B Cell Antigen Receptor Stimulation Research Articles

B cell receptor-induced IL-10 production from neonatal mouse CD19+CD43- cells depends on STAT5-mediated IL-6 secretion.

Newborns are unable to reach the adult-level humoral immune response partly due to the potent immunoregulatory role of IL-10. Increased IL-10 production by neonatal B cells has been attributed to the larger population of IL-10-producting CD43+ B-1 cells in neonates. Here, we show that neonatal mouse CD43- non-B-1 cells also produce substantial amounts of IL-10 following B cell antigen receptor (BCR) activation. In neonatal mouse CD43- non-B-1 cells, BCR engagement activated STAT5 under the control of phosphorylated forms of signaling molecules Syk, Btk, PKC, FAK, and Rac1. Neonatal STAT5 activation led to IL-6 production, which in turn was responsible for IL-10 production in an autocrine/paracrine fashion through the activation of STAT3. In addition to the increased IL-6 production in response to BCR stimulation, elevated expression of IL-6Rα expression in neonatal B cells rendered them highly susceptible to IL-6-mediated STAT3 phosphorylation and IL-10 production. Finally, IL-10 secreted from neonatal mouse CD43- non-B-1 cells was sufficient to inhibit TNF-α secretion by macrophages. Our results unveil a distinct mechanism of IL-6-dependent IL-10 production in BCR-stimulated neonatal CD19+CD43- B cells.

TNF receptor-associated factor 3 restrains B-cell receptor signaling in normal and malignant B cells

TRAF3 has diverse signaling functions, which vary by cell type. Uniquely in B lymphocytes, TRAF3 inhibits homeostatic survival. Highlighting the role of TRAF3 as a tumor suppressor, loss-of-function TRAF3 mutations are associated with human B-cell malignancies, while B-cell-specific deletion of TRAF3 in mice leads to autoimmunity and lymphoma development. The role of TRAF3 in inhibiting noncanonical NF-κB activation, CD40 and BAFF-R signaling to B cells is well documented. In contrast, TRAF3 enhances many T-cell effector functions, through associating with and enhancing signaling by the T-cell receptor (TCR)-CD28 complex. The present study was designed to determine the role of TRAF3 in signaling via the B-cell antigen receptor (BCR). The BCR is crucial for antigen recognition, survival, proliferation, and antibody production, and defects in BCR signaling can promote abnormal survival of malignant B cells. Here, we show that TRAF3 is associated with both CD79B and the BCR-activated kinases Syk and Btk following BCR stimulation. BCR-induced phosphorylation of Syk and additional downstream kinases was increased in TRAF3−/− B cells, with regulation observed in both follicular and marginal zone B-cell subsets. BCR stimulation of TRAF3−/− B cells resulted in increased surface expression of MHC-II, CD80, and CD86 molecules. Interestingly, increased survival of TRAF3−/− primary B cells was resistant to inhibition of Btk, while TRAF3-deficient malignant B-cell lines showed enhanced sensitivity. TRAF3 serves to restrain normal and malignant BCR signaling, with important implications for its role in normal B-cell biology and abnormal survival of malignant B cells.

Tumor necrosis factor receptor associated factor 3 (TRAF3) regulates B cell antigen receptor (BCR) signaling

Abstract Tumor necrosis factor receptor associated factor 3 (TRAF3) is an intracellular adaptor protein that plays multiple cell type- and context-dependent roles. In B cells, TRAF3 negatively regulates signaling by several receptors important to survival and activation, including the B cell activating factor receptor (BAFF-R), CD40, toll-like receptors and the IL-6 receptor. Deletions or inactivating mutations of TRAF3 are common in the human B cell malignancies multiple myeloma and B cell lymphoma (BCL). Enhanced homeostatic B cell survival in B cell-specific TRAF3-deficient mice (B-Traf3−/−) causes enlarged secondary lymphoid organs, spontaneous germinal centers, and autoantibody production. Highlighting the role TRAF3 plays as a B cell tumor suppressor, B-Traf3−/− mice develop BCL at an increased rate as they age. The B cell antigen receptor (BCR) is crucial for B cell antigen recognition, survival, proliferation, and antibody production. Defects in BCR signaling can lead to malignancy, and many BCLs are dependent upon BCR signaling for survival. TRAF3 regulates the signaling of several pathways that interact with the BCR pathway, but the role of TRAF3 in regulating BCR signals is unknown, a knowledge gap we are currently addressing. Our initial studies show that TRAF3 associates with CD79b of the BCR complex. Phosphorylation of Syk, a proximal BCR signaling target, is increased after BCR stimulation in the absence of TRAF3, with increased downstream phosphorylation and activation of Btk, PLCγ2, Akt, Erk, and canonical NF-κB. This suggests that TRAF3 plays an important role as a regulator of BCR signaling. Ongoing studies are examining the mechanisms of this regulation.

