ARPC1B binds WASP to control actin polymerization and curtail tonic signaling in B cells

Abstract CXCL13 is a homeostatic chemokine for lymphocyte homing and positioning within follicles of secondary lymphoid tissues, acting through its cognate receptor, CXCR5. Moreover, the CXCR5-CXCL13 axis plays a unique role in trafficking and homing of B1 cells. Here, we report that chronic lymphocytic leukemia (CLL) B cells express high levels of functional CXCR5. CXCR5 expression levels were similar on CLL B cells and normal CD5+ B cells, and higher compared with normal CD5− B cells, follicular B-helper T cells (TFH cells), or neoplastic B cells from other B-cell neoplasias. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis, and prolonged activation of p44/42 mitogen-activated protein kinases. Anti-CXCR5 antibodies, pertussis toxin, and wortmannin inhibited chemotaxis to CXCL13, demonstrating the importance of Gi proteins and PI3 kinases for CXCR5 signaling. Moreover, CLL patients had significantly higher CXCL13 serum levels than volunteers, and CXCL13 levels correlated with β2 microglobulin. We detected CXCL13 mRNA expression by nurselike cells, and high levels of CXCL13 protein in supernatants of CLL nurselike cell cultures. By immunohistochemistry, we detected CXCL13+ expression by CD68+ macrophages in situ within CLL lymph nodes. These data suggest that CXCR5 plays a role in CLL cell positioning and cognate interactions between CLL and CXCL13-secreting CD68+ accessory cells in lymphoid tissues.

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CD20 is dispensable for B-cell receptor signaling but is required for proper actin polymerization, adhesion and migration of malignant B cells

PLoS ONE ◽

10.1371/journal.pone.0229170 ◽

2020 ◽

Vol 15 (3) ◽

pp. e0229170

Author(s):

Veronika Kozlova ◽

Aneta Ledererova ◽

Adriana Ladungova ◽

Helena Peschelova ◽

Pavlina Janovska ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Actin Polymerization ◽

Cell Receptor ◽

B Cell Receptor ◽

Receptor Signaling ◽

B Cell Receptor Signaling ◽

And Migration ◽

Cell Receptor Signaling

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Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v94.11.3658.423k11_3658_3667 ◽

1999 ◽

Vol 94 (11) ◽

pp. 3658-3667 ◽

Cited By ~ 6

Author(s):

Jan A. Burger ◽

Meike Burger ◽

Thomas J. Kipps

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Pertussis Toxin ◽

Stromal Cells ◽

Stromal Cell ◽

Actin Polymerization ◽

Kinase Inhibitor ◽

Critical Role ◽

Lymphocytic Leukemia ◽

Marrow Stromal Cells

Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

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Expression of the chemokine receptor CCR2 on immature B cells negatively regulates their cytoskeletal rearrangement and migration

Blood ◽

10.1182/blood-2003-11-4013 ◽

2004 ◽

Vol 104 (4) ◽

pp. 933-941 ◽

Cited By ~ 28

Author(s):

Liat Flaishon ◽

Shirly Becker-Herman ◽

Gili Hart ◽

Yoram Levo ◽

William A. Kuziel ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

Chemokine Receptor ◽

Actin Polymerization ◽

Signaling Cascade ◽

Mature Stage ◽

Negative Regulators ◽

Immature B Cells ◽

And Migration ◽

Chemokine Receptor Ccr2

AbstractImmature B cells are targeted to specific areas in the spleen, where a fraction of these cells receive signals that induce them to mature and participate in the immune response. In this study, we show that the C-C chemokine receptor 2 (CCR2) is transcribed in immature B cells, while its message is dramatically down-regulated at the mature stage. CCR2-deficient cells exhibit up-regulation of chemokine-induced actin polymerization, migration, and homing to the lymph nodes of immature B cells. In addition, we demonstrate that control of homing by CCR2 is mediated by its ligand, CCL2/JE, which is secreted by B cells and down-regulates the stromal derived factor-1 (SDF-1) signaling cascade. Thus, this study describes an additional, previously uncharacterized, role for CCR2 and its ligand as negative regulators of the homing of immature B cells.

