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Development and pharmacological validation of novel methods of B cell activation in rat whole blood.Journal of Pharmacological and... 2015Whole blood functional assays are pharmacologically relevant in the drug discovery process to evaluate potency in a relevant biological matrix, to support establishment...
INTRODUCTION
Whole blood functional assays are pharmacologically relevant in the drug discovery process to evaluate potency in a relevant biological matrix, to support establishment of PK/PD relationships and to aid in human dose predictions. However development of B cell activation assays by BCR ligation in rat whole blood has not been previously described. The aim of the present study was to develop novel methods of B cell activation in rat whole blood.
METHODS
B cell activation in rat whole blood was evaluated by measuring CD86 up-regulation via flow cytometry. Rat B cells in whole blood were stimulated with dextran-coupled anti-IgD or a combination of anti-IgD and TLR9 agonist. BTK, SYK, and PI3Kδ inhibitors were added to rat whole blood prior to activation with dextran-coupled anti-IgD or anti-IgD and TLR9 agonist combination for pharmacological validation of the assay.
RESULTS
Both methods of stimulation in rat whole blood evoked robust B cell activation in a uni-modal fashion. Highly selective inhibitors of BTK, SYK, and PI3Kδ dose-dependently attenuated B cell activity evoked by both dextran-coupled anti-IgD and combined anti-IgD and TLR9 agonist. Compound potencies and rank order determined by the two assays were comparable.
DISCUSSION
Two novel methods were developed to stimulate B cells in rat whole blood, that have the potential to be used to support drug discovery efforts in the therapeutic targeting of B cells. Furthermore, we pharmacologically validated these whole blood assays using highly selective inhibitors of BTK, SYK, and PI3Kδ, signaling kinases which are downstream of the B cell receptor.
Topics: Animals; B-Lymphocytes; Blood; Dose-Response Relationship, Drug; Female; Lymphocyte Activation; Protein Kinase Inhibitors; Rats; Rats, Inbred Lew; Structure-Activity Relationship
PubMed: 25545336
DOI: 10.1016/j.vascn.2014.12.006 -
Frontiers in Immunology 2019B lymphocytes, as a central part of adaptive immune responses, have the ability to fight against an almost unlimited numbers of pathogens. Impairment of B cell...
B lymphocytes, as a central part of adaptive immune responses, have the ability to fight against an almost unlimited numbers of pathogens. Impairment of B cell development, activation and differentiation to antibody secreting plasma cells can lead to malignancy, allergy, autoimmunity and immunodeficiency. However, the impact of environmental factors, such as hyperosmolality or osmotic stress caused by varying salt concentrations in different lymphoid organs, on these processes is not well-understood. Here, we report that B cells respond to osmotic stress in a biphasic manner. Initially, increased osmolality boosted B cell activation and differentiation as shown by an untimely downregulation of Pax5 as well as upregulation of CD138. However, in the second phase, we observed an increase in cell death and impaired plasmablast differentiation. Osmotic stress resulted in impaired class switch to IgG1, inhibition of phosphorylation of p38 mitogen-activated kinase and a delayed NFAT5 response. Overall, these findings demonstrate the importance of microenvironmental hyperosmolality and osmotic stress caused by NaCl for B cell activation and differentiation.
Topics: Animals; B-Lymphocytes; Cell Differentiation; Cells, Cultured; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C57BL; Osmolar Concentration; Phosphorylation; p38 Mitogen-Activated Protein Kinases
PubMed: 31057551
DOI: 10.3389/fimmu.2019.00828 -
European Journal of Immunology Mar 2022BOB.1/OBF.1 is a lymphocyte-specific transcriptional co-activator of octamer-dependent transcription. It regulates the expression of genes important for lymphocyte...
