scholarly journals Visualization of Negative Signaling in B Cells by Quantitative Confocal Microscopy

2001 ◽  
Vol 21 (24) ◽  
pp. 8615-8625 ◽  
Author(s):  
Hyewon Phee ◽  
William Rodgers ◽  
K. Mark Coggeshall
Keyword(s):  
B Cells ◽  
B Cell ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Ex Vivo ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Dominant Negative Mutant ◽  
Mutant Form

ABSTRACT Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcγRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcγRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5′-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcγRII−/− but not gamma chain−/− splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were corecruited to the BCR, regardless of the presence or absence of FcγRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcγRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP−/− animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcγRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.

scholarly journals RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis

2004 ◽  
Vol 279 (19) ◽  
pp. 19523-19530 ◽  
Author(s):  
Benoit Guilbault ◽  
Robert J. Kay
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wehi 231 ◽  
Induced Apoptosis

RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-κB and Bcl-xL, which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-κB was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-κB suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-κB and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter.

scholarly journals Lytic Replication and Reactivation from B cells Is Not Required for Maintaining Gammaherpesvirus Latency in vivo

2021 ◽  
Author(s):  
Arundahti Gupta ◽  
Shana Michelle Owens ◽  
Darby G. Oldenburg ◽  
Douglas W White ◽  
James Craig Forrest
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Ex Vivo ◽  
Antiviral Drug ◽  
Lytic Replication ◽  
Viral Reactivation ◽  
Transcription Activator ◽  
Murine Gammaherpesvirus ◽  
Latently Infected

Gammaherpesviruses (GHVs) are lymphotropic tumor viruses with a biphasic infectious cycle. Lytic replication at the primary site of infection is necessary for GHVs to spread throughout the host and establish latency in distal sites. Dissemination is mediated by infected B cells that traffic hematogenously from draining lymph nodes to peripheral lymphoid organs, such as the spleen. B cells serve as the major reservoir for viral latency, and it is hypothesized that periodic reactivation from latently infected B cells contributes to maintaining long-term chronic infection. While fundamentally important to an understanding of GHV biology, aspects of B cell infection in latency establishment and maintenance are incompletely defined, especially roles for lytic replication and reactivation in this cell type. To address this knowledge gap and overcome limitations of replication-defective viruses, we generated a recombinant murine gammaherpesvirus 68 (MHV68) in which ORF50, the gene that encodes the essential immediate-early replication and transcription activator protein (RTA), was flanked by loxP sites to enable conditional ablation of lytic replication by ORF50 deletion in cells that express Cre recombinase. Following infection of mice that encode Cre in B cells with this virus, splenomegaly and viral reactivation from splenocytes were significantly reduced, however the number of latently infected splenocytes was equivalent to WT MHV68. Despite ORF50 deletion, MHV68 latency was maintained over time in spleens of mice at levels approximating WT, reactivation-competent MHV68. Stimulation of polyclonal B cell activation and proliferation by treating mice with lipopolysaccharide (LPS), which promotes MHV68 reactivation ex vivo, yielded equivalent increases in the number of latently infected cells for both ORF50-deleted and WT MHV68, even when mice were simultaneously treated with the antiviral drug cidofovir. Together, these data demonstrate that lytic replication in B cells is not required for MHV68 latency establishment and maintenance and further indicate that B cell proliferation, and not reactivation per se, is a major mechanism for maintaining latent viral genomes in the host.

Aberrant Splicing of the SLP65 SH2 Domain Enables Pre-B Cell Transformation and Compromises the Leukemia-Suppressive Function of the Pre-B Cell Receptor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 294-294
Author(s):  
Cihangir Duy ◽  
Lars Klemm ◽  
Rahul Nahar ◽  
Peter van Essen ◽  
Mieke Sprangers ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Full Length ◽  
Aberrant Splicing ◽  
Cell Stage ◽  
Suppressive Function

