoma cell line produced by fusing OKT3-secreting hybridoma cells with hybridoma cells that secrete 1D10. 1D10 is an MoAb that recognizes an antigen found on a majority of malignant human B cells that has not been detected to a significant degree on normal resting or activated lymphocytes. High performance liquid chromatography (HPLC) was used to separate bsab from monospecific antibodies that were also present in the hybrid-hybridoma antibody product. The bsab was then evaluated in vitro for its ability to induce lysis of malignant B cells by activated T cells. The bsab consistently induced extensive lysis in vitro of 1D10 (+) cells, including both cell lines and cells obtained from patients with a variety of B-cell malignancies. No such effect was seen with activated T cells alone or activated T cells with monospecific antibody. No increased lysis was seen with 1D10 (-) cell lines. The bsab also mediated lysis of malignant B cells by autologous T cells. We conclude bsab containing an OKT3 arm and a 1D10 arm can induce T-cell-mediated lysis in a manner that is both potent and specific. This supports further evaluation of this bsab as a potential immunotherapy of B-cell malignancy.

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