scholarly journals Induction of In Vivo Antipolysaccharide Immunoglobulin Responses to Intact Streptococcus pneumoniae Is More Heavily Dependent on Btk-Mediated B-Cell Receptor Signaling than Antiprotein Responses

2006 ◽  
Vol 74 (2) ◽  
pp. 1419-1424 ◽  
Author(s):  
Abdul Q. Khan ◽  
Goutam Sen ◽  
Shuling Guo ◽  
Owen N. Witte ◽  
Clifford M. Snapper
Keyword(s):  
Streptococcus Pneumoniae ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Relative Role ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Extracellular Bacterium

ABSTRACT The relative role of Btk-dependent B-cell receptor (BCR) signaling in the induction of antipolysaccharide (anti-PS) and antiprotein immunoglobulin (Ig) responses to an intact extracellular bacterium in vivo is unknown. Btklow mice exhibit reduced BCR signaling but largely restore B-cell development. Btklow mice immunized with intact Streptococcus pneumoniae elicit reduced anti-PS but normal antiprotein Ig responses. Immunization of Btklow mice with PS-protein conjugate in saline results in an even more profound defect in the anti-PS but not antiprotein response, which is largely restored by use of a CpG-containing oligodeoxynucleotide as an adjuvant. These data demonstrate a greater dependence on Btk-mediated BCR signaling for physiologic anti-PS relative to antiprotein responses, as well as the existence of a compensatory Toll-like-receptor-mediated signaling pathway naturally triggered in response to intact bacterial pathogens.

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.

High ZAP-70 and Differential Expression of B-Cell Receptor Related Genes in Chronic Lymphocytic Leukemia with V3-21 Gene Usage.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 773-773
Author(s):  
Dirk Kienle ◽  
Alexander Kröber ◽  
Dirk Winkler ◽  
Daniel Mertens ◽  
Annett Habermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mutation Status ◽  
Bcr Signaling ◽  
Gene Usage ◽  
Lower Expression

Abstract V3-21 gene usage defines a distinct genetic subgroup of chronic lymphocytic leukemia (CLL) characterized by a poor clinical outcome regardless of the VH mutation status. V3-21 cases exhibit a highly characteristic B-cell receptor (BCR) structure as demonstrated by homologous CDR3 sequences and a restricted use of VL genes implicating a common antigen involved in tumor pathogenesis of this specific CLL subgroup. To investigate the role of antigenic stimulation in the pathogenesis of V3-21 using CLL, we analyzed the quantitative expression of genes involved in BCR signaling (ZAP-70, SYK, BLNK, LYN, PI3K, PLCG2, FOS), B-cell activation (TRAF3, STAT6, NFKB), and cell cycle or apoptosis control (ATM, BCL-2, BAX, CDK4, CCND1, CCND2, CCND3, p27, E2F1, MYC) in V3-21 cases in comparison to VH mutated (VH MUT) and VH unmutated (VH UM) cases not using the V3-21 gene. To obtain native expression signatures we studied a non-CD19-purified (nPU) cohort (V3-21: 18 cases, equally divided into VH mutated and VH unmutated cases; VH MUT: 17; VH UM: 19) and, for verification, a CD19-purified (PU) cohort (V3-21: 10 cases, equally divided into VH mutated and unmutated; VH MUT: 12; VH UM: 16) to exclude a contamination of the results by non-tumor cells. All cases were analyzed by FISH for +3q, 6q-, +8q, 11q-, +12q, 13q-, 17p-, and t(11;14) to avoid major imbalances of genomic alterations between the subgroups under study. As expected, ZAP-70 expression was higher in VH UM as compared to VH MUT cases in the nPU (p=0.007) as well as the PU cohort (p=0.009). V3-21 cases showed a higher ZAP-70 expression as compared to VH MUT (nPU: p=0.033; PU: p=0.038). This applied also when restricting this comparison to V3-21 mutated cases (nPU: p=0.018). Median ZAP-70 expression in the PU cohort was 1.15 in VH MUT vs. 7.69 in VH UM cases, as compared to 7.05 in V3-21 cases (V3-21 mutated cases: 10.69; V3-21 unmutated: 6.7). Other genes differentially expressed between the V3-21 and VH MUT subgroups in nPU cases were PI3K (p=0.048), PLCG2 (p=0.007), CCND2 (p=0.003), p27 (p=0.003), BCL-2 (p=0.025), and ATM (p=0.006). In addition, a set of genes was detected with a differential expression between V3-21 and VH UM (nPU) including PLCG2 (p=0.014), NFKB (p=0.023), CCND2 (p=0.001), p27 (0.002), and BAX (p=0.028). Notably, except for ZAP-70, all of the differentially expressed genes showed a lower expression in V3-21 as compared to the other subgroups. When comparing the V3-21 mutated and V3-21 unmutated subgroups (nPU), there were no significant gene expression differences except for CDK4, which showed a lower expression in V3-21 unmutated cases. Therefore, cases with V3-21 usage appear to show a rather homogeneous gene expression pattern independently of the VH mutation status, which can be distinguished from VH MUT and VH UM cases not using V3-21. The expression differences observed suggest a role of differential BCR signaling in the pathogenesis of this distinct CLL subgroup. Deregulation of cell cycle, apoptosis, and candidate genes such as ATM indicate the involvement of additional pathways in the pathogenesis of CLL cases using V3-21.