Abstract The linker molecule SLP65 is critical for pre-B cell receptor-mediated differentiation signals during early B cell development. Germline mutations of the SLP65 gene in mice and humans result in a profound differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblast leukemia (ALL) cells are phenotypically arrested at an early pre-B cell stage in the vast majority of cases. In about 2–5% of cases with ALL, mutations and deletions of the SLP65 gene are found (Sprangers et al., 2006; Mullighan et al., 2007). In a comprehensive study of SLP65 function in human ALL cells, we identified numerous aberrant SLP65 splice variants, which all lack one or more exons that encode the C-terminal SLP65 SH2 domain. A splice variant lacking exon 16, which encodes the central part of the SH2 domain was particularly abundant in ALL but not in normal pre-B cells (EMBL/GenBank Accession number AM180337). The SH2 domain may have critical function because it represents the main ligand for the non-ITAM Y204 in the Igα (CD79A) signaling chain of the pre-B cell receptor (Patterson et al., 2006) and thereby connects the pre-B cell receptor to downstream signaling molecules. To examine a potential role of the SLP65 SH2 domain during the process of malignant transformation in ALL, we measured mRNA levels of full-length SLP65 vs SLP65 splice variants, in which the SH2 domain was deleted (SLP65ΔSH2) in normal pre-B cells and primary human ALL cells by quantitative RT-PCR. While normal pre-B cells express full-length SLP65 at high and SLP65ΔSH2 at very low levels, the SLP65/SLP65ΔSH2 ratio was reduced in E2A-PBX1-driven ALL and particularly low in BCR-ABL1-driven ALL. Given that SLP65ΔSH2 is overexpressed in ALL cells at the expense of full-length SLP65, we investigated biochemical properties of SLP65ΔSH2. To this end, we transduced Slp65−/− pre-B cells with either full-length Slp65, SLP65ΔSH2 or a GFP empty vector control. As opposed to full-length SLP65, SLP65ΔSH2 does not interact with Igα and does not confer tyrosine phosphorylation of BTK downstream of the pre-B cell receptor. Also Ca2+-release in response to pre-B cell receptor engagement and induction of differentiation was drastically reduced in Slp65−/− pre-B cells that were reconstituted with SLP65ΔSH2 instead of SLP65. Co-expression experiments with SLP65 showed that SLP65ΔSH2 has a dominant-negative function and impairs pre-B cell receptor signaling. We next transformed Slp65−/− pre-B cells with a retroviral BCR-ABL1 vector and tested whether SLP65 and SLP65ΔSH2 have a different impact on BCR-ABL1-mediated pre-B cell transformation and BCR-ABL1-driven leukemic growth. Reconstitution of SLP65 expression in Slp65−/− BCR-ABL1-transformed pre-B ALL cells resulted in cell death and nine days after transduction more than 90% of the SLP65-transduced cells had undergone apoptosis. Conversely, reconstitution of Slp65−/− BCR-ABL1-transformed pre-B ALL cells with SLP65ΔSH2 had no significant effect on viability of the leukemia cells. In a titration experiment, we reconstituted SLP65−/−BCR-ABL1-driven ALL cells with SLP65 and SLP65ΔSH2 at various ratios and thereby confirmed that SLP65ΔSH2 has a dominant-negative effect and compromises the leukemia-suppressive function of the pre-B cell receptor. We next tested whether aberrant splicing also interferes with the tumor suppressor function of SLP65 in vivo: To this end, we engrafted SLP65−/−BCR-ABL1- driven ALL cells that were transduced with either SLP65-GFP, SLP65ΔSH2 or GFP into sublethally irradiated NOD/SCID mice. After 24 days, mice that were engrafted with ALL cells transduced with SLP65ΔSH2 or GFP became terminally leukemic, whereas mice injected with SLP65-transduced ALL cells showed no signs of disease. At this time, all mice were sacrificed and analyzed. Whereas mice with SLP65ΔSH2- or GFP-transduced ALL cells had enlarged spleens and substantial leukemic infiltration of the bone marrow (79 and 81% leukemia cell content), leukemic infitration was reduced if SLP65-transduced ALL cells were injected (1.8% leukemia infiltration). Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor.

scholarly journals Subcellular topography modulates actin dynamics and signaling in B-cells

2018 ◽  
Vol 29 (14) ◽  
pp. 1732-1742 ◽  
Author(s):  
Christina M. Ketchum ◽  
Xiaoyu Sun ◽  
Alexandra Suberi ◽  
John T. Fourkas ◽  
Wenxia Song ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Signaling ◽  
B Cell ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Calcium Oscillations ◽  
Actin Dynamics ◽  
B Cell Activation ◽  
B Cell Signaling