BOB.1/OBF.1 is a lymphocyte-specific transcriptional co-activator of octamer-dependent transcription. It regulates the expression of genes important for lymphocyte physiology together with the Oct-1 and Oct-2 transcription factors. So far, BOB.1/OBF.1 has been studied in conventional knockout mice, whereby a function of BOB.1/OBF.1 in B but also in T cells was described. The main characteristic of BOB.1/OBF.1-deficient mice is the complete absence of germinal centers. However, it is entirely unsolved at which stage of B-cell development BOB.1/OBF.1 expression is essential for germinal center formation. Still, it is not known whether defects observed late in B-cell development of BOB.1/OBF.1-deficient mice are merely a consequence of defective early B-cell development. To answer the question, whether BOB.1/OBF.1 expression is required before or during the process of germinal center formation, we established a mouse system, which allows the conditional deletion of BOB.1/OBF.1 at different stages of B-cell development. Our data reveal a requirement for BOB.1/OBF.1 during both early antigen-independent and late antigen-dependent B-cell development, and further a requirement for efficient germinal center reaction during complete B-cell ontogeny. By specifically deleting BOB.1/OBF.1 in germinal center B cells, we provide evidence that the failure to form germinal centers is a germinal center B-cell intrinsic defect and not exclusively a consequence of defective early B-cell maturation.
Topics: Animals; B-Lymphocytes; Cell Differentiation; Germinal Center; Lymphocyte Activation; Mice; Octamer Transcription Factor-2; Trans-Activators; Transcription Factors
PubMed: 34918350
DOI: 10.1002/eji.202149333 -
Journal of Molecular Medicine (Berlin,... Sep 2020Atherosclerosis is the leading cause of cardiovascular mortality and morbidity worldwide and is described as a complex disease involving several different cell types and... (Review)
Review
Atherosclerosis is the leading cause of cardiovascular mortality and morbidity worldwide and is described as a complex disease involving several different cell types and their molecular products. Recent studies have revealed that atherosclerosis arises from a systemic inflammatory process, including the accumulation and activities of various immune cells. However, the immune system is a complicated network made up of many cell types, hundreds of bioactive cytokines, and millions of different antigens, making it challenging to readily define the associated mechanism of atherosclerosis. Nevertheless, we previously reported a potential persistent inflammatory process underlying atherosclerosis development, centered on a pathological humoral immune response between commensal microbes and activated subpopulations of substantial B cells in the vicinity of the arterial adventitia. Accumulating evidence has indicated the importance of gut microbiota in atherosclerosis development. Commensal microbiota are considered important regulators of immunity and metabolism and also to be possible antigenic sources for atherosclerosis development. However, the interplay between gut microbiota and metabolism with regard to the modulation of atherosclerosis-associated immune responses remains poorly understood. Here, we review the mechanisms by which the gut microbiota may influence atherogenesis, with particular focus on humoral immunity and B cells, especially the gut-immune-B2 cell axis. Graphical abstract Under high-fat and high-calorie conditions, signals driven by the intestinal microbiota via the TLR signaling pathway cause B2 cells in the spleen to become functionally active and activated B2 cells then modify responses such as antibody production (generation of active antibodies IgG and IgG3), thereby contributing to the development of atherosclerosis. On the other hand, intestinal microbiota also resulted in recruitment and ectopic activation of B2 cells via the TLR signaling pathway in perivascular adipose tissue (PVAT), and, subsequently, an increase in circulating IgG and IgG3 led to the enhanced disease development. This is a potential link between microbiota alterations and B cells in the context of atherosclerosis.
Topics: Animals; Atherosclerosis; B-Lymphocytes; Cardiovascular Diseases; Disease Susceptibility; Dysbiosis; Gastrointestinal Diseases; Gastrointestinal Microbiome; Humans; Immune System
PubMed: 32737524
DOI: 10.1007/s00109-020-01936-5 -
Nature Communications Nov 2017O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) catalyzes O-GlcNAc modification. O-GlcNAcylation is increased after cross-linking of the B-cell receptor (BCR),...