Loss of B cell Ezrin in lupus-prone Lyn-deficient mice downregulates B cell activation and autoimmune pathology

Abstract Systemic lupus erythematosus (SLE) is an aggressive autoimmune disorder that afflicts 1.5 million Americans. Human GWAS studies have reported a strong association between the Src family tyrosine kinase Lyn and incidence of SLE, and B cells from SLE patients exhibit decreased Lyn expression. Genetic deletion of Lyn in mice results in loss of peripheral B cell tolerance and development of human SLE-like autoimmunity, characterized by hyperactive B cell antigen receptor (BCR) signaling, autoantibody production and glomerulonephritis. However, the molecular regulators of B cell pathogenesis in Lyn-deficient mice have not been fully characterized. We have previously shown that the membrane-cytoskeleton linker protein ezrin regulates multiple aspects of B cell function through its dynamic switching between phosphorylated and dephosphorylated states. We observed that ezrin is hyperphosphorylated in Lyn-deficient B cells, suggesting that it may impact B cell activation in this context. To test this hypothesis, we generated conditional deletion of ezrin in the B cells of Lyn-deficient mice (hereafter referred to as DKO mice). Compared to Lyn-deficient mice, the DKO mice exhibited a significant decrease in splenomegaly and reduction in autoantibodies against a variety of autoantigens. Glomerulonephritis and deposition of IgG and complement factor C3 in the kidneys of DKO mice were also markedly reduced. The DKO B cells were hyporesponsive to BCR stimulation and displayed a calcium mobilization pattern reminiscent of that observed in anergic B cells. Our data demonstrate that ezrin regulates B cell hyperactivation and associated pathology in the absence of Lyn, and suggest that ezrin may be a novel molecular target in SLE.

State of the art in microRNA as diagnostic and therapeutic biomarkers in chronic lymphocytic leukemia.

Early diagnostic is one of the most important steps in cancer therapy which helps to design and choose a better therapeutic approach. The finding of biomarkers in various levels including genomics, transcriptomics, and proteomics levels could provide better treatment for various cancers such as chronic lymphocytic leukemia (CLL). The CLL is the one of main lymphoid malignancies which is specified by aggregation of mature B lymphocytes. Among different biomarkers (e.g., CD38, chromosomes abnormalities, ZAP-70, TP53, and microRNA [miRNA]), miRNAs have appeared as new diagnostic and therapeutic biomarkers in patients with the CLL disease. Multiple lines of evidence indicated that deregulation of miRNAs could be associated with pathological events which are present in the CLL. These molecules have an effect on a variety of targets such as Bcl2, c-fos, c-Myc, TP53, TCL1, and STAT3 which play critical roles in the CLL pathogenesis. It has been shown that expression of miRNAs could lead to the activation of B cells and B cell antigen receptor (BCR). Moreover, exosomes containing miRNAs are one of the other molecules which could contribute to BCR stimulation and progression of CLL cells. Hence, miRNAs and exosomes released from CLL cells could be used as potential diagnostic and therapeutic biomarkers for CLL. This critical review focuses on a very important aspect of CLL based on biomarker discovery covers the pros and cons of using miRNAs as important diagnostics and therapeutics biomarkers for this deadly disease.

