Specific In Vitro IgM Responses of Human B Cells: A Complex Regulatory Network Modulated by Antigen

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

Download Full-text

Obesity Accelerates Age-Associated Defects in Human B Cells Through a Metabolic Reprogramming Induced by the Fatty Acid Palmitate

Frontiers in Aging ◽

10.3389/fragi.2021.828697 ◽

2022 ◽

Vol 2 ◽

Author(s):

Daniela Frasca ◽

Maria Romero ◽

Denisse Garcia ◽

Alain Diaz ◽

Bonnie B. Blomberg

Keyword(s):

Fatty Acid ◽

B Cells ◽

Critical Role ◽

Metabolic Reprogramming ◽

Autoimmune Antibodies ◽

Human B Cells ◽

Initial Hypothesis ◽

First Time ◽

Culture Supernatants

We have measured the secretion of autoimmune antibodies in plasma samples and in culture supernatants of blood-derived B cells from four groups of individuals: young lean (YL), elderly lean (EL), young obese (YO) and elderly obese (EO). We found secretion comparable in YO and EL individuals, suggesting that obesity accelerates age-associated defects in circulating B cells. To define at least one possible molecular pathway involved, we used an in vitro model in which B cells from YL and EL individuals have been stimulated with the Fatty Acid (FA) palmitate, the most common saturated FA in the human body. The rationale to use palmitate is that there is a chronic increase in circulating levels of palmitate, due to increased spontaneous lipolysis occurring during aging and obesity, and this may induce autoimmune B cells. Results herein show that in vitro incubation of B cells from YL and EL individuals with the FA palmitate induces mRNA expression of T-bet, the transcription factor for autoimmune antibodies, as well as secretion of autoimmune IgG antibodies, with B cells from YL individuals looking similar to B cells from EL individuals, confirming our initial hypothesis. The generation of autoimmune B cells in the presence of the FA palmitate was found to be associated with a metabolic reprogramming of B cells from both YL and EL individuals. These results altogether show the critical role of the FA palmitate in inducing human B cell immunosenescence and show for the first time the importance of metabolic pathways in this process.

Download Full-text

B lymphocyte binding to E- and P-selectins is mediated through the de novo expression of carbohydrates on in vitro and in vivo activated human B cells.

Journal of Clinical Investigation ◽

10.1172/jci117500 ◽

1994 ◽

Vol 94 (4) ◽

pp. 1585-1596 ◽

Cited By ~ 24

Author(s):

A A Postigo ◽

M Marazuela ◽

F Sánchez-Madrid ◽

M O de Landázuri

Keyword(s):

B Cells ◽

B Lymphocyte ◽

De Novo ◽

Human B Cells

Download Full-text

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.

Download Full-text

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.

Download Full-text

The effect of RU 41740 on the in vitro maturation of human B-cells

International Journal of Immunopharmacology ◽

10.1016/0192-0561(88)90009-4 ◽

1988 ◽

Vol 10 (7) ◽

pp. 851-854 ◽

Cited By ~ 2

Author(s):

A. Durandy ◽

P. Smets ◽

R. Zalisz ◽

C. Griscelli

Keyword(s):

B Cells ◽

In Vitro Maturation ◽

Human B Cells

Download Full-text

Inhibition of Interleukin-4- and CD40-induced IgE Germline Gene Promoter Activity by 2′-Aminoethoxy-modified Triplex-forming Oligonucleotides

Journal of Biological Chemistry ◽

10.1074/jbc.m010260200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11759-11765 ◽

Cited By ~ 16

Author(s):

Adrian M. Stütz ◽

Jutta Hoeck ◽

Francois Natt ◽

Bernard Cuenoud ◽

Maximilian Woisetschläger

Keyword(s):

