31 results on '"Hasbold J"'
Search Results
2. The logic of intercellular communication in the immune system
- Author
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HODGKIN, P D, RUSH, J, GETT, A V, BARTELL, G, and HASBOLD, J
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- 1998
3. Properties of mouse CD40: differential expression of CD40 epitopes on dendritic cells and epithelial cells
- Author
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VAN DEN BERG, T. K., HASBOLD, J., DE LAVALETTE, RENARDEL C., DÖPP, E. A., DIJKSTRA, C. D., and KLAUS, G. G. B.
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- 1996
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4. Quantitative analysis of lymphocyte differentiation and proliferationin vitrousing carboxyfluorescein diacetate succinimidyl ester
- Author
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Hasbold, J, primary, Gett, AV, additional, Rush, JS, additional, Deenick, E, additional, Avery, D, additional, Jun, J, additional, and Hodgkin, PD, additional
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- 1999
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5. The effects of IFN-gamma on CD40-mediated activation of B cells from X-linked immunodeficient or normal mice.
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Johnson-Léger, C, primary, Hasbold, J, additional, Holman, M, additional, and Klaus, G G, additional
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- 1997
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6. Properties of mouse CD40: differential expression of CD40 epitopes on dendritic cells and epithelial cells
- Author
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BERG, T. K., primary, HASBOLD, J., additional, LAVALETTE, C. RENARDEL, additional, DÖPP, E. A., additional, DIJKSTRA, C. D., additional, and KLAUS, G. G. B., additional
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- 1996
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7. Hypercross-linking surface IgM or IgD receptors on mature B cells induces apoptosis that is reversed by costimulation with IL-4 and anti-CD40.
- Author
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Parry, S L, primary, Hasbold, J, additional, Holman, M, additional, and Klaus, G G, additional
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- 1994
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8. Dynamic changes in Id3 and E-protein activity orchestrate germinal center and plasma cell development.
- Author
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Gloury R, Zotos D, Zuidscherwoude M, Masson F, Liao Y, Hasbold J, Corcoran LM, Hodgkin PD, Belz GT, Shi W, Nutt SL, Tarlinton DM, and Kallies A
- Subjects
- Animals, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors genetics, Cell Differentiation genetics, Inhibitor of Differentiation Proteins genetics, Mice, Mice, Knockout, Positive Regulatory Domain I-Binding Factor 1, Receptors, CXCR4 genetics, Receptors, CXCR4 immunology, Transcription Factor 4, Transcription Factors genetics, Transcription Factors immunology, X-Box Binding Protein 1 genetics, X-Box Binding Protein 1 immunology, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors immunology, Basic Helix-Loop-Helix Transcription Factors immunology, Cell Differentiation immunology, Germinal Center immunology, Inhibitor of Differentiation Proteins immunology, Plasma Cells immunology
- Abstract
The generation of high-affinity antibodies requires germinal center (GC) development and differentiation of long-lived plasma cells in a multilayered process that is tightly controlled by the activity of multiple transcription factors. Here, we reveal a new layer of complexity by demonstrating that dynamic changes in Id3 and E-protein activity govern both GC and plasma cell differentiation. We show that down-regulation of Id3 in B cells is essential for releasing E2A and E2-2, which in a redundant manner are required for antigen-induced B cell differentiation. We demonstrate that this pathway controls the expression of multiple key factors, including Blimp1, Xbp1, and CXCR4, and is therefore critical for establishing the transcriptional network that controls GC B cell and plasma cell differentiation., (© 2016 Gloury et al.)
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- 2016
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9. The transcription factors IRF8 and PU.1 negatively regulate plasma cell differentiation.
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Carotta S, Willis SN, Hasbold J, Inouye M, Pang SH, Emslie D, Light A, Chopin M, Shi W, Wang H, Morse HC 3rd, Tarlinton DM, Corcoran LM, Hodgkin PD, and Nutt SL
- Subjects
- Animals, Cell Line, Cluster Analysis, Gene Expression Profiling, Gene Expression Regulation, Humans, Immunoglobulin Class Switching genetics, Interferon Regulatory Factors metabolism, Mice, Mice, Transgenic, Plasma Cells immunology, Protein Binding, Proto-Oncogene Proteins metabolism, Trans-Activators metabolism, Cell Differentiation genetics, Interferon Regulatory Factors genetics, Plasma Cells cytology, Plasma Cells metabolism, Proto-Oncogene Proteins genetics, Trans-Activators genetics
- Abstract
Activated B cells undergo immunoglobulin class-switch recombination (CSR) and differentiate into antibody-secreting plasma cells. The distinct transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors: those that maintain the B cell program, including BCL6 and PAX5, and plasma cell-promoting factors, such as IRF4 and BLIMP-1. We show that the complex of IRF8 and PU.1 controls the propensity of B cells to undergo CSR and plasma cell differentiation by concurrently promoting the expression of BCL6 and PAX5 and repressing AID and BLIMP-1. As the PU.1-IRF8 complex functions in a reciprocal manner to IRF4, we propose that concentration-dependent competition between these factors controls B cell terminal differentiation., (© 2014 Carotta et al.)
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- 2014
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10. Activation-induced B cell fates are selected by intracellular stochastic competition.