Self-Enforcing Feedback Activation Between BCL6 and Tonic Pre-B Cell Receptor Signaling in Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 284-284
Author(s):  
Huimin Geng ◽  
Christian Hurtz ◽  
Dirk Baumjohann ◽  
Zhengshan Chen ◽  
Wei-Yi Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Tyrosine Kinases ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Bcr Signaling ◽  
Btk Inhibitor ◽  
Bcl6 Expression ◽  
Targeting Strategy

Abstract Background and hypothesis: Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival signals emanating from a functional (pre-) B cell receptor (BCR). While recent work successfully introduced BCR signaling inhibitors into patient care for various subtypes of mature B cell lymphoma, it is not known whether pre-BCR signaling represents a therapeutic target in pre-B ALL and in which cytogenetic subsets targeting of pre-BCR signaling will be effective. In this study we demonstrated that ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR signalling. We identified a novel mechanism of self-enforcing feedback activation between the transcription factor BCL6 and tonic pre-BCR signaling in pre-BCR+ ALL and proposed a dual targeting strategy of both BCL6 and pre-BCR related tyrosine kinases for the treatment of patients with pre-BCR+ ALL. Results: Flow cytometry analysis of surface pre-BCR expression (λ5, VpreB), cytoplasmic μ heavy chain (μHC) expression and intracellular Ca2+ signal in 29 patient-derived pre-B ALL xenograft samples and cell lines showed pre-BCR expression and activity in a subset of pre-B ALL, including all TCF3-PBX1 cases studied (n=4) and two cases with deletions at 6q21. Studying 830 pre-B ALL cases from four clinical trials (MDACC, St. Jude, COG P9906 and ECOG E2993), tonic pre-BCR signaling and constitutive PI3K-AKT activation was found in 112 cases (13.5%), including 93% TCF3-PBX1 (53 of 57), del (6)(q21) (7 of 7), PBX1 (1q23) duplication (4 of 4), MLL-rearrangement (3 of 86), hyperdiploid (2 of 43) and other (43 of 406) pre-B ALL cases. In other major ALL subtypes, we found no evidence for pre-BCR expression and activity, including BCR-ABL1 (0 of 196) and ETV6-RUNX1 (0 of 31). We found frequent 1q23 (PBX1) duplication, TCF3-PBX1 or other PBX1-rearrangement, 6q21 (PRDM1) deletion in ALL cells with tonic pre-BCR signaling. Development of a genetic mouse model for inducible ablation of Bcl6. Pre-BCR-induced activation of BCL6 relieves PRDM1-mediated repression of pre-BCR signaling components and positively regulates pre-BCR signaling output at the transcriptional level. The clinical data (COG P9906, ECOG E2993) revealed that high mRNA levels of BCL6 at the time of diagnosis is predictive of poor clinical outcome specifically in patients with pre-BCR+ ALL but not ALL cells lacking pre-BCR expression. These findings suggest an important role of BCL6 as a cofactor of pre-BCR signaling in a large subset of ALL. To directly test the role of Bcl6- and pre-BCR interactions, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Deletion of Bcl6 in mouse pre-BCR+ ALL and expression of a dominate-negative form of BCL6 in human primary pre-BCR+ALL cells, both rapidly induced cell death, indicating BCL6 cooperates with the pre-BCR in leukemic transformation. Cooperation between pre-BCR and BCL6 signaling. Inhibition of BCL6 via the specific BCL6 inhibitor RI-BPI showed compromised colony formation and induced cell cycle arrest. Interestingly, constitutive BCL6 expression was sensitive to inhibition of SYK and SRC tyrosine kinases downstream of the pre-BCR. Treating 6 pre-BCR+ and 8 pre-BCR- patient-derived ALL samples with the SYK inhibitor (PRT06207), BTK inhibitor (Ibrutinib) or a broader SRC and BTK inhibitor Dasatinib, we observed remarkably decreased BCL6 expression and increased apoptosis in pre-BCR+ but not pre-BCR- ALL cells. In vivo treatments with Dasatinib prevented leukemia initiation and significantly prolonged survival of the recipient mice that were injected with primary pre-BCR+ ALL cells, compared to non-treatment or Nilotinib-treatment. These data demonstrate that both inhibition of BCL6 and pre-BCR signaling selectively killed patient-derived pre-BCR+ ALL cells. Conclusions: Our study identified two distinct subtypes of pre-B ALL that fundamentally differ with respect to pre-BCR signaling. Tonic pre-BCR signaling engages a BCL6-dependent, self-enforcing amplification loop. Based on these findings, we propose a dual targeting strategy of BCL6 and pre-BCR tyrosine kinases for the treatment of patients with pre-BCR+ALL. Disclosures No relevant conflicts of interest to declare.