B-cell signaling activation is most effectively triggered by the binding of B-cell receptors (BCRs) to membrane-bound antigens. In vivo, B-cells encounter antigen on antigen-presenting cells (APC), which possess complex surfaces with convoluted topographies, a fluid membrane and deformable cell bodies. However, whether and how the physical properties of antigen presentation affect B-cell activation is not well understood. Here we use nanotopographic surfaces that allow systematic variation of geometric parameters to show that surface features on a subcellular scale influence B-cell signaling and actin dynamics. Parallel nanoridges with spacings of 3 microns or greater induce actin intensity oscillations on the ventral cell surface. Nanotopography-induced actin dynamics requires BCR signaling, actin polymerization, and myosin contractility. The topography of the stimulatory surface also modulates the distribution of BCR clusters in activated B-cells. Finally, B-cells stimulated on nanopatterned surfaces exhibit intracellular calcium oscillations with frequencies that depend on topography. Our results point to the importance of physical aspects of ligand presentation, in particular, nanotopography for B-cell activation and antigen gathering.

scholarly journals Igβ tyrosine residues contribute to the control of B cell receptor signaling by regulating receptor internalization

2006 ◽  
Vol 203 (7) ◽  
pp. 1785-1794 ◽  
Author(s):  
Anna Gazumyan ◽  
Amy Reichlin ◽  
Michel C. Nussenzweig
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
B Cell Receptor ◽  
Receptor Internalization ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
B1 Cells

Immunoglobulin (Ig)α and Igβ initiate B cell receptor (BCR) signaling through immune receptor tyrosine activation motifs (ITAMs) that are targets of SH2 domain–containing kinases. To examine the function of Igβ ITAM tyrosine resides in mature B cells in vivo, we exchanged these residues for alanine by gene targeting (IgβAA). Mutant mice showed normal development of all B cell subtypes with the exception of B1 cells that were reduced by fivefold. However, primary B cells purified from IgβAA mice showed significantly decreased steady-state and ligand-mediated BCR internalization and higher levels of cell surface IgM and IgD. BCR cross-linking resulted in decreased Src and Syk activation but paradoxically enhanced and prolonged BCR signaling, as measured by cellular tyrosine phosphorylation, Ca++ flux, AKT, and ERK activation. In addition, B cells with the ITAM mutant receptor showed an enhanced response to a T-independent antigen. Thus, Igβ ITAM tyrosines help set BCR signaling threshold by regulating receptor internalization.

scholarly journals Pre-Clinical Blocking of PD-L1 molecule, which expression is down regulated by NF-κB, JAK1/JAK2 and BTK inhibitors, induces regression of activated B-cell lymphoma

2018 ◽  
Author(s):  
Christelle Vincent-Fabert ◽  
Lilian Roland ◽  
Ursula Zimber-Strobl ◽  
Jean Feuillard ◽  
Nathalie Faumont
Keyword(s):  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Kinase Inhibitors ◽  
Tumor Regression ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
One Year ◽  
Inhibitory Molecules

AbstractEscape from immune control must be important in the natural course of B-cell lymphomas, especially for those with activation of NF-κB. The pre-clinical L.CD40 transgenic mouse model is characterized by B-cell specific CD40 signaling responsible for NF-κB continuous activation with a spleen monoclonal B-cell tumor after one year in 60% of cases. L.CD40 tumors B-cells expressed high levels of PD-L1. This expression was dependent on activation of either NF-κB, JAK1/JAK2 or BTK pathways since ex vivo treatment with the inhibitory molecules PHA-408, ruxolitinib and ibrutinib led to decrease of its expression. Treatment of L.CD40 lymphomatous mice with an anti-PD-L1 monoclonal antibody induced tumor regression with decreased spleen content, activation and proliferation rate of B-cells as well as a marked increase in T cell activation, as assessed by CD62L and CD44 expression. These results highlight the interest of therapies targeting the PD-1/PD-L1 axis in activated lymphomas with PD-L1 expression, with possible synergies with tyrosine kinase inhibitors.

scholarly journals Capture of plasma membrane fragments from target cells by trogocytosis requires signaling in T cells but not in B cells

Blood ◽  
2008 ◽  
Vol 111 (12) ◽  
pp. 5621-5628 ◽  
Author(s):  
Anne Aucher ◽  
Eddy Magdeleine ◽  
Etienne Joly ◽  
Denis Hudrisier
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Intracellular Signaling ◽  
Cell Biology ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Cell Receptor ◽  
Target Cells