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Duffy KR, Wellard CJ, Markham JF, Zhou JH, Holmberg R, Hawkins ED, Hasbold J, Dowling MR, and Hodgkin PD
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- Animals, Cell Death, Cell Differentiation, Cell Division, Female, Immunoglobulin Class Switching, Mice, Models, Immunological, Stochastic Processes, B-Lymphocytes cytology, B-Lymphocytes immunology, Lymphocyte Activation
- Abstract
In response to stimulation, B lymphocytes pursue a large number of distinct fates important for immune regulation. Whether each cell's fate is determined by external direction, internal stochastic processes, or directed asymmetric division is unknown. Measurement of times to isotype switch, to develop into a plasmablast, and to divide or to die for thousands of cells indicated that each fate is pursued autonomously and stochastically. As a consequence of competition between these processes, censorship of alternative outcomes predicts intricate correlations that are observed in the data. Stochastic competition can explain how the allocation of a proportion of B cells to each cell fate is achieved. The B cell may exemplify how other complex cell differentiation systems are controlled.
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- 2012
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11. The genetic network controlling plasma cell differentiation.
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Nutt SL, Taubenheim N, Hasbold J, Corcoran LM, and Hodgkin PD
- Subjects
- B-Lymphocytes immunology, Humans, Models, Biological, Transcription, Genetic, B-Lymphocytes cytology, Cell Differentiation, Gene Regulatory Networks, Plasma Cells cytology
- Abstract
Upon activation by antigen, mature B cells undergo immunoglobulin class switch recombination and differentiate into antibody-secreting plasma cells, the endpoint of the B cell developmental lineage. Careful quantitation of these processes, which are stochastic, independent and strongly linked to the division history of the cell, has revealed that populations of B cells behave in a highly predictable manner. Considerable progress has also been made in the last few years in understanding the gene regulatory network that controls the B cell to plasma cell transition. The mutually exclusive transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors, those that maintain the B cell program, including Pax5, Bach2 and Bcl6, and those that promote and facilitate plasma cell differentiation, notably Irf4, Blimp1 and Xbp1. In this review, we discuss progress in the definition of both the transcriptional and cellular events occurring during late B cell differentiation, as integrating these two approaches is crucial to defining a regulatory network that faithfully reflects the stochastic features and complexity of the humoral immune response., (2011 Elsevier Ltd. All rights reserved.)
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- 2011
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12. Oct2 enhances antibody-secreting cell differentiation through regulation of IL-5 receptor alpha chain expression on activated B cells.
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Emslie D, D'Costa K, Hasbold J, Metcalf D, Takatsu K, Hodgkin PO, and Corcoran LM
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- Animals, Antibody-Producing Cells drug effects, Antibody-Producing Cells immunology, B-Lymphocytes cytology, B-Lymphocytes drug effects, Cell Differentiation drug effects, Cell Differentiation immunology, Cell Line, Eosinophils drug effects, Eosinophils immunology, Gene Expression Regulation drug effects, Interleukin-5 pharmacology, Interleukin-5 Receptor alpha Subunit genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Octamer Transcription Factor-2 deficiency, Octamer Transcription Factor-2 genetics, Positive Regulatory Domain I-Binding Factor 1, RNA, Messenger metabolism, Repressor Proteins genetics, Repressor Proteins immunology, Spleen cytology, T-Lymphocytes drug effects, T-Lymphocytes immunology, Transcription Factors genetics, Transcription Factors immunology, Antibody-Producing Cells cytology, B-Lymphocytes immunology, Interleukin-5 Receptor alpha Subunit immunology, Octamer Transcription Factor-2 immunology
- Abstract
Mice lacking a functional gene for the Oct2 transcriptional activator display several developmental and functional deficiencies in the B lymphocyte lineage. These include defective B cell receptor (BCR) and Toll-like receptor 4 signaling, an absence of B-1 and marginal zone populations, and globally reduced levels of serum immunoglobulin (Ig) in naive and immunized animals. Oct2 was originally identified through its ability to bind to regulatory regions in the Ig loci, but genetic evidence has not supported an essential role for Oct2 in the expression of Ig genes. We describe a new Oct2-mediated role in B cells. Oct2 augments the ability of activated B cells to differentiate to antibody-secreting plasma cells (ASCs) under T cell-dependent conditions through direct regulation of the gene encoding the alpha chain of the interleukin (IL) 5 receptor. Ectopic expression of IL-5Ralpha in oct2-deficient B cells largely restores their ability to differentiate to functional ASCs in vitro but does not correct other phenotypic defects in the mutants, such as the maturation and specialization of peripheral B cells, which must therefore rely on distinct Oct2 target genes. IL-5 augments ASC differentiation in vitro, and we show that IL-5 directly activates the plasma cell differentiation program by enhancing blimp1 expression.
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- 2008
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13. Initiation of plasma-cell differentiation is independent of the transcription factor Blimp-1.
