scholarly journals DNA rearrangement can account for in vitro switching to IgG1.

1993 ◽  
Vol 178 (4) ◽  
pp. 1381-1390 ◽  
Author(s):  
C C Chu ◽  
E E Max ◽  
W E Paul
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Cultured Cells ◽  
Surface Expression ◽  
Immunoglobulin M ◽  
Interleukin 4 ◽  
Class Switching ◽  
Dna Deletion ◽  
Alternative Processing

During immune responses, B lymphocytes may switch from the expression of immunoglobulin M (IgM) to the expression of another isotype (e.g., IgG, IgE, IgA). In stable hybridomas and myelomas expressing a "switched" (S) isotype, DNA deletions between S mu and a "downstream" S region (S region recombination) have been found. In primary B cells, studies of the molecular basis of switching have been limited by the ability to sensitively quantitate the amount of DNA deletion; such studies would be of interest because other nondeletional mechanisms (trans-splicing, alternative processing of a long transcript) have been proposed to account for isotype switching in certain circumstances. We have applied the digestion-circularization polymerase chain reaction (DC-PCR) technique to measure the amount of S region recombination that occurs in the course of class switching in primary B lymphocytes. Resting B cells were cultured in lipopolysaccharide (LPS) and interleukin 4 (IL-4) to stimulate switching to IgG1. These cells begin to express membrane IgG1 at day 2.5 of culture and reach maximum expression by day 4.5. DNA was prepared from cultured cells and analyzed for S mu-S gamma 1 rearrangement by DC-PCR. Chimeric switch regions, indicating S mu-S gamma 1 recombination, were detected in amounts that, in most cases, correlated with surface expression. Furthermore, when cells were sorted on the basis of surface IgG1 expression, a mean of at least one S mu-S gamma 1 rearrangement per cell was seen in five out of seven experiments. In general, the IgG1+ cells obtained at 4.5 and 5.5 d of culture had close to 2 S mu-S gamma 1 rearrangements per cell. In IgG1- cells, S mu-S gamma 1 rearrangements were detectable, but at frequencies substantially lower that in IgG1+ cells. Thus, these results indicate that DNA deletion accompanies class switching in normal B cells stimulated with LPS and IL-4.

scholarly journals AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1046.1-1046
Author(s):  
L. Schlicher ◽  
P. Kulig ◽  
M. Murphy ◽  
M. Keller
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Activation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
B Cell Activation ◽  
Human B Cells ◽  
Circulating Lymphocytes

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

The development of functional B lymphocytes in conditional PU.1 knock-out mice

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 2083-2090 ◽  
Author(s):  
Matthew Polli ◽  
Aleksandar Dakic ◽  
Amanda Light ◽  
Li Wu ◽  
David M. Tarlinton ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Fetal Liver ◽  
Cell Receptor ◽  
Interleukin 4 ◽  
Knock Out ◽  
B Lineage ◽  
And Function

Abstract An abundance of research has entrenched the view that the Ets domain containing transcription factor PU.1 is fundamental to the development and function of B lymphocytes. In this study, we have made use of a conditional PU.1 allele to test this notion. Complete deletion of PU.1 resulted in the loss of B cells and all other lineage-positive cells in the fetal liver and death between E18.5 and birth; however, specific deletion of PU.1 in the B lineage had no effect on B-cell development. Furthermore, deletion of PU.1 in B cells did not compromise their ability to establish and maintain an immune response. An increased level of apoptosis was observed in vitro upon B-cell receptor (BCR) cross-linking; however, this was partially rescued by interleukin-4 (IL-4). These findings suggest that PU.1 is not essential for the development of functional B lymphocytes beyond the pre-B stage. (Blood. 2005;106:2083-2090)

scholarly journals Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells.

1994 ◽  
Vol 179 (2) ◽  
pp. 425-438 ◽  
Author(s):  
M P Cooke ◽  
A W Heath ◽  
K M Shokat ◽  
Y Zeng ◽  
F D Finkelman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Transgenic Mice ◽  
B Lymphocytes ◽  
Molecular Mechanisms ◽  
Interleukin 4 ◽  
Th Cells

The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells.

scholarly journals Transcriptional Repression of Stat6-Dependent Interleukin-4-Induced Genes by BCL-6: Specific Regulation of Iɛ Transcription and Immunoglobulin E Switching

1999 ◽  
Vol 19 (10) ◽  
pp. 7264-7275 ◽  
Author(s):  
Miera B. Harris ◽  
Chih-Chao Chang ◽  
Michael T. Berton ◽  
Nika N. Danial ◽  
Jandong Zhang ◽  
...  
Keyword(s):  
B Cells ◽  
Target Genes ◽  
Germ Line ◽  
Immunoglobulin E ◽  
Interleukin 4 ◽  
Target Sequence ◽  
Class Switching ◽  
Mobility Shift ◽  
Specific Regulation