An HSP90 Inhibitor, PU-H71, Antagonizes Stroma-Induced Pro-Survival Effects in CLL through Its Inhibition of Multi-Component B-Cell Receptor Signaling Pathway

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5289-5289
Author(s):  
Ailin Guo ◽  
Pin Lu ◽  
Chaojie Zhen ◽  
Gabriela Chiosis ◽  
Yue Lynn Wang
Keyword(s):  
B Cell ◽  
Signaling Pathway ◽  
Cell Receptor ◽  
Hsp90 Inhibitor ◽  
B Cell Receptor ◽  
Receptor Signaling ◽  
Bcr Signaling ◽  
Receptor Signaling Pathway ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

Abstract Background: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B-cells in the hematopoietic system. The B-cell receptor (BCR) plays an essential role in the pathogenesis of CLL and many components of the BCR signaling pathway are known clients of HSP90. HSP90 is a highly conserved molecular chaperone that ensures the proper folding and stabilization of its client proteins. In this study, we investigated whether PU-H71 a novel purine-scaffold HSP90 Inhibitor, has anti-tumor activity in CLL by destabilizing BCR signaling pathway constituents. Design: Fresh CLL cells were isolated and cultured ex vivo with or without stromal co-culture. Molecular and cellular events were studied in PU-H71-treated and control CLL cells. Results: Immunoblotting revealed that a significantly higher amount of HSP90 is present in CLL cells than in peripheral blood mononuclear cells (PBMC), suggesting the chaperone is pathogenically relevant. We found that PU-H71 caused the death of CLL cells in a dose and time dependent manner while the viability of either PBMC or normal B lymphocytes were not affected. PU-H71 induced apoptosis resulting in CLL cell death as it caused mitochondrial cytochrome C release and a decrease in the abundance of several anti-apoptotic proteins. Interestingly, PU-H71 has the ability to counteract the pro-survival effects of the stroma and caused apoptosis in CLL cells co-cultured with stroma. To gain mechanistic insights into how PU-H71 acts, we examined the BCR signaling pathway. We found that the amounts of several key components of the pathway were reduced by PU-H71 treatment. This occurred even in the presence of stromal co-culture. The results suggest that PU-H71 antagonizes the function of HSP90 leading to the destabilization of the BCR signaling transducers. A chemical pull-down experiment revealed the co-existence of the BCR components and HSP90 in the same complex, suggesting these BCR constituents are indeed clients of HSP90 in CLL cells. Further, specific genetic knock-down of the signal transducers by siRNA confirmed their key roles in mediating the survival of CLL cells. Conclusions: PU-H71 antagonizes stroma-induced pro-survival effects in CLL through its inhibition of the B-cell receptor signaling pathway. Our results suggest that PU-H71 may serve as a useful therapy against CLL and is worth further clinical development. Disclosures No relevant conflicts of interest to declare.