Abstract Upon recognition of their respective cellular partners, T and B cells acquire their antigens by a process of membrane capture called trogocytosis. Here, we report that various inhibitors of actin polymerization or of kinases involved in intracellular signaling partially or fully inhibited trogocytosis by CD8+ and CD4+ T cells, whereas they had no effect on trogocytosis by B cells. Similarly, trogocytosis by T cells was inhibited at 4°C, whereas in B cells it was independent of temperature, indicating that trogocytosis by B cells does not rely on active processes. By contrast, most inhibitors we tested impaired both T-cell and B-cell activation. The differential effect of inhibitors on T-cell and B-cell trogocytosis was not due to the higher affinity of the B-cell receptor for its cognate antigen compared with the affinity of the T-cell receptor for its own antigen, but it correlated tightly with the abilities of T cells and B cells to form conjugates with their target cells in the presence of inhibitors. Trogocytosis thus has different requirements in different cell types. Moreover, the capture of membrane antigen by B cells is identified as a novel signaling-independent event of B-cell biology.

scholarly journals OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics

2018 ◽  
Vol 2018 ◽  
pp. 1-14 ◽  
Author(s):  
Kristien Van Belle ◽  
Jean Herman ◽  
Mark Waer ◽  
Ben Sprangers ◽  
Thierry Louat
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Cell Function ◽  
Ex Vivo ◽  
Cell Activity ◽  
Chemical Library ◽  
Activation Assay

B cells are pathogenic in various disease processes and therefore represent an interesting target for the development of novel immunosuppressants. In the search for new therapeutic molecules, we utilized an in vitro B cell activation assay with ODN2006-stimulated Namalwa cells to screen a chemical library of small molecules for B cell modulating effects. OSU-T315, described as an inhibitor of integrin-linked kinase (ILK), was hereby identified as a hit. On human and murine primary B cells, OSU-T315 potently suppressed the proliferation and the production of antibodies and cytokines upon stimulation, suggesting that ILK could be a promising target in the modulation of B cell activity. Mice with B cell-specific knockout of ILK were generated. Surprisingly, knockout of ILK in murine B cells did not affect B cell function as assessed by several in vivo and ex vivo B cell assays and did not alter the B cell immunosuppressive activity of OSU-T315. In conclusion, OSU-T315 displays potency as B cell modulator, probably through a mechanism of action independent of ILK, and might serve as lead drug molecule for the development of novel B cell-selective drugs.

scholarly journals B cells from patients with chronic GVHD are activated and primed for survival via BAFF-mediated pathways

Blood ◽  
2012 ◽  
Vol 120 (12) ◽  
pp. 2529-2536 ◽  
Author(s):  
Jessica L. Allen ◽  
Matthew S. Fore ◽  
Jenna Wooten ◽  
Philip A. Roehrs ◽  
Nazmim S. Bhuiya ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Ex Vivo ◽  
Cell Metabolism ◽  
Chronic Gvhd ◽  
B Cell Activation ◽  
Hematopoietic Stem ◽  
Survival Pathways ◽  
Therapeutic Development

Abstract Recent data reveal an important role for B cells in the pathogenesis of chronic GVHD (cGVHD). Patients with cGVHD have delayed B-cell reconstitution and elevated BAFF to B-cell ratios compared to patients without cGVHD. The mechanisms promoting and sustaining B-cell activation in this disease, however, remain unknown. As BAFF increases murine B-cell metabolism and survival and maintains autoreactive B-cell clones, we performed ex vivo analyses of peripheral B cells from 51 patients who either had or did not have active cGVHD and were greater than 1 year from the time of allogeneic hematopoietic stem cell transplantation. We found that B cells from patients with active cGVHD were in a heightened metabolic state and were resistant to apoptosis. Exogenous BAFF treatment amplified cell size and survival in B cells from these patients. We found significantly increased signaling through ERK and AKT that associated with decreased levels of proapoptotic Bim, suggesting a mechanistic link between elevated BAFF levels and aberrant B-cell survival. Thus, we identify a role for BAFF in the pathogenesis of cGVHD and define B-cell activation and survival pathways suitable for novel therapeutic development in cGVHD.

scholarly journals Innate stimulation of B cells ex vivo enhances antibody secretion and identifies tumour-reactive antibodies from cancer patients

2021 ◽  
Author(s):  
Panagiotis Karagiannis ◽  
Isabel Correa ◽  
Jitesh Chauhan ◽  
Anthony Cheung ◽  
Diana Dominguez-Rodriguez ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Antibody Production ◽  
Cell Activation ◽  
Ex Vivo ◽  
Human B Cells ◽  
B Cell Survival ◽  
B Cell Precursors ◽  
Stimulation Of

Abstract Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognising cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B cell activation factor (BAFF) and the toll like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation and antibody production. Combined IL-17+BAFF+CpG prolonged B cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19 +CD20 +CD27 +IgD - B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha (FOLR)) led to secreted antibodies recognising the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B cell precursors (CD27 -CD38 -IgD -), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses.

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