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Kallies A, Hasbold J, Fairfax K, Pridans C, Emslie D, McKenzie BS, Lew AM, Corcoran LM, Hodgkin PD, Tarlinton DM, and Nutt SL
- Subjects
- Animals, Antibodies immunology, Antigen-Presenting Cells cytology, Antigen-Presenting Cells immunology, Antigen-Presenting Cells metabolism, B-Lymphocytes metabolism, Cells, Cultured, DNA metabolism, Gene Expression Regulation, Immunoglobulin G metabolism, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation genetics, PAX5 Transcription Factor metabolism, Plasma Cells immunology, Positive Regulatory Domain I-Binding Factor 1, Protein Binding, Repressor Proteins genetics, Transcription Factors deficiency, Transcription Factors genetics, Cell Differentiation immunology, Plasma Cells cytology, Plasma Cells metabolism, Repressor Proteins metabolism, Transcription Factors metabolism
- Abstract
Blimp-1 is considered an essential regulator of the terminal differentiation of B cells into antibody-secreting plasma cells. We show here that Rag1-/- mice reconstituted with fetal liver cells homozygous for a DNA-binding-deficient mutant of Prdm1 (the gene encoding Blimp-1) lack a defined plasma-cell compartment, yet show detectable amounts of all immunoglobulin isotypes. In vitro analysis revealed that Blimp-1 is not required for the initiation of antibody secretion but is essential for subsequent high immunoglobulin production. Blimp-1-independent differentiation was blocked at a preplasmablast stage characterized by decreased Pax5 expression and the activation of plasma-cell genes. Analysis of Blimp-1-sufficient differentiation revealed a phase prior to Blimp-1 expression in which several genes normally repressed by Pax5 are re-expressed, suggesting that plasma-cell differentiation is initiated by the inhibition of Pax5 function. Our results indicate that full plasma-cell differentiation but not commitment to the plasma-cell fate requires the expression of functional Blimp-1.
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- 2007
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14. Decision criteria for resolving isotype switching conflicts by B cells.
- Author
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Deenick EK, Hasbold J, and Hodgkin PD
- Subjects
- Animals, Female, Flow Cytometry, Male, Mice, Antibody Formation immunology, B-Lymphocytes immunology, Immunoglobulin Class Switching immunology, Interferon-gamma immunology, Interleukin-4 immunology, Transforming Growth Factor beta immunology
- Abstract
Isotype switching by B cells is highly regulated by a group of cytokines including IL-4, IFN-gamma and TGF-beta. A B cell can only express one isotype at a time; however, during an immune response it may be exposed to combinations of stimuli that provide it with conflicting switching instructions. To determine how such cytokine-induced isotype switch conflicts would be resolved, the responses of B cells exposed to multiple cytokines were investigated. To eliminate complications arising from simultaneous effects of switching cytokines on proliferation, division number was used as a reference framework to monitor switching rate. The results show a clear hierarchy in which IFN-gamma is dominant over IL-4, and both IL-4 and IFN-gamma are dominant over TGF-beta. These studies reveal how B cells possess a set of logical decision criteria for dealing with pathogens that invoke a range of different stimuli.
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- 2005
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15. Differential requirement for OBF-1 during antibody-secreting cell differentiation.
- Author
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Corcoran LM, Hasbold J, Dietrich W, Hawkins E, Kallies A, Nutt SL, Tarlinton DM, Matthias P, and Hodgkin PD
- Subjects
- Animals, Antibody-Producing Cells metabolism, Cells, Cultured, DNA Primers, DNA-Binding Proteins metabolism, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Genetic Complementation Test, Lipopolysaccharides, Mice, Mice, Inbred C57BL, Mice, Knockout, PAX5 Transcription Factor, Plasma Cells metabolism, Positive Regulatory Domain I-Binding Factor 1, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-bcl-6, Repressor Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Trans-Activators metabolism, Transcription Factors metabolism, Antibody-Producing Cells physiology, Cell Differentiation physiology, Gene Expression Regulation, Trans-Activators physiology
- Abstract
Resting B cells can be cultured to induce antibody-secreting cell (ASC) differentiation in vitro. A quantitative analysis of cell behavior during such a culture allows the influences of different stimuli and gene products to be measured. The application of this analytical system revealed that the OBF-1 transcriptional coactivator, whose loss impairs antibody production in vivo, has two effects on ASC development. Although OBF-1 represses early T cell-dependent (TD) differentiation, it is also critical for the completion of the final stages of ASC development. Under these conditions, the loss of OBF-1 blocks the genetic program of ASC differentiation so that Blimp-1/prdm1 induction fails, and bcl-6, Pax5, and AID are not repressed as in control ASC. Retroviral complementation confirmed that OBF-1 was the critical entity. Surprisingly, when cells were cultured in lipopolysaccharide to mimic T cell-independent conditions, OBF-1-null B cells differentiated normally to ASC. In the OBF-1(-/-) ASC generated under either culture regimen, antibody production was normal or only modestly reduced, revealing that Ig genes are not directly dependent on OBF-1 for their expression. The differential requirement for OBF-1 in TD ASC generation was confirmed in vivo. These studies define a new regulatory role for OBF-1 in determining the cell-autonomous capacity of B cells to undergo terminal differentiation in response to different immunological signals.
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- 2005
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16. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression.