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WASp Is Crucial for the Unique Architecture of the Immunological Synapse in Germinal Center B-Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.646077 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yanan Li ◽

Anshuman Bhanja ◽

Arpita Upadhyaya ◽

Xiaodong Zhao ◽

Wenxia Song

Keyword(s):

B Cells ◽

B Cell ◽

Lipid Bilayers ◽

Germinal Center ◽

Actin Polymerization ◽

Immunological Synapse ◽

Somatic Hypermutation ◽

Affinity Maturation ◽

Membrane Protrusions ◽

Germinal Center B Cells

B-cells undergo somatic hypermutation and affinity maturation in germinal centers. Somatic hypermutated germinal center B-cells (GCBs) compete to engage with and capture antigens on follicular dendritic cells. Recent studies show that when encountering membrane antigens, GCBs generate actin-rich pod-like structures with B-cell receptor (BCR) microclusters to facilitate affinity discrimination. While deficiencies in actin regulators, including the Wiskott-Aldrich syndrome protein (WASp), cause B-cell affinity maturation defects, the mechanism by which actin regulates BCR signaling in GBCs is not fully understood. Using WASp knockout (WKO) mice that express Lifeact-GFP and live-cell total internal reflection fluorescence imaging, this study examined the role of WASp-mediated branched actin polymerization in the GCB immunological synapse. After rapid spreading on antigen-coated planar lipid bilayers, GCBs formed microclusters of phosphorylated BCRs and proximal signaling molecules at the center and the outer edge of the contact zone. The centralized signaling clusters localized at actin-rich GCB membrane protrusions. WKO reduced the centralized micro-signaling clusters by decreasing the number and stability of F-actin foci supporting GCB membrane protrusions. The actin structures that support the spreading membrane also appeared less frequently and regularly in WKO than in WT GCBs, which led to reductions in both the level and rate of GCB spreading and antigen gathering. Our results reveal essential roles for WASp in the generation and maintenance of unique structures for GCB immunological synapses.

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The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.649433 ◽

2021 ◽

Vol 9 ◽

Author(s):

Madison Bolger-Munro ◽

Kate Choi ◽

Faith Cheung ◽

Yi Tian Liu ◽

May Dang-Lawson ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Actin Filaments ◽

Actin Polymerization ◽

Cell Spreading ◽

B Cell Antigen Receptor ◽

Actin Remodeling ◽

Immune Synapse ◽

Cell Antigen ◽

Bcr Signaling

When B cells encounter membrane-bound antigens, the formation and coalescence of B cell antigen receptor (BCR) microclusters amplifies BCR signaling. The ability of B cells to probe the surface of antigen-presenting cells (APCs) and respond to APC-bound antigens requires remodeling of the actin cytoskeleton. Initial BCR signaling stimulates actin-related protein (Arp) 2/3 complex-dependent actin polymerization, which drives B cell spreading as well as the centripetal movement and coalescence of BCR microclusters at the B cell-APC synapse. Sustained actin polymerization depends on concomitant actin filament depolymerization, which enables the recycling of actin monomers and Arp2/3 complexes. Cofilin-mediated severing of actin filaments is a rate-limiting step in the morphological changes that occur during immune synapse formation. Hence, regulators of cofilin activity such as WD repeat-containing protein 1 (Wdr1), LIM domain kinase (LIMK), and coactosin-like 1 (Cotl1) may also be essential for actin-dependent processes in B cells. Wdr1 enhances cofilin-mediated actin disassembly. Conversely, Cotl1 competes with cofilin for binding to actin and LIMK phosphorylates cofilin and prevents it from binding to actin filaments. We now show that Wdr1 and LIMK have distinct roles in BCR-induced assembly of the peripheral actin structures that drive B cell spreading, and that cofilin, Wdr1, and LIMK all contribute to the actin-dependent amplification of BCR signaling at the immune synapse. Depleting Cotl1 had no effect on these processes. Thus, the Wdr1-LIMK-cofilin axis is critical for BCR-induced actin remodeling and for B cell responses to APC-bound antigens.

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Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v94.11.3658 ◽

1999 ◽

Vol 94 (11) ◽

pp. 3658-3667 ◽

Cited By ~ 338

Author(s):

Jan A. Burger ◽

Meike Burger ◽

Thomas J. Kipps

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Pertussis Toxin ◽

Stromal Cells ◽

Stromal Cell ◽

Actin Polymerization ◽

Kinase Inhibitor ◽

Critical Role ◽

Lymphocytic Leukemia ◽

Marrow Stromal Cells

Abstract Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

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Activation of alpha-2-adrenoceptors results in an increase in F-actin formation in HIT-T15 pancreatic B-cells

Biochemical Journal ◽

10.1042/bj3070169 ◽

1995 ◽

Vol 307 (1) ◽

pp. 169-174 ◽

Cited By ~ 16

Author(s):

H C Cable ◽

A el-Mansoury ◽

N G Morgan

Keyword(s):