51 - The BCR Redox Machinery

Through the B cell antigen receptor (BCR), mature B cells recognize the presence of antigens in the external world and adapt their responses. The signaling output from this receptor is tightly controlled so as to prevent its hypo- or hyper-stimulation. Reactive oxygen species (ROS) can amplify BCR signaling, by inhibiting its intracellular negative regulators, i.e. protein tyrosine phosphatases. However, ROS fluxes and signals - most likely dependent on close proximity - have never been systematically studied. We recently showed that the NADPH oxidase complex 2 (NOX2) produces ROS extracellularly in B lymphocytes and that the water channel aquaporin 8 (AQP8) is consequently needed for their passage across the membrane for BCR signal amplification. On aquaporin 8 silencing, indeed, inducible B lymphoma cells respond poorly to BCR stimulation. Their differentiation is severely impaired, as demonstrated by retarded onset of IgM polymerization, low amounts of IgM secretion, and prolonged BCR expression on the cell surface. Thus, efficient induction of B cell activation and differentiation requires intact H 2 O 2 fluxes across the plasma membrane for signal amplification. The following step would be to understand whether there are physical connections between BCR, NOXes and AQPs. Are these connections induced or reinforced by antigen binding? To answer these questions, we are currently characterizing by PLA techniques the spatial organization and interaction of redox-regulators on the plasma membrane of resting and activated B cells.

Kidins220/ARMS binds to the B cell antigen receptor and regulates B cell development and activation.

B cell antigen receptor (BCR) signaling is critical for B cell development and activation. Using mass spectrometry, we identified a protein kinase D-interacting substrate of 220 kD (Kidins220)/ankyrin repeat-rich membrane-spanning protein (ARMS) as a novel interaction partner of resting and stimulated BCR. Upon BCR stimulation, the interaction increases in a Src kinase-independent manner. By knocking down Kidins220 in a B cell line and generating a conditional B cell-specific Kidins220 knockout (B-KO) mouse strain, we show that Kidins220 couples the BCR to PLCγ2, Ca(2+), and extracellular signal-regulated kinase (Erk) signaling. Consequently, BCR-mediated B cell activation was reduced in vitro and in vivo upon Kidins220 deletion. Furthermore, B cell development was impaired at stages where pre-BCR or BCR signaling is required. Most strikingly, λ light chain-positive B cells were reduced sixfold in the B-KO mice, genetically placing Kidins220 in the PLCγ2 pathway. Thus, our data indicate that Kidins220 positively regulates pre-BCR and BCR functioning.

In Vivo Evidence That Ibrutininb Deregulates Chemokine Receptor CXCR4 Surface Membrane Expression and Signaling, Along with Inhibiting B Cell Antigen Receptor Signaling, As Causes for Defective Homing and Impaired Retention of CLL Cells in Tissues