ABSTRACT The BCL-6 proto-oncogene encodes a POZ/zinc-finger transcription factor that is expressed in B cells and a subset of CD4+ T cells within germinal centers. Recent evidence suggests that BCL-6 can act as a sequence-specific repressor of transcription, but the target genes for this activity have not yet been identified. The binding site for BCL-6 shares striking homology to the sites that are the target sequence for the interleukin-4 (IL-4)-induced Stat6 (signal transducers and activators of transcription) signaling molecule. Electrophoretic mobility shift assays demonstrate that BCL-6 can bind, with different affinities, to several DNA elements recognized by Stat6. Expression of BCL-6 can repress the IL-4-dependent induction of immunoglobulin (Ig) germ line ɛ transcripts, but does not repress the IL-4 induction of CD23 transcripts. Consistent with the role of BCL-6 in modulating transcription from the germ line ɛ promoter, BCL-6−/−mice display an increased ability to class switch to IgE in response to IL-4 in vitro. These animals also exhibit a multiorgan inflammatory disease characterized by the presence of a large number of IgE+ B cells. The apparent dysregulation of IgE production is abolished in BCL-6−/− Stat6−/− mice, indicating that BCL-6 regulation of Ig class switching is dependent upon Stat6 signaling. Thus, BCL-6 can modulate the transcription of selective Stat6-dependent IL-4 responses, including IgE class switching in B cells.

scholarly journals SELECTIVE TRIGGERING OF HUMAN T AND B LYMPHOCYTES IN VITRO BY POLYCLONAL MITOGENS

1974 ◽  
Vol 140 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Melvyn Greaves ◽  
George Janossy ◽  
Michael Doenhoff
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Human Lymphocytes ◽  
Culture Conditions ◽  
Pokeweed Mitogen ◽  
Cell Populations ◽  
T And B Cells ◽  
Activated T Cells ◽  
Lymphocyte Responses

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

scholarly journals Interleukin 4 induces selective production of interleukin 6 from normal human B lymphocytes.

1989 ◽  
Vol 170 (4) ◽  
pp. 1463-1468 ◽  
Author(s):  
E B Smeland ◽  
H K Blomhoff ◽  
S Funderud ◽  
M R Shalaby ◽  
T Espevik
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Interleukin 6 ◽  
Specific Effect ◽  
Interleukin 4 ◽  
Tnf Alpha ◽  
Ifn Gamma ◽  
Normal Human ◽  
Tnf Secretion

In this paper we have shown that extensively purified human B lymphocytes respond to IL-4 treatment with a marked production of IL-6. Addition of anti-mu potentiated the effect of IL-4 on IL-6 production. Other cytokines tested like TNF-alpha and-beta, IFN-gamma, IL-1, IL-2, and IL-5 did not induce IL-6 secretion when given to resting B cells. Although B cells generally also produced TNF-alpha and TNF-beta upon stimulation, IL-4 did not induce TNF secretion and seemingly had a specific effect on IL-6 production.

scholarly journals Autologous stimulation of human lymphocyte subpopulation.

1975 ◽  
Vol 142 (5) ◽  
pp. 1327-1333 ◽  
Author(s):  
G Opelz ◽  
M Kiuchi ◽  
M Takasugi ◽  
P I Terasaki
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Lymphocytes ◽  
Human Lymphocyte ◽  
Lymphocyte Subpopulation ◽  
High Background ◽  
Background Stimulation ◽  
Most Probable Explanation ◽  
Stimulation Of

The background stimulation universally seen when lymphocytes are cultured in vitro has been shown to be markedly lowered by reducing the proportion of B lymphocytes. B-rich fractions of lymphocytes had extremely high background stimulation. It is concluded that stimulation of T cells, probably by autologous B cells, provides the most probable explanation for the findings described.

scholarly journals Interleukin-13 in Combination With CD40 Ligand Potently Inhibits Apoptosis in Human B Lymphocytes: Upregulation of Bcl-xL and Mcl-1

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4415-4424 ◽  
Author(s):  
Jon Lømo ◽  
Heidi Kiil Blomhoff ◽  
Sten Eirik Jacobsen ◽  
Stanislaw Krajewski ◽  
John C. Reed ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Cell Apoptosis ◽  
Cell Types ◽  
Cd40 Ligand ◽  
Interleukin 13

Abstract Interleukin-13 (IL-13) is a novel T-cell–derived cytokine with IL-4–like effects on many cell types. In human B lymphocytes, IL-13 induces activation, stimulates proliferation in combination with anti-IgM or anti-CD40 antibodies, and directs Ig isotype switching towards IgE and IgG4 isotypes. We show here that IL-13 also regulates human B-cell apoptosis. IL-13 reduced spontaneous apoptosis of peripheral blood B cells in vitro, as shown by measurement of DNA fragmentation using the TUNEL and Nicoletti assays. The inhibition of cell death by IL-13 alone was significant but modest, but was potently enhanced in combination with CD40 ligand (CD40L), a survival stimulus for B cells by itself. Interestingly, IL-13 increased the expression of CD40 on peripheral blood B cells, providing a possible mechanism for the observed synergy. IL-13 alone was a less potent inhibitor of apoptosis than IL-4. Moreover, there was no additive effect of combining IL-4 and IL-13 at supraoptimal concentrations, which is consistent with the notion that the IL-4 and IL-13 binding sites share a common signaling subunit. The combination of IL-13 with CD40L augmented the expression of the Bcl-2 homologues Bcl-xL and Mcl-1, suggesting this as a possible intracellular mechanism of induced survival. By contrast, levels of Bcl-2, and two other Bcl-2 family members, Bax and Bak, remained unaltered. Given the importance of the CD40-CD40L interaction in B-cell responses, these results suggest a significant role of IL-13 in the regulation of B-cell apoptosis.