- Author
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Kallies A, Hasbold J, Tarlinton DM, Dietrich W, Corcoran LM, Hodgkin PD, and Nutt SL
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- Animals, Antibody-Producing Cells metabolism, Cell Differentiation, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Phenotype, Positive Regulatory Domain I-Binding Factor 1, Repressor Proteins physiology, Transcription Factors physiology, Gene Expression Regulation, Plasma Cells cytology, Repressor Proteins genetics, Transcription Factors genetics
- Abstract
Plasma cells comprise a population of terminally differentiated B cells that are dependent on the transcriptional regulator B lymphocyte--induced maturation protein 1 (Blimp-1) for their development. We have introduced a gfp reporter into the Blimp-1 locus and shown that heterozygous mice express the green fluorescent protein in all antibody-secreting cells (ASCs) in vivo and in vitro. In vitro, these cells display considerable heterogeneity in surface phenotype, immunoglobulin secretion rate, and Blimp-1 expression levels. Importantly, analysis of in vivo ASCs induced by immunization reveals a developmental pathway in which increasing levels of Blimp-1 expression define developmental stages of plasma cell differentiation that have many phenotypic and molecular correlates. Thus, maturation from transient plasmablast to long-lived ASCs in bone marrow is predicated on quantitative increases in Blimp-1 expression.
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- 2004
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17. Evidence from the generation of immunoglobulin G-secreting cells that stochastic mechanisms regulate lymphocyte differentiation.
- Author
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Hasbold J, Corcoran LM, Tarlinton DM, Tangye SG, and Hodgkin PD
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- Animals, B-Lymphocytes cytology, CD40 Ligand immunology, Female, Flow Cytometry, Immunoglobulin G biosynthesis, Interleukin-4 immunology, Interleukin-5 immunology, Male, Mice, Mice, Inbred CBA, Stochastic Processes, B-Lymphocytes immunology, Cell Differentiation immunology, Immunoglobulin Class Switching immunology, Immunoglobulin G immunology
- Abstract
Naive B lymphocytes undergo isotype switching and develop into immunoglobulin-secreting cells to generate the appropriate class and amount of antibody necessary for effective immunity. Although this seems complex, we report here that the generation of immunoglobulin G-secreting cells from naive precursors is highly predictable. The probabilities of isotype switching and development into secreting cells change with successive cell divisions and interleave independently. Cytokines alter the probability of each differentiation event, while leaving intact their independent assortment. As a result, cellular heterogeneity arises automatically as the cells divide. Stochastic division-linked regulation of heterogeneity challenges the conventional paradigms linking distinct phenotypes to unique combinations of signals and has the potential to simplify our concept of immune complexity considerably.
- Published
- 2004
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18. B cell receptor-independent stimuli trigger immunoglobulin (Ig) class switch recombination and production of IgG autoantibodies by anergic self-reactive B cells.
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Phan TG, Amesbury M, Gardam S, Crosbie J, Hasbold J, Hodgkin PD, Basten A, and Brink R
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- Animals, Autoantigens physiology, Cell Lineage, Cell Movement, Immunoglobulin Heavy Chains genetics, Immunoglobulin Variable Region genetics, Immunophenotyping, Lipopolysaccharides pharmacology, Lymphocyte Activation, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Transgenic, Muramidase, Autoantibodies biosynthesis, B-Lymphocytes immunology, Immunoglobulin Class Switching, Immunoglobulin G biosynthesis, Receptors, Antigen, B-Cell physiology, Self Tolerance immunology
- Abstract
In both humans and animals, immunoglobulin (Ig)G autoantibodies are less frequent but more pathogenic than IgM autoantibodies, suggesting that controls over Ig isotype switching are required to reinforce B cell self-tolerance. We have used gene targeting to produce mice in which hen egg lysozyme (HEL)-specific B cells can switch to all Ig isotypes (SWHEL mice). When crossed with soluble HEL transgenic (Tg) mice, self-reactive SWHEL B cells became anergic. However, in contrast to anergic B cells from the original nonswitching anti-HEL x soluble HEL double Tg model, self-reactive SWHEL B cells also displayed an immature phenotype, reduced lifespan, and exclusion from the splenic follicle. These differences were not related to their ability to Ig class switch, but instead to competition with non-HEL-binding B cells generated by VH gene replacement in SWHEL mice. When activated in vitro with B cell receptor (BCR)-independent stimuli such as anti-CD40 monoclonal antibody plus interleukin 4 or lipopolysaccharide (LPS), anergic SWHEL double Tg B cells proliferated and produced IgG anti-HEL antibodies as efficiently as naive HEL-binding B cells from SWHEL Ig Tg mice. These results demonstrate that no intrinsic constraints to isotype switching exist in anergic self-reactive B cells. Instead, production of IgG autoantibodies is prevented by separate controls that reduce the likelihood of anergic B cells encountering BCR-independent stimuli. That bacteria-derived LPS could circumvent these controls may explain the well-known association between autoantibody-mediated diseases and episodes of systemic infection.
- Published
- 2003
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19. Cross-linking surface Ig delays CD40 ligand- and IL-4-induced B cell Ig class switching and reveals evidence for independent regulation of B cell proliferation and differentiation.