BTK Inhibition Targets in Vivo CLL Proliferation Through Its Effects On B-Cell Receptor Signaling Activity.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2903-2903
Author(s):  
Y. Lynn Wang ◽  
Shuhua Cheng ◽  
Jiao Ma ◽  
Ailin Guo ◽  
Pin Lu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Ex Vivo ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
B Cell Receptor Signaling ◽  
Serial Samples ◽  
Cell Receptor Signaling

Abstract Abstract 2903 Purpose: Bruton tyrosine kinase (BTK) is a component of the B-cell receptor signaling pathway. Ibrutinib (previously known as PCI-32765), a first in class, covalent BTK inhibitor, has demonstrated significant clinical activity against CLL in early clinical trials. Understanding the molecular mechanisms of action would shed light on CLL pathophysiology and provide additional opportunities for the development of new therapies. Experimental Design: The anti-tumor activity of ibrutinib in CLL has been investigated previously using either an ex vivo approach or a mouse model (Herman et.al, Blood. 2011;117:6287–96 and Ponader et.al, Blood. 2012;119:1182–9). In this study, we have chosen, instead, a patient-oriented in vivo approach by using samples from an ongoing phase 1b trial of ibrutinib (NCT01105247). We prospectively collected serial samples from CLL patients (n=14) before and at several time points after the initiation of therapy and analyzed them for cellular and molecular signaling events. Results: We demonstrated that levels of the phosphorylated BTK protein (p-BTK) in CLL cells from treatment-naïve patients were significantly higher than in normal B cells, explaining why CLL cells are more susceptible to BCR inhibition than normal B cells. Response assessments, performed at the end of cycle 2 (∼Day 56), demonstrated nodal responses in all patients by CT scan. Ex vivo apoptosis did occur but required high concentrations of ibrutinib (>500 nM). In addition, in vivo apoptosis was rarely observed in serial peripheral blood samples collected from treated patients. With these serial samples, we found that the population of Ki67+ cells were gradually decreased over a 28-day ibrutinib treatment course. Using a newly established co-culture system that induces CLL proliferation in vitro, the analysis of several parameters, including Ki-67 expression, cell growth and bromodeoxyuridine (BrdU) incorporation (shown in the figure), revealed that the proliferation of CLL cells was directly inhibited by ibrutinib (200 nM). Furthermore, activities of BTK and downstream signaling events, such as the phosphorylation of PLCg2, AKT and ERK, were all suppressed over time in ibrutinib-treated patients. Conclusions: With primarily an in vivo approach, we have demonstrated that the blockage of cell proliferation was a major effect of ibrutinib against leukemic CLL cells. Blocking cell proliferation via inhibition of BTK-mediated signaling concurs with clinical responses in ibrutinib-treated CLL patients. Disclosures: Leonard: Pharmacyclics Inc.: Consultancy, Honoraria. Buggy:Pharmacyclics: Employment, Equity Ownership.

scholarly journals Phospho-proteomic analysis of mantle cell lymphoma cells suggests a pro-survival role of B-cell receptor signaling

2011 ◽  
Vol 34 (2) ◽  
pp. 141-153 ◽  
Author(s):  
Chiara Pighi ◽  
Ting-Lei Gu ◽  
Irene Dalai ◽  
Stefano Barbi ◽  
Claudia Parolini ◽  
...  
Keyword(s):  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Proteomic Analysis ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Mantle Cell ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

scholarly journals Interplay between HGAL and Grb2 proteins regulates B-cell receptor signaling

2019 ◽  
Vol 3 (15) ◽  
pp. 2286-2297
Author(s):  
Xiaoyu Jiang ◽  
Xiaoqing Lu ◽  
Yu Zhang ◽  
Leda Lacaria ◽  
Brett J. Schuchardt ◽  
...  
Keyword(s):  
B Cell ◽  
Binding Site ◽  
Synapse Formation ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Putative Binding Site