CD40 inhibits B cell apoptosis by upregulating bcl-xL expression and blocking oxidant accumulation

1997 ◽  
Vol 272 (3) ◽  
pp. C950-C956 ◽  
Author(s):  
W. Fang ◽  
K. A. Nath ◽  
M. F. Mackey ◽  
R. J. Noelle ◽  
D. L. Mueller ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cd40 Ligand ◽  
Antioxidant Response ◽  
Immunoglobulin M ◽  
Inhibitory Protein ◽  
Wehi 231 ◽  
Immature Mouse

Signaling through the CD40 receptor on human and murine B lymphocytes is necessary for germinal center formation and immunoglobulin class switching in vivo and rescues B cells from apoptosis triggered by cross-linking of surface immunoglobulin M in vitro. Ligation of CD40 on the immature mouse B cell line WEHI-231 with recombinant CD40 ligand (CD40L) was found to protect cells from apoptosis after gamma irradiation, as well as that following treatment with the sphingomyelin ceramide or compounds that deplete intracellular glutathione. CD40 signaling led to a rapid increase in the expression of the apoptosis inhibitory protein Bcl-xL. In addition, the apoptosis-induced accumulation of intracellular oxidants in WEHI-231 B cells was rapidly diminished by CD40 crosslinking. This antioxidant response was observed within 1 h and coincided with a preservation of intracellular thiols. These findings indicate that CD40 signaling induces a generalized cellular resistance to apoptosis characterized by an upregulation of Bcl-xL and changes in the intracellular redox potential.

Circulating CD24hiCD38hi regulatory B cells correlate inversely with the ThEM17 cell frequency in granulomatosis with polyangiitis patients

Rheumatology ◽  
2019 ◽  
Vol 58 (8) ◽  
pp. 1361-1366 ◽  
Author(s):  
Anouk von Borstel ◽  
Lucas L Lintermans ◽  
Peter Heeringa ◽  
Abraham Rutgers ◽  
Coen A Stegeman ◽  
...  
Keyword(s):  
B Cells ◽  
Granulomatosis With Polyangiitis ◽  
Cultured Cells ◽  
Effector Memory ◽  
Regulatory B Cells ◽  
Cell Frequency ◽  
Th1 Cell ◽  
Th Cells ◽  
Cpg Oligodeoxynucleotides

Abstract Objectives To investigate whether there is a direct relation between expanded proportions of Th17 effector memory (ThEM17) cells and regulatory B cells (Bregs) in peripheral blood of granulomatosis with polyangiitis (GPA) patients. Methods Frequencies of Bregs and ThEM17 cells, as well as ThEM1 cells, were determined by flow cytometry in blood samples from 42 GPA patients in remission and 18 matched healthy controls (HCs). The Breg frequency was defined as CD24hiCD38hiCD19+ cells. ThEM17 cells were defined as CCR6+CXCR3-CCR4+ cells and ThEM1 cells as CCR6-CXCR3+CCR4- cells within the CD3+CD4+CD45RO+CCR7- population. In addition, CD3+CD4+ Th cells from 9 GPA patients were co-cultured in vitro with either total B cells or a Breg-depleted B cell fraction. Cultured cells were stimulated with Staphylococcus Enterotoxin B (SEB) and CpG-oligodeoxynucleotides (CpG-ODN). Th17- (IL-17+) and Th1 cell (IFNγ+) frequencies were determined at baseline and day 5 upon restimulation with phorbol myristate acetate (PMA) and Ca-I. Results A decreased Breg frequency was found in treated GPA patients, whereas an increased ThEM17 cell frequency was observed in treated and untreated GPA patients compared with HCs. Additionally, a decreased ThEM1 cell frequency was seen in untreated GPA patients compared with HCs. In untreated GPA patients circulating Breg frequencies correlated negatively with ThEM17 cells (r = −0.533; P = 0.007) and positively with ThEM1 cells (r = −0.473; P = 0.015). The co-culture experiments revealed a significant increase in the frequency of IL-17+ Th cells in Breg-depleted samples (median: 3%; range: 1–7.5%) compared with Breg-undepleted samples (P = 0.002; undepleted samples median: 2.1%; range: 0.9–6.4%), whereas no difference in the frequency of IFNγ+ Th cells in Breg-depleted cultures was observed (undepleted median: 11.8%; range: 2.8–21% vs Breg-depleted median: 12.2%; range: 2.6–17.6%). Conclusion Bregs modulate ThEM17 responses in GPA patients. Future studies should elaborate on clinical and therapeutical implications of the Breg-Th17 interaction in GPA patients.

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