- Author
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Rush JS, Hasbold J, and Hodgkin PD
- Subjects
- Adjuvants, Immunologic pharmacology, Animals, Antibodies, Anti-Idiotypic pharmacology, Antibodies, Monoclonal pharmacology, Antigens, Differentiation, B-Lymphocyte biosynthesis, B-Lymphocytes metabolism, CD40 Ligand physiology, Cell Differentiation immunology, Cell Division immunology, Cells, Cultured, Female, Immunoglobulin D immunology, Immunoglobulin E biosynthesis, Immunoglobulin G biosynthesis, Male, Mice, Mice, Inbred CBA, Recombinant Proteins pharmacology, B-Lymphocytes cytology, B-Lymphocytes immunology, CD40 Ligand pharmacology, Cross-Linking Reagents metabolism, Down-Regulation immunology, Immunoglobulin Class Switching immunology, Interleukin-4 pharmacology, Receptors, Antigen, B-Cell metabolism
- Abstract
T cells stimulate B cells to divide and differentiate by providing activating signals in the form of inducible membrane-bound molecules and secreted cytokines. Provision of these signals in vitro reproduces many of the consequences of T-B collaboration in the absence of any form of Ag stimulation. Although clearly not obligatory, Ag signals appear to play an important regulatory role in numerous aspects of the B cell response. To examine directly the effect of an Ag signal, naive B cells were stimulated in the presence of rCD40 ligand, with or without IL-4 in the presence or absence of different anti-Ig mAbs. Anti-Ig mAbs exerted variable effects on the B cell division rate, from enhancement to no effect to inhibition. In contrast, all anti-Ig mAbs tested inhibited division-linked isotype switching to IgG1 and IgE. Thus, B cell Ag receptor ligands could modify the rates of B cell expansion and class switching independently. The ability of anti-Ig reagents to modify class switching suggests the B cell Ag receptor may play an important role in the selection of Ig isotypes during T cell-dependent humoral immune responses to Ags of different physical structure.
- Published
- 2002
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20. Flow cytometric analysis of cell division history using dilution of carboxyfluorescein diacetate succinimidyl ester, a stably integrated fluorescent probe.
- Author
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Lyons AB, Hasbold J, and Hodgkin PD
- Subjects
- Animals, Antigens, Surface immunology, Antigens, Surface metabolism, Antimetabolites metabolism, B-Lymphocytes cytology, B-Lymphocytes immunology, Bromodeoxyuridine metabolism, Cell Separation, Cells, Cultured, Cytokines metabolism, DNA metabolism, Fluoresceins chemistry, Fluorescent Dyes chemistry, Humans, Immunoglobulins metabolism, Mice, Signal Processing, Computer-Assisted, Spleen cytology, Succinimides chemistry, T-Lymphocytes cytology, T-Lymphocytes immunology, Cell Division physiology, Flow Cytometry methods, Fluoresceins metabolism, Fluorescent Dyes metabolism, Succinimides metabolism
- Published
- 2001
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21. Flow cytometric cell division tracking using nuclei.
- Author
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Hasbold J and Hodgkin PD
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- Animals, Bromodeoxyuridine metabolism, Cell Fractionation, Dactinomycin analogs & derivatives, Female, Fluoresceins metabolism, Fluorescent Dyes, Male, Mice, Mice, Inbred CBA, Proliferating Cell Nuclear Antigen, Staining and Labeling, Succinimides metabolism, Cell Division physiology, Cell Nucleus metabolism, Flow Cytometry methods
- Abstract
Background: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions., Methods: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining., Results: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined., Conclusions: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle., (Copyright 2000 Wiley-Liss, Inc.)
- Published
- 2000
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22. Switching to IgG3, IgG2b, and IgA is division linked and independent, revealing a stochastic framework for describing differentiation.
- Author
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Deenick EK, Hasbold J, and Hodgkin PD
- Subjects
- Animals, B-Lymphocytes cytology, B-Lymphocytes immunology, Cell Differentiation immunology, Cell Division immunology, Cells, Cultured, Female, Lipopolysaccharides pharmacology, Lymphocyte Activation immunology, Lymphocyte Count, Male, Mice, Mice, Inbred CBA, Stochastic Processes, Time Factors, Transforming Growth Factor beta pharmacology, Immunoglobulin A biosynthesis, Immunoglobulin Class Switching immunology, Immunoglobulin G biosynthesis
- Abstract
LPS was used to induce switching of B cells to IgG3 and, in the presence of TGF-beta, to IgG2b and IgA. Switching to all three isotypes increased with division number according to a consistent relationship that was independent of time in culture. The mode of activation altered the relationship with division, as CD40 ligand increased switching to IgA and decreased switching to IgG2b and IgG3 when measured per division. This division-linked switching behavior could be described by Gaussian probability distributions centered around a mean division number. The divisions at which switching to IgG3 and IgG2b occurred overlapped, raising the possibility that the two switching mechanisms were linked. However, when IgG3+ and IgG3- B cells were sorted and placed back in culture, they switched to IgG2b at an equivalent rate, indicating that alternative switching decisions were made independently within a single cell. As a consequence, isotype switching could be predicted at the population level by standard probability laws. Therefore, division number provides a framework for a stochastic description of differentiation that may be widely applicable.
- Published
- 1999
23. Integrating signals from IFN-gamma and IL-4 by B cells: positive and negative effects on CD40 ligand-induced proliferation, survival, and division-linked isotype switching to IgG1, IgE, and IgG2a.