O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) catalyzes O-GlcNAc modification. O-GlcNAcylation is increased after cross-linking of the B-cell receptor (BCR), but the physiological function of this reaction is unknown. Here we show that lack of Ogt in B-cell development not only causes severe defects in the activation of BCR signaling, but also perturbs B-cell homeostasis by enhancing apoptosis of mature B cells, partly as a result of impaired response to B-cell activating factor. O-GlcNAcylation of Lyn at serine 19 is crucial for efficient Lyn activation and Syk interaction in BCR-mediated B-cell activation and expansion. Ogt deficiency in germinal center (GC) B cells also results in enhanced apoptosis of GC B cells and memory B cells in an immune response, consequently causing a reduction of antibody levels. Together, these results demonstrate that B cells rely on O-GlcNAcylation to maintain homeostasis, transduce BCR-mediated activation signals and activate humoral immunity.
Topics: Acetylglucosamine; Animals; B-Lymphocytes; HEK293 Cells; Homeostasis; Humans; Immunity, Humoral; Immunoglobulin G; Lymphocyte Activation; Male; Mice, Knockout; Mice, Transgenic; N-Acetylglucosaminyltransferases; Serine; Syk Kinase; src-Family Kinases
PubMed: 29187734
DOI: 10.1038/s41467-017-01677-z -
Current Opinion in Immunology Aug 2021B cell activating factor (BAFF or BLyS), an important cytokine for B cell survival and humoral immune responses, is targeted in the clinic for the treatment of systemic... (Review)
Review
B cell activating factor (BAFF or BLyS), an important cytokine for B cell survival and humoral immune responses, is targeted in the clinic for the treatment of systemic lupus erythematosus. This review focuses on the structure, function and inhibition profiles of membrane-bound BAFF, soluble BAFF 3-mer and soluble BAFF 60-mer, all of which have distinct properties. BAFF contains a loop region not required for receptor binding but essential for receptor activation via promotion of BAFF-to-BAFF contacts. This loop region additionally allows formation of BAFF 60-mer, in which epitopes of the BAFF inhibitor belimumab are inaccessible. If 60-mer forms in humans, it is predicted to be short-lived and to act locally because adult serum contains a BAFF 60-mer dissociating activity. Cord blood contains elevated levels of BAFF, part of which displays attributes of 60-mer, suggesting a role for this form of BAFF in the development of foetal or neonate B cells.
Topics: Antibodies, Monoclonal, Humanized; B-Cell Activating Factor; B-Lymphocytes; Humans; Immunosuppressive Agents
PubMed: 34182216
DOI: 10.1016/j.coi.2021.06.009 -
Proceedings of the National Academy of... Sep 1997The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about...
The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.
Topics: B-Lymphocyte Subsets; B-Lymphocytes; Cell Differentiation; Enzyme Activation; Enzyme Induction; Humans; Immunologic Memory; Palatine Tonsil; Telomerase; Telomere
PubMed: 9380719
DOI: 10.1073/pnas.94.20.10827 -
PLoS Genetics Feb 2016Hematopoietic stem cells are capable of self-renewal or differentiation along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage...
Hematopoietic stem cells are capable of self-renewal or differentiation along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for blood progenitor cells is whether to adopt the lymphoid or myeloid fate. Previous work had shown that myocyte enhancer factor 2C (MEF2C) is indispensable for the lymphoid fate decision, yet the specific mechanism of action remained unclear. Here, we have identified early B cell factor-1 (EBF1) as a co-regulator of gene expression with MEF2C. A genome-wide survey of MEF2C and EBF1 binding sites identified a subset of B cell-specific genes that they target. We also determined that the p38 MAPK pathway activates MEF2C to drive B cell differentiation. Mef2c knockout mice showed reduced B lymphoid-specific gene expression as well as increased myeloid gene expression, consistent with MEF2C's role as a lineage fate regulator. This is further supported by interaction between MEF2C and the histone deacetylase, HDAC7, revealing a likely mechanism to repress the myeloid transcription program. This study thus elucidates both activation and repression mechanisms, identifies regulatory partners, and downstream targets by which MEF2C regulates lymphoid-specific differentiation.