B Cells ◽

Critical Role ◽

Gene Promoter ◽

Gene Induction ◽

Modified Oligonucleotides ◽

Germline Gene ◽

Triplex Formation ◽

Human B Cells ◽

Dna Triplex

Elevated levels of IgE are intimately associated with a number of allergic diseases, such as allergic rhinitis or asthma. Therefore, prevention of IgE production in human B-cells represents an attractive therapeutic target. IL-4-induced IgE germline gene transcription represents a crucial early step during IgE isotype switch differentiation. Gene induction is orchestrated by the coordinated action of the transcription factors STAT6 (signal transducer and activator of transcription), NF-κB, PU.1, and C/EBP. This study shows that 2′-aminoethoxy-modified oligonucleotides, which partially overlap with the STAT6 and the adjacent PU.1/NF-κB binding site, inhibit DNA binding of all three proteins with high affinity in a dose- and time-dependent fashionin vitro. Loss of protein binding correlated strongly with increasing DNA triplex formation. Importantly, the oligomers also effectively displaced pre-bound recombinant NF-κB p50 from double-stranded DNAin vitro. Functionally, the oligonucleotides led to a selective inhibition of IL-4-induced reporter gene activity from a construct driven by the IgE germline gene promoter in human B-cells. These data confirm the critical role of this cytokine-responsive regulatory region in IgE germline gene induction and further support the concept of specific modulation of gene expression by DNA triplex formation induced with chemically modified oligonucleotides.

Download Full-text

Effect of RU 41740 on the in vitro maturation of human B cells

International Journal of Immunopharmacology ◽

10.1016/0192-0561(85)90365-0 ◽

1985 ◽

Vol 7 (3) ◽

pp. 369

Author(s):

A. Durandy ◽

P. Smets ◽

R. Zalisz ◽

C. Griscelli

Keyword(s):

B Cells ◽

In Vitro Maturation ◽

Human B Cells

Download Full-text

Production of autoantibodies by CD5-expressing B lymphocytes from patients with chronic lymphocytic leukemia.

Journal of Experimental Medicine ◽

10.1084/jem.169.1.255 ◽

1989 ◽

Vol 169 (1) ◽

pp. 255-268 ◽

Cited By ~ 200

Author(s):

Z M Sthoeger ◽

M Wakai ◽

D B Tse ◽

V P Vinciguerra ◽

S L Allen ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Lymphocytes ◽

Autoimmune Disorders ◽

Lymphoproliferative Disorders ◽

Lymphocytic Leukemia ◽

Antigenic Specificity ◽

Human B Cells ◽

Well Differentiated

CD5-expressing B lymphocytes from patients with selected chronic lymphoproliferative disorders were used to determine whether monoclonal populations of CD5+ human B cells produce autoantibodies. CD5+ B cells from 19 patients with chronic lymphocytic leukemia (CLL) and one with diffuse well-differentiated lymphocytic lymphoma (DWDL) were cultured, with and without mitogenic stimulation, to obtain Ig from these cells. 17 of the 20 samples produced Ig in vitro. mAb from nine of the 17 patients were reactive with either IgG, ssDNA, or dsDNA. In every instance, the autoantibodies displayed monotypic L chain usage that correlated precisely with the L chain expressed on the CD5+ leukemic B cell surface. These monoclonal autoantibodies varied in their degree of antigenic specificity; some were quite specific, reacting with only one antigen, whereas others were polyspecific, reacting with two or all three autoantigens tested. Three features distinguish these autoantibodies from those observed in prior studies of CD5+ B cells. First, they are clearly the products of monoclonal populations of CD5+ cells; second, several react with dsDNA, a specificity not previously reported and often seen in association with significant autoimmune disorders; and third, two of the monoclonal autoantibodies secreted by the CD5+ clones were of the IgG class. Although not all of the Ig-producing, CD5-expressing clones elaborated mAbs reactive with the autoantigens tested, greater than 50% did. It is possible that with a broader autoantigenic panel or with larger quantities of CLL/DWDL-derived Ig, even more autoantibody-producing clones might be identified. These studies may have important implications for the antigenic specificity of subsets of human B lymphocytes as well as for lymphoproliferative and autoimmune disorders in general.

Download Full-text