- Author
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Hasbold J, Hong JS, Kehry MR, and Hodgkin PD
- Subjects
- Adjuvants, Immunologic antagonists & inhibitors, Adjuvants, Immunologic physiology, Animals, B-Lymphocytes cytology, B-Lymphocytes metabolism, CD40 Antigens metabolism, CD40 Ligand, Cell Division immunology, Cell Survival immunology, Cells, Cultured, Female, Interleukin-4 antagonists & inhibitors, Ligands, Lymphocyte Count, Male, Membrane Glycoproteins antagonists & inhibitors, Mice, Mice, Inbred CBA, B-Lymphocytes immunology, Immunoglobulin Class Switching immunology, Immunoglobulin E biosynthesis, Immunoglobulin G biosynthesis, Interferon-gamma physiology, Interleukin-4 physiology, Membrane Glycoproteins physiology, Signal Transduction immunology
- Abstract
IL-4 and IFN-gamma each have potent effects on B cell responses as well as strong mutual antagonism. Here we have examined the quantitative effects of these cytokines on CD40 ligand-induced B cell proliferation, cell survival, and division-linked isotype switching. Both IL-4 (strongly) and IFN-gamma (weakly) enhanced the number of B cells found in culture by reducing the average time cells take to enter the first division cycle and by promoting B cell survival. When added in combination, the net effect of IL-4 and IFN-gamma on time to division and survival was a response intermediate between that of the two cytokines alone, indicating a partial antagonism of IL-4 by IFN-gamma. By modulating both time to division and cell survival, these small effects of IFN-gamma are amplified and give rise to large reductions in cell number in the presence of IL-4. At higher concentrations, IFN-gamma had minor inhibitory effects on IL-4-induced isotype switching to IgG1 and greater effects on IgE. A reciprocal relation was observed between the ability to inhibit IgE at late cell divisions vs induction of IgG2a. In contrast, IL-4 did not prevent switching to IgG2a induced by IFN-gamma alone. Therefore, antagonism between IFN-gamma and IL-4 is observed at multiple levels and over different concentration ranges, resulting in complex net outcomes. The evolutionary significance of this complexity is discussed.
- Published
- 1999
24. Generation of splenic follicular structure and B cell movement in tumor necrosis factor-deficient mice.
- Author
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Cook MC, Körner H, Riminton DS, Lemckert FA, Hasbold J, Amesbury M, Hodgkin PD, Cyster JG, Sedgwick JD, and Basten A
- Subjects
- Animals, Cell Movement, Germinal Center physiology, Humans, Lymphotoxin-alpha physiology, Mice, Mice, Inbred C57BL, Rats, Tumor Necrosis Factor-alpha deficiency, B-Lymphocytes physiology, Spleen cytology, Tumor Necrosis Factor-alpha physiology
- Abstract
Secondary lymphoid tissue organogenesis requires tumor necrosis factor (TNF) and lymphotoxin alpha (LTalpha). The role of TNF in B cell positioning and formation of follicular structure was studied by comparing the location of newly produced naive recirculating and antigen-stimulated B cells in TNF-/- and TNF/LTalpha-/- mice. By creating radiation bone marrow chimeras from wild-type and TNF-/- mice, formation of normal splenic B cell follicles was shown to depend on TNF production by radiation-sensitive cells of hemopoietic origin. Reciprocal adoptive transfers of mature B cells between wild-type and knockout mice indicated that normal follicular tropism of recirculating naive B cells occurs independently of TNF derived from the recipient spleen. Moreover, soluble TNF receptor-IgG fusion protein administered in vivo failed to prevent B cell localization to the follicle or the germinal center reaction. Normal T zone tropism was observed when antigen-stimulated B cells were transferred into TNF-/- recipients, but not into TNF/LTalpha-/- recipients. This result appeared to account for the defect in isotype switching observed in intact TNF/LTalpha-/- mice because TNF/LTalpha-/- B cells, when stimulated in vitro, switched isotypes normally. Thus, TNF is necessary for creating the permissive environment for B cell movement and function, but is not itself responsible for these processes.
- Published
- 1998
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25. Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4.
- Author
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Hasbold J, Lyons AB, Kehry MR, and Hodgkin PD
- Subjects
- Animals, CD40 Ligand, Cell Division, Dose-Response Relationship, Drug, Down-Regulation, Gene Expression Regulation, Immunoglobulin D metabolism, Immunoglobulin M metabolism, Mice, Mice, Inbred CBA, Receptors, Antigen, B-Cell metabolism, Up-Regulation, B-Lymphocytes immunology, Immunoglobulin E genetics, Immunoglobulin G genetics, Interleukin-4 pharmacology, Membrane Glycoproteins pharmacology
- Abstract
CD40 ligand (CD40L) and IL-4 are sufficient to induce resting murine B cells to divide and switch isotypes from IgM and IgD to IgG1 and IgE. Tracking of cell division following (5- and 6) carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling revealed that B cells expressed IgG1 after three cell divisions, and IgE after five. The probability of isotype switching at each division was independent of both time after stimulation and of the dose of CD40L. IL-4 concentration regulated the number of divisions that preceded isotype switching. Loss of surface IgM and IgD was also related to cell division and appeared to be differentially regulated. B cell proliferation was typically asynchronous with the proportion of cells in consecutive divisions being markedly affected by the concentration of CD40L and IL-4. Simultaneous (5-bromo)-2'-deoxyuridine labeling and CFSE staining revealed that B cells in each division cycle were dividing at the same rate. Therefore, division cycle asynchrony resulted from dose-dependent variation in the time taken to enter the first division cycle. These results suggest that T-dependent B cell expansion is linked to predictable functional changes that may, in part, explain why IgE is produced in response to prolonged antigenic stimulation.