Abstract Human germinal center (GC)–associated lymphoma (HGAL) is an adaptor protein expressed in GC B cells. HGAL regulates cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. Herein, we demonstrate phosphorylation of HGAL by Syk and Lyn kinases at tyrosines Y80, Y86, Y106Y107, Y128, and Y148. The HGAL YEN motif (amino acids 107-109) is similar to the phosphopeptide motif pYXN used as a binding site to the growth factor receptor–bound protein 2 (Grb2). We demonstrate by biochemical and molecular methodologies that HGAL directly interacts with Grb2. Concordantly, microscopy studies demonstrate HGAL-Grb2 colocalization in the membrane central supramolecular activation clusters (cSMAC) following BCR activation. Mutation of the HGAL putative binding site to Grb2 abrogates the interaction between these proteins. Further, this HGAL mutant localizes exclusively in the peripheral SMAC and decreases the rate and intensity of BCR accumulation in the cSMAC. Furthermore, we demonstrate that Grb2, HGAL, and Syk interact in the same complex, but Grb2 does not modulate the effects of HGAL on Syk kinase activity. Overall, the interplay between the HGAL and Grb2 regulates the magnitude of BCR signaling and synapse formation.

The Syk inhibitor fostamatinib disodium (R788) inhibits tumor growth in the Eμ- TCL1 transgenic mouse model of CLL by blocking antigen-dependent B-cell receptor signaling

Blood ◽  
2010 ◽  
Vol 116 (23) ◽  
pp. 4894-4905 ◽  
Author(s):  
Mirza Suljagic ◽  
Pablo G. Longo ◽  
Sara Bennardo ◽  
Emerald Perlas ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
Mouse Model ◽  
B Cell ◽  
Transgenic Mouse ◽  
Cell Receptor ◽  
Transgenic Mouse Model ◽  
B Cell Receptor ◽  
Promising Therapeutic Approach ◽  
Bcr Signaling ◽  
Syk Inhibitor

Abstract Inhibition of antigen-dependent B-cell receptor (BCR) signaling is considered a promising therapeutic approach in chronic lymphocytic leukemia (CLL), but experimental in vivo evidence to support this view is still lacking. We have now investigated whether inhibition of BCR signaling with the selective Syk inhibitor fostamatinib disodium (R788) will affect the growth of the leukemias that develop in the Eμ-TCL1 transgenic mouse model of CLL. Similarly to human CLL, these leukemias express stereotyped BCRs that react with autoantigens exposed on the surface of senescent or apoptotic cells, suggesting that they are antigen driven. We show that R788 effectively inhibits BCR signaling in vivo, resulting in reduced proliferation and survival of the malignant B cells and significantly prolonged survival of the treated animals. The growth-inhibitory effect of R788 occurs despite the relatively modest cytotoxic effect in vitro and is independent of basal Syk activity, suggesting that R788 functions primarily by inhibiting antigen-dependent BCR signals. Importantly, the effect of R788 was found to be selective for the malignant clones, as no disturbance in the production of normal B lymphocytes was observed. Collectively, these data provide further rationale for clinical trials with R788 in CLL and establish the BCR-signaling pathway as an important therapeutic target in this disease.

scholarly journals A B Cell Receptor with Two Igα Cytoplasmic Domains Supports Development of Mature But Anergic B Cells

2004 ◽  
Vol 199 (6) ◽  
pp. 855-865 ◽  
Author(s):  
Amy Reichlin ◽  
Anna Gazumyan ◽  
Hitoshi Nagaoka ◽  
Kathrin H. Kirsch ◽  
Manfred Kraus ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cytoplasmic Domain ◽  
B Cell Receptor ◽  
B1a Cells ◽  
Bcr Signaling ◽  
Membrane Bound

B cell receptor (BCR) signaling is mediated through immunoglobulin (Ig)α and Igβ a membrane-bound heterodimer. Igα and Igβ are redundant in their ability to support early B cell development, but their roles in mature B cells have not been defined. To examine the function of Igα–Igβ in mature B cells in vivo we exchanged the cytoplasmic domain of Igα for the cytoplasmic domain of Igβ by gene targeting (Igβc→αc mice). Igβc→αc B cells had lower levels of surface IgM and higher levels of BCR internalization than wild-type B cells. The mutant B cells were able to complete all stages of development and were long lived, but failed to differentiate into B1a cells. In addition, Igβc→αc B cells showed decreased proliferative and Ca2+ responses to BCR stimulation in vitro, and were anergic to T-independent and -dependent antigens in vivo.

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