Topics: B-Lymphocytes; Cell Differentiation; Cell Line; Cell Lineage; Hematopoietic Stem Cells; Histone Deacetylases; Humans; Immunoprecipitation; MEF2 Transcription Factors; Myeloid Cells; Phosphorylation; Precursor Cells, B-Lymphoid; Protein Transport; Subcellular Fractions; Trans-Activators; Transcription, Genetic; Transcriptional Activation; p38 Mitogen-Activated Protein Kinases
PubMed: 26900922
DOI: 10.1371/journal.pgen.1005845 -
PloS One 2018Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation. Several members of the NKL subclass are deregulated...
Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation. Several members of the NKL subclass are deregulated in T-cell progenitors and support leukemogenesis. We have recently described particular expression patterns of nine NKL homeobox genes in early hematopoiesis and T-cell development. Here, we screened NKL homeobox gene activities in normal B-cell development and extended the NKL-code to include this lymphoid lineage. Analysis of public expression profiling datasets revealed that HHEX and NKX6-3 were the only members differentially active in naïve B-cells, germinal center B-cells, plasma cells and memory B-cells. Subsequent examination of different types of B-cell malignancies showed both aberrant overexpression of NKL-code members and ectopic activation of subclass members physiologically silent in lymphopoiesis including BARX2, DLX1, EMX2, NKX2-1, NKX2-2 and NKX3-2. Based on these findings we performed detailed studies of the B-cell specific NKL homeobox gene NKX6-3 which showed enhanced activity in patient subsets of follicular lymphoma, mantle cell lymphoma and diffuse large B-cell lymphoma (DLBCL), and in three DLBCL cell lines to serve as in vitro models. While excluding genomic and chromosomal rearrangements at the locus of NKX6-3 (8p11) promoter studies demonstrated that B-cell factors MYB and PAX5 activated NKX6-3 transcription. Furthermore, aberrant BMP7/SMAD1-signalling and deregulated expression of chromatin complex components AUTS2 and PCGF5 promoted NKX6-3 activation. Finally, NKL homeobox genes HHEX, HLX, MSX1 and NKX6-3 were expressed in B-cell progenitors and generated a regulatory gene network in cell lines which we propose may provide physiological support for NKL-code formation in early B-cell development. Together, we identified an NKL-code in B-cell development whose violation may deregulate differentiation and promote malignant transformation.
Topics: B-Lymphocytes; Cell Differentiation; Cell Line; Gene Expression; Gene Expression Profiling; Genes, Homeobox; Homeobox Protein Nkx-2.2; Homeodomain Proteins; Humans; Lymphoma; Nuclear Proteins; Transcription Factors
PubMed: 30308041
DOI: 10.1371/journal.pone.0205537 -
Trends in Immunology May 2011Mature B cells generate protective immunity by undergoing immunoglobulin (Ig) class switching and somatic hypermutation, two Ig gene-diversifying processes that usually... (Review)
Review
Mature B cells generate protective immunity by undergoing immunoglobulin (Ig) class switching and somatic hypermutation, two Ig gene-diversifying processes that usually require cognate interactions with T cells that express CD40 ligand. This T cell-dependent pathway provides immunological memory but is relatively slow to occur. Thus, it must be integrated with a faster, T cell-independent pathway for B cell activation through CD40 ligand-like molecules that are released by innate immune cells in response to microbial products. Here, we discuss recent advances in our understanding of the interplay between the innate immune system and B cells, particularly at the mucosal interface. We also review the role of innate signals in the regulation of Ig diversification and production.
Topics: B-Cell Activating Factor; B-Lymphocytes; Gene Expression Regulation; Genes, Immunoglobulin; Immunity, Innate; Ligands; T-Lymphocytes; Toll-Like Receptors; Tumor Necrosis Factor Ligand Superfamily Member 13
PubMed: 21419699
DOI: 10.1016/j.it.2011.02.004