- Published
- 1998
- Full Text
- View/download PDF
26. The importance of efficacy and partial agonism in evaluating models of B lymphocyte activation.
- Author
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Hodgkin PD, Chin SH, Bartell G, Mamchak A, Doherty K, Lyons AB, and Hasbold J
- Subjects
- Animals, Cell Division, Cells, Cultured, Humans, Immunoglobulin Class Switching, Interleukin-4 pharmacology, Models, Biological, B-Lymphocytes immunology, Lymphocyte Activation
- Abstract
Immunologists have developed a range of in vitro techniques for probing the receptor mediated response of cells comprising the immune system. An important and ubiquitous method is the use of antibodies in either soluble or aggregated form to engage cell surface receptors and transmit a signal. Models of cell and molecular interactions, derived from the use of these antibodies, form the basis of our efforts to understand and explain the corresponding in vivo systems. However, interpreting in vitro experiments and distinguishing between alternative models is difficult. This complexity is illustrated here using B cell stimulation by surface immunoglobulin and CD40. The fluorescent cell labelling dye carboxyfluorescein, diacetate, succinimidyl ester (CFSE) is used to show that many anti-Ig and CD40 stimulatory agents, used to assess the role of B cells and lymphokines, are partial agonists. By modelling each step in B cell signalling, activation and division it is possible to show that small changes in signal contributed by a second receptor can generate numerous distinct dose response curves that are highly dependent on the "efficacy" of signal transmission by the primary ligand and the number of cell divisions taken in culture. Differences in dose response curves become particularly striking if the primary activating stimulus is a partial agonist. Although exemplified here with B cell stimulation the conclusions are applicable to other in vitro activation systems and suggest ways to improve both the design and interpretation of in vitro experiments.
- Published
- 1997
- Full Text
- View/download PDF
27. Properties of mouse CD40: the role of homotypic adhesion in the activation of B cells via CD40.
- Author
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Klaus GG, Holman M, and Hasbold J
- Subjects
- Animals, Antibodies, Monoclonal immunology, CD40 Antigens, Cell Adhesion, Cell Adhesion Molecules physiology, DNA biosynthesis, Immunophenotyping, Lymphocyte Function-Associated Antigen-1 physiology, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Antigens, CD physiology, Antigens, Differentiation, B-Lymphocyte physiology, B-Lymphocytes immunology, Lymphocyte Activation
- Abstract
Stimulation of human B cells via CD40 is known to induce their homotypic aggregation. We show here that anti-mouse CD40 monoclonal antibodies (mAb) also induce B cells to form large, spherical, extremely stable clusters. This clustering is markedly enhanced by co-stimulation with either interleukin-4 (IL-4) or anti-immunoglobulin (Ig). The aggregation is slow in onset, and is largely (but not completely) abrogated by anti-LFA-1 mAb, but not by mAb directed against other potentially important adhesion molecules on B cells. Anti-LFA-1 mAb also partially suppressed DNA synthesis induced by anti-CD40, but not by other B cell mitogens, suggesting that clustering is an important component of B cell activation via CD40. This concept is supported by analyses of the phenotype of clustered B cells: the cells within clusters express higher levels of various activation markers, and also more of them are in cell cycle than non-clustered cells. These results therefore suggest that CD40 stimulation may either induce B cells to secrete soluble factors which act in an autocrine way to promote B cell activation, or that clustering generates cell contact-mediated signals which are important in the activation cascade.
- Published
- 1994
- Full Text
- View/download PDF
28. Properties of mouse CD40: cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies.
- Author
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Hasbold J, Johnson-Léger C, Atkins CJ, Clark EA, and Klaus GG
- Subjects
- Animals, Animals, Newborn, Antigens, CD biosynthesis, Antigens, Differentiation, B-Lymphocyte biosynthesis, B7-1 Antigen immunology, Bone Marrow Cells, CD40 Antigens, Cell Differentiation immunology, Enzyme-Linked Immunosorbent Assay, Female, Flow Cytometry, Histocompatibility Antigens Class II immunology, Male, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Rats, Rats, Inbred Strains, Receptors, IgE immunology, T-Lymphocyte Subsets immunology, Antibodies, Monoclonal immunology, Antigens, CD immunology, Antigens, Differentiation, B-Lymphocyte immunology, B-Lymphocytes immunology
- Abstract
We describe here the derivation of a rat monoclonal antibody (mAb) against mouse CD40 (designated 3/23), which stains 45-50% of spleen cells of adult mice, approximately 90% of which are B cells. Interestingly, some 5-10% of both CD4+ and CD8+ T cells in the spleens of (some, but not all) adult, unimmunized mice are also CD40+, whereas CD40+ cells were not detectable in the thymus, even following collagenase digestion. Some 35-40% of lymphoid cells in the bone marrow of adult mice are CD40+ and virtually all of these are B220+, and hence of the B cell lineage: triple-color flow cytometry showed that CD40 is expressed at low levels on some 30% of pre-B cells, at intermediate levels on 80% of immature B cells and on essentially all mature B cells in the bone marrow. These results, therefore, suggest that in the mouse CD40 is expressed relatively late during the process of B cell differentiation. The mAb induced marked up-regulation of major histocompatibility complex class II molecules, CD23 and B7.2 antigens on mature B cells. It also stimulated modest levels of DNA synthesis in mature B cells by itself: this was markedly enhanced by suboptimal concentrations of mitogenic (but not non-mitogenic) anti-mu and anti-delta mAb, and moderately enhanced by co-stimulation with interleukin-4. Hypercross-linking of CD40 (using biotinylated mAb and avidin) also enhanced the proliferative response to anti-CD40.