Plasma Membrane ◽

Insulin Secretion ◽

B Cells ◽

Fluorescence Microscopy ◽

Pertussis Toxin ◽

Actin Polymerization ◽

Secretory Granules ◽

Rhodamine Phalloidin ◽

Late Step ◽

Pancreatic B Cells

1. Alpha-2-adrenoceptor agonists, such as noradrenaline, are potent inhibitors of insulin secretion, and it has been suggested that they control a late step in the pathway of exocytosis. We have investigated whether this could be related to a change in the extent of actin polymerization in the pancreatic B-cell, since actin microfilaments are implicated in regulating the access of secretory granules to the plasma membrane prior to exocytosis. 2. Cultured HIT-T15 pancreatic B-cells responded to noradrenaline with an increase in F-actin content, as judged by a rise in the fluorescence output after probing of the cells with phalloidin (a toxin which binds specifically to F-actin) conjugated to rhodamine. The response to noradrenaline was rapid, dose-dependent and sustained and could be reproduced by the highly selective alpha-2-agonist UK14,304. Examination of HIT-T15 cells by fluorescence microscopy after treatment with rhodamine-phalloidin, revealed a significant localization of F-actin immediately adjacent to the plasma membrane. The pattern of F-actin distribution in the cells was not altered dramatically by noradrenaline, although the intensity of staining close to the plasma membrane appeared to be slightly reduced. 3. The increase in F-actin content induced by noradrenaline and UK14,304 was inhibited significantly by the alpha-2-antagonist idazoxan but not by the alpha-1-selective antagonist prazosin. Pretreatment of HIT-T15 cells with pertussis toxin did not lead to any direct alteration in F-actin content, although the toxin significantly modified the responses induced by noradrenaline and UK14,304. In each case, cells incubated for 24 h with pertussis toxin responded to the alpha-2-agonist with an enhanced fluorescence output, indicating that F-actin levels had increased still further. This did not correlate with any gross change in the distribution of F-actin as judged by fluorescence microscopy. 4. The results demonstrate that alpha-2-adrenoceptors are coupled to control of actin polymerization in HIT-T15 cells. They suggest that regulation of F-actin formation could be a component of the mechanism by which alpha-2-agonists mediate inhibition of insulin secretion.

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Compared to Adult Peripheral Blood T Cells, Cord Blood T Cells Show Enhanced Immunological Recognition of Chronic Lymphocytic Leukemia Tumor Cells.

Blood ◽

10.1182/blood.v112.11.2333.2333 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2333-2333

Author(s):

Alan G. Ramsay ◽

Dong-Xia Xing ◽

William K. Decker ◽

Jared K. Burks ◽

William G. Wierda ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cord Blood ◽

Peripheral Blood ◽

Actin Polymerization ◽

Synapse Formation ◽

Immunological Synapse ◽

Lymphocytic Leukemia ◽

Adult Peripheral Blood

Abstract Following allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion (DLI) from adult peripheral blood (APB), chronic lymphocytic leukemia (CLL) cells are good targets of a graft-versus-leukemia effect. However, some patients eligible for this treatment do not have a suitable allogeneic donor and CLL B cells have been shown to be dysfunctional antigen-presenting cells (APCs) for allogeneic APB T cells. As a result, allogeneic APB T cells show suppressed immunological synapse formation with CLL cells. Umbilical cord blood (CB) is a promising source of hematopoietic cells for allogeneic transplantation and can be obtained from matched unrelated donors with greater tolerance for incompletely HLA-matched recipients. Moreover, we have successfully expanded CB T cells ex vivo (anti-CD3/CD28 beads and rIL-2) using a protocol that retains a naïve and diverse immune population including central memory cells. In this present study we used confocal microscopy to visualize F-actin polymerization to assess immunological synapse formation of CB T cells compared to APB T cells with CLL B cells with and without superantigen as APCs. Our results identify the ability of unexpanded and expanded CB CD4 and CD8 T cells to form F-actin immune synapses with CLL B cells and of note, CB was more effective than unexpanded or expanded APB T cells (p<0.05). Of interest, the expansion protocol maintained immune synapse formation with a trend towards increased F-actin polymerization. As control, we examined the ability of unexpanded and expanded T cells to form F-actin synapses with allogeneic healthy B cells with or without superantigen as APCs and found no significant difference between CB and APB as a source of T cells. Our results demonstrate that CB T cells have an enhanced ability to recognize CLL B cells as allogeneic APCs compared to APB T cells and provide important and exciting pre-clinical data for the potential use of expanded CB T cells in the setting of CB transplantation in CLL.

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Visualization of Negative Signaling in B Cells by Quantitative Confocal Microscopy

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8615-8625.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8615-8625 ◽

Cited By ~ 42

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.

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