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has been approved by the FDA for the treatment of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Patients receiving this treatment often develop lymphocytosis and concomitant reduced organomegaly. These actions of ibrutinib are believed due to egress of CLL cells from lymphoid compartments, although the mechanism(s) responsible for this are not clear.Overexpressed surface membrane chemokine receptor CXCR4 (smCXCR4) is a hallmark of CLL cells and is involved in CLL cell migration and interaction with protective niches. Surface expression of CXCR4 is regulated by phosphorylation at serine/theronine residues in the receptor’s cytoplasmic tail. Kinases including GRK and PIM are known to regulate phosphorylation and surface expression of CXCR4. The transcript and protein levels of BTK, GRK and PIM also correlate with smCXCR4 expression.Here we studied the effect of ibrutinib on CLL cell distribution using TCL1-192 cells, a clonal murine cell line that mimics aggressive CLL and involves active B-cell antigen receptor (BCR) signaling. Similar to patients, TCL1-192-bearing mice receiving ibrutinib developed almost immediately (≤ 1 hour) lymphocytosis that consisted of both non-divided and recently-divided cells. While TCL1-192 cells overexpress smCXCR4, ibrutinib promoted lymphocytosis was associated with a fall in smCXCR4 to a level similar to that on normal B cells from wild type C3H/B6 mice. Reduced levels of smCXCR4 were also observed at the later timepoints when mice still had the continued release of cells into circulation even though lymphocytosis was no longer evident. Despite this change in smCXCR4 expression, total CXCR4 was not changed after ibrutinib treatment.Importantly, cells obtained from ibrutinib treated animals that had reduced levels of smCXCR4 also failed to respond to their ligand CXCL12 in vitro, as measured by absence of smCXCR4 internalization or calcium mobilization. Furthermore, re-expression of smCXCR4 after withdrawal of CXCL12 was not observed in treated cells. We therefore next studied in vitro the mechanism whereby ibrutinib lowered smCXCR4 expression. Western blot analysis using cells treated with ibrutinib at the dose ≥0.1μM at 37⁰C for 2 hours in the presence of CXCL12 showed significantly reduced levels of phosphorylated CXCR4 at Ser339. Phosphorylation of Ser339 in the CXCR4 intracellular domain is known to be essential for normal receptor recycling, hyper-phosphorylated CXCR4 on Ser339 is associated with overexpressed smCXCR4 in CLL patient cells. Correlating with this, TCL1-192 cells treated with ibrutinib at 0.1μM not only de-phosphorylated CXCR4 on Ser339, but also significantly enhanced internalization of smCXCR4 after CXCL12 stimulation.We then examined the kinases responsible for the de-phosphorylation of CXCR4. Western blot analysis of CLL cells from animals treated with ibrutinib for 4 weeks showed minimal levels of total BTK protein. There was a direct positive correlation in between BTK protein expression and smCXCR4 expression. In addition to BTK, reduced levels of kinases known to regulate CXCR4 phosphorylation on Ser339 were also observed in cells collected from ibrutinib treated mice. Together, these results suggest blocking BTK, directly or indirectly causes de-phosphorylation of CXCR4, resulting in impaired smCXCR4 recycling and blocked CXCL12 signaling.The function of CXCR4 is dependent on its location on the cell membrane. Impaired CXCR4 recycling after ibrutinib treatment would lead to defective cell trafficking. Indeed, cell transfer studies involving TCL1-192 cells previously treated with ibrutinib indicated that persistent decrease in smCXCR4, which was ibrutinib-dependent, blocked the return of CLL cells to solid tissue niches. Finally, ibrutinib-fed mice eventually developed lower lymphocyte counts, reduced organomegaly, and prolonged survival. Ibrutinib-fed mice also had defective response to BCR stimulation, including abolished calcium mobilization, blocked cell proliferation and survival after anti-IgM antibody stimulation. Together, the data provide direct in vivo evidence of ibrutinib impairing CLL cell homing and retention and suggest a mechanism by which BTK targeted therapy affects not only BCR signaling but also CXCR4 phosphorylation and signaling, with the latter contributing to defective smCXCR4 expression. DisclosuresChang:Pharmacyclics, Inc: Employment, Equity Ownership. Chang:Pharmacyclics: Employment. Buggy:Pharmacyclics: Employment. Burger:Pharmacyclics: Consultancy, Honoraria, Research Funding.

The Novel Kinase Inhibitor PRT062070 (Cerdulatinib) Demonstrates Efficacy in Models of Autoimmunity and B-Cell Cancer

The heterogeneity and severity of certain autoimmune diseases and B-cell malignancies warrant simultaneous targeting of multiple disease-relevant signaling pathways. Dual inhibition of spleen tyrosine kinase (SYK) and Janus kinase (JAK) represents such a strategy and may elicit several benefits relative to selective kinase inhibition, such as gaining control over a broader array of disease etiologies, reducing probability of selection for bypass disease mechanisms, and the potential that an overall lower level suppression of individual targets may be sufficient to modulate disease activity. To this end, we provide data on the discovery and preclinical development of PRT062070 [4-(cyclopropylamino)-2-({4-[4-(ethylsulfonyl)piperazin-1-yl]phenyl}amino)pyrimidine-5-carboxamide hydrochloride], an orally active kinase inhibitor that demonstrates activity against SYK and JAK. Cellular assays demonstrated specific inhibitory activity against signaling pathways that use SYK and JAK1/3. Limited inhibition of JAK2 was observed, and PRT062070 did not inhibit phorbol 12-myristate 13-acetate-mediated signaling or activation in B and T cells nor T-cell antigen receptor-mediated signaling in T cells, providing evidence for selectivity of action. Potent antitumor activity was observed in a subset of B-cell lymphoma cell lines. After oral dosing, PRT062070 suppressed inflammation and autoantibody generation in a rat collagen-induced arthritis model and blocked B-cell activation and splenomegaly in a mouse model of chronic B-cell antigen receptor stimulation. PRT062070 is currently under evaluation in a phase I dose escalation study in patients with B-cell leukemia and lymphoma (NCT01994382), with proof-of-concept studies in humans planned to assess therapeutic potential in autoimmune and malignant diseases.

IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell-dependent antigen.

The importance of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in B cells was investigated using Nfkb1(SSAA/SSAA) mice, in which this NF-κB signaling pathway is blocked. Nfkb1(SSAA) mutation had no effect on the development and homeostasis of follicular mature (FM) B cells. However, analysis of mixed bone marrow chimeras revealed that Nfkb1(SSAA/SSAA) FM B cells were completely unable to mediate T cell-dependent antibody responses. Nfkb1(SSAA) mutation decreased B cell antigen receptor (BCR) activation of NF-κB in FM B cells, which selectively blocked BCR stimulation of cell survival and antigen-induced differentiation into plasmablasts and germinal center B cells due to reduced expression of Bcl-2 family proteins and IRF4, respectively. In contrast, the antigen-presenting function of FM B cells and their BCR-induced migration to the follicle T cell zone border, as well as their growth and proliferation after BCR stimulation, were not affected. All of the inhibitory effects of Nfkb1(SSAA) mutation on B cell functions were rescued by normalizing NF-κB activation genetically. Our study identifies critical B cell-intrinsic functions for IKK-induced NF-κB1 p105 proteolysis in the antigen-induced survival and differentiation of FM B cells, which are essential for T-dependent antibody responses.

Concomitant, T-Independent TLR9-Mediated and BCR-Mediated Activation Provides Signals For Optimal Telomerase Induction In Chronic Lymphocytic Leukemia Cells Regardless Of IGHV Mutation Status

Chronic lymphocytic leukemia (CLL) is characterized by the clonal amplification of CD5-expressing B cells that appear to develop and evolve based on signals from the microenvironment. In vitro and in vivo evidence suggests that the B-cell antigen receptor (BCR) and Toll-like receptors (TLRs) may be keys to this stimulation. Because clonal turnover can lead to the release of naked nuclear material into the cellular microenvironment, these remnants of dying/dead cells may contribute to disease progression by repeated low level T-independent activation of CLL cells through the combination of the BCR and TLRs. To test this hypothesis, we assessed TLR9-driven or BCR + TLR9-driven CLL B-cell activation, focusing on its impact on telomerase activation in CLL cells, which is known to be important in the disease and which we have shown to be selectively activated by BCR stimulation in Ig V-unmutated (U-CLL) clones but not in Ig V-mutated (M-CLL) clones.B cells, isolated by negative selection from peripheral blood of IgM+ CLL patients and cryopreserved until use, were cultured for 16 hr without/ with TLR9 agonist, ODN 2006, alone and were assayed for apoptosis using Annexin V and flow cytometry. To study the relative contribution of simultaneous TLR9 activation and BCR activation, B cells were exposed to ODN2006 alone or HB57dex (monoclonal anti IgM Ab conjugated onto dextran) alone or a combination of the two reagents. Extracts from cells cultured for a period of 3 days were assayed for functional telomerase activity using TRAP. Parallel cultures of B cells exposed to the same stimuli were harvested at day 3 and assayed for cell activation and proliferation, which was assessed by 3H thymidine incorporation. CLL cells cultured with ODN2006 exhibited significant apoptosis within 16 hours in 6/12 cases. However at day 3, the same stimulus elicited significant increases in percentages of CD69-expressing cells and densities of HLA-DR in all CLL cases studied. As compared to BCR activation, which upregulates telomerase activity in U-CLL only, TLR9-mediated activation of CLL induced telomerase activation in all CLL cases. Furthermore, ODN2006 elicited significantly higher induction of telomerase activity in M-CLL cases compared to U-CLL cases (p=0.01). In addition, in M-CLL cases, simultaneous activation via TLR9 and BCR significantly upregulated the telomerase activity (p=0.05) that was induced by TLR9 activation alone. IRAK-1/4 inhibitor down modulated both TLR9 mediated and TLR9 +BCR mediated telomerase activity to a greater extent in M-CLL cases than in U-CLL cases. TLR9 activation of CLL cells induced a 3.75 + 0.8 fold (range 1.1 to 19.6; n=32) increase in cell proliferation. When segregated by Ig V mutation, U-CLL cells (n=16) responded significantly better (6.0 + 1.6 fold) compared to M-CLL cells (2.1 + 0.3 fold, n=16; p=0.03). However, co-stimulation of cells via their BCR significantly increased TLR-mediated responses only in M-CLL cases (from 2.3 + 0.4 fold to 5.4 + 1.7 fold; p=0.05). IRAK-1/4 inhibitor did not exert a significant effect on TLR9 mediated cell proliferation in either the U-CLL or M-CLL cases. Co-culture of CLL cells with human stromal cells, HS5, further upregulated the concerted TLR9 + BCR induced proliferative responses in 70% of the cases studied.Together, these results indicate that simultaneous stimulation of CLL cells via both their TLR9 and BCR molecules positively impacts on telomerase activity in all patients studied. Since telomerase is crucial in maintaining longevity of repeatedly stimulated cells, this could represent a mechanism for worse clinical outcome in CLL. These studies stress the need for devising therapeutic agents or combinations thereof to effectively target multiple pathways downstream of these signaling receptors and to ultimately eradicate newly evolving CLL cells. Disclosures:No relevant conflicts of interest to declare.

Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator

Monitoring the pattern of intracellular Ca2+ signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca2+ indicators (GECIs) are used for monitoring intracellular Ca2+ changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca2+ signals using GECIs. We here compared the Ca2+ signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca2+ responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca2+ signals.

Protein kinase N1, a cell inhibitor of Akt kinase, has a central role in quality control of germinal center formation.

Germinal centers (GCs) are specialized microenvironments in secondary lymphoid organs where high-affinity antibody-producing B cells are selected based on B-cell antigen receptor (BCR) signal strength. BCR signaling required for normal GC selection is uncertain. We have found that protein kinase N1 (PKN1, also known as PRK1) negatively regulates Akt kinase downstream of the BCR and that this regulation is necessary for normal GC development. PKN1 interacted with and inhibited Akt1 kinase and transforming activities. Pkn1(-/-) B cells were hyperresponsive and had increased phosphorylated Akt1 levels upon BCR stimulation. In the absence of immunization or infection, Pkn1(-/-) mice spontaneously formed GCs and developed an autoimmune-like disease with age, which was characterized by autoantibody production and glomerulonephritis. More B cells, with fewer somatic BCR gene V region hypermutations were selected in Pkn1(-/-) GCs. These results indicate that PKN1 down-regulation of BCR-activated Akt activity is critical for normal GC B-cell survival and selection.

Characterization of a new ARID family transcription factor (Brightlike/ARID3C) that co-activates Bright/ARID3A-mediated immunoglobulin gene transcription

Two members, Bright/ARID3A and Bdp/ARID3B, of the ARID (AT-Rich Interaction Domain) transcription family are distinguished by their ability to specifically bind to DNA and to self-associate via a second domain, REKLES. Bright and Bdp positively regulate immunoglobulin heavy chain gene (IgH) transcription by binding to AT-rich motifs within Matrix Associating Regions (MARs) residing within a subset of VH promoters and the Eμ intronic enhancer. In addition, REKLES provides Bright nuclear export function, and a small pool of Bright is directed to plasma membrane sub-domains/lipid rafts where it associates with and modulates signaling of the B cell antigen receptor (BCR). Here, we characterize a third, highly conserved, physically condensed ARID3 locus, Brightlike/ARID3C. Brightlike encodes two alternatively spliced, SUMO-I-modified isoforms that include or exclude (Δ6) the REKLES-encoding exon 6. Brightlike transcripts and proteins are expressed preferentially within B lineage lymphocytes and coordinate with highest Bright expression in activated follicular B cells. Brightlike, but not BrightlikeΔ6, undergoes nuclear-cytoplasmic shuttling with a fraction localizing within lipid rafts following BCR stimulation. Brightlike, but not BrightlikeΔ6, associates with Bright in solution, at common DNA binding sites in vitro, and is enriched at Bright binding sites in chromatin. Although possessing little transactivation capacity of its own, Brightlike significantly co-activates Bright-dependent IgH transcription with maximal activity mediated by the unsumoylated form. In sum, this report introduces Brightlike as an additional functional member of the family of ARID proteins, which should be considered in regulatory circuits, previously ascribed to be mediated by Bright.

The ERM family protein Ezrin associates with the JNK signaling complex to regulate its activation and antigen-dependent B cell survival. (109.7)

Abstract Antigen binding to the B cell antigen receptor (BCR) results in initiation of a highly orchestrated series of enzymatic reactions and protein-protein interactions that transmit the signals for B cell survival and proliferation. The highly conserved Ezrin-Radixin-Moesin (ERM) family members are thought to regulate lymphocyte activation via their membrane-actin crosslinking property, which is in turn regulated by phosphorylation of a conserved threonine residue at their C-terminus. Here, we report a novel signal transduction axis that links antigen-dependent tyrosine phosphorylation of ezrin with activation of the c-Jun N-terminal kinase (JNK). BCR ligation induces phosphorylation of Y353 in ezrin, a residue that is not conserved in other ERM proteins. Phosphorylation of Y353 is dependent on Src and Syk family kinase activity, occurs preferentially on the open conformer of ezrin, and correlates with the strength of BCR signaling in mature B cell subsets. Tyrosine phosphorylated ezrin associates with components of the JNK signaling complex in response to BCR stimulation. A non-phosphorylatable (Y353F) mutant of ezrin acts in a dominant negative manner to specifically impair antigen-induced JNK activation, and reduces B cell survival. Our results demonstrate a unique and novel signaling role for ezrin in regulating antigen-dependent JNK activation and B cell survival.

Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro‐survival microenvironmental signals

As antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c-Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33·7% at 48 h, with dasatinib sensitivity correlating with inhibition of Syk(Y348) phosphorylation. Dasatinib inhibited calcium flux, phosphatidylinositol-3-kinase and mitogen-activated protein kinase activation following BCR crosslinking, and blocked the Mcl-1-dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro-apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)-4 (CD154L/IL-4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co-culture to both fludarabine and chlorambucil, the addition of CD154 and IL-4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17-DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL-4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigen-independent microenvironmental signals.

B cell antigen receptor-induced activation of an IRAK4-dependent signaling pathway revealed by a MALT1-IRAK4 double knockout mouse model

BackgroundThe B cell antigen receptor (BCR) and pathogen recognition receptors, such as Toll-like receptor 4 (TLR4), act in concert to control adaptive B cell responses. However, little is known about the signaling pathways that integrate BCR activation with intrinsic TLR4 stimulation. Antigen receptors initialize activation of the inducible transcription factor nuclear factor-κB (NF-κB) via recruitment of the membrane-associated guanylate kinase caspase recruitment domain protein 11 (CARD11), the adapter molecule B cell CLL/lymphoma 10 (BCL10), and the "paracaspase" mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) into lipid rafts. Upon BCR triggering, this activation strictly depends on BCL10, but not on MALT1, leading to the hypothesis that a MALT1-independent NF-κB activation pathway contributes to BCR-induced NF-κB activation downstream of BCL10. The identity of this pathway has remained elusive.ResultsUsing genetic and biochemical approaches, we demonstrate that the IRAK4- and IRAK1-dependent TLR signaling branch is activated upon BCR triggering to induce partial NF-κB activation. BCR-induced MALT1-independent IκB degradation and B cell proliferation were inhibited in MALT1/IRAK4 double knockout B cells. Moreover, IRAK1 was recruited into lipid rafts upon BCR stimulation and activated following transient recruitment of IRAK4.ConclusionWe propose that