- Published
- 1994
- Full Text
- View/download PDF
29. Plastic-immobilized anti-mu or anti-delta antibodies induce apoptosis in mature murine B lymphocytes.
- Author
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Parry SL, Holman MJ, Hasbold J, and Klaus GG
- Subjects
- Animals, Antigens, CD physiology, Antigens, Differentiation, B-Lymphocyte physiology, CD40 Antigens, DNA biosynthesis, Female, Interleukin-4 pharmacology, Lipopolysaccharides pharmacology, Male, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Plastics, RNA biosynthesis, Rats, Antibodies, Anti-Idiotypic immunology, Antibodies, Monoclonal immunology, Apoptosis, B-Lymphocytes physiology
- Abstract
It is widely accepted that extensive cross-linking of surface immunoglobulin (sIg) receptors on mature B cells promotes their activation and progression through the cell cycle. A commonly employed method to maximize receptor cross-linking via anti-receptor antibodies is to immobilize them on tissue culture plastic. We show here that immobilizing monoclonal anti-mu or anti-delta antibodies, which are mitogenic in solution, on plastic abrogates their capacity to induce DNA synthesis in mature murine B cells, even in the presence of interleukin-4 (IL-4). The cells do become abortively activated, as evidenced by up-regulation of major histocompatibility complex class II antigen levels, but subsequently virtually all of them die, manifesting DNA fragmentation characteristic of apoptosis. The induction of apoptosis is abrogated by the inclusion of either IL-4 or anti-CD40 antibodies in the cultures, with the two stimuli acting in concert. We believe that the system represents a polyclonal model of clonal deletion tolerance in mature B cells, such as may be induced under physiological conditions by antigens with repeating epitopes.
- Published
- 1994
- Full Text
- View/download PDF
30. B cells from CBA/N mice do not proliferate following ligation of CD40.
- Author
-
Hasbold J and Klaus GG
- Subjects
- Animals, CD40 Antigens, CD40 Ligand, Cells, Cultured, Female, Immunologic Deficiency Syndromes immunology, Lymphocyte Activation immunology, Male, Membrane Glycoproteins, Mice, Mice, Inbred CBA, Recombinant Fusion Proteins, Spleen cytology, T-Lymphocytes physiology, Antigens, CD physiology, Antigens, Differentiation, B-Lymphocyte physiology, B-Lymphocytes physiology
- Abstract
The CBA/N mouse carries the X-linked immunodeficiency xid, resulting in defective B cell development. B cells from these animals cannot mount antibody responses to type 2 T-independent antigens, and do not synthesize DNA when stimulated with anti-immunoglobulin (Ig) antibodies which are mitogenic for normal B cells. The primary antibody responses of CBA/N mice to T-dependent antigens have also been reported to be abnormal. Here we describe the results of experiments which demonstrate that the B cells from these animals respond abnormally to ligation of CD40, a B cell surface molecule now known to play a key role during T cell-B cell interactions, via its interaction with the counterligand (CD40L) expressed by activated T cells. Hence, xid B cells fail to proliferate when cultured with preactivated T helper type 2 (Th2)T cells (known to express CD40L), with a soluble CD40L-CD8 fusion protein, or in response to monoclonal antibodies to CD40, even in the presence of IL-4 and/or anti-Ig reagents. However, xid B cells do receive abortive activation signals following ligation of CD40, as manifested by up-regulation of class II major histocompatibility complex and CD23 antigens. Since the xid defect has now been identified as a point mutation in the protein tyrosine kinase Btk, our results point to an important role for this kinase in the downstream signaling cascade elicited in response to ligation of either surface Ig or CD40.
- Published
- 1994
- Full Text
- View/download PDF
31. Anti-immunoglobulin antibodies induce apoptosis in immature B cell lymphomas.
- Author
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Hasbold J and Klaus GG
- Subjects
- Antibodies, Monoclonal pharmacology, Cell Division drug effects, Cell Survival, Cells, Cultured, DNA Damage, Dose-Response Relationship, Drug, Electrophoresis, Agar Gel, Humans, In Vitro Techniques, Lipopolysaccharides pharmacology, Antibodies, Anti-Idiotypic immunology, B-Lymphocytes immunology, Immunoglobulin M immunology, Lymphoma, B-Cell immunology
- Abstract
WEHI-231 and CH31 are phenotypically immature sIgM+ murine B cell lymphomas whose growth is inhibited by anti-immunoglobulin (Ig) antibodies. These lines have therefore been used as models for studying the role of surface Ig receptors in the induction of B cell tolerance. We show here that anti-mu antibodies induce DNA cleavage into oligonucleosomal fragments characteristic of programmed cell death (apoptosis) in both cell lines, although WEHI-231 cells are less susceptible than CH31. This effect was reversed by lipopolysaccharide, in agreement with the known effects of lipopolysaccharide on anti-Ig-induced growth inhibition. These results therefore indicate that these lymphomas afford a potentially interesting model to study the mechanisms of programmed cell death induced by ligation of the antigen receptors on normal B cells.
- Published
- 1990
- Full Text
- View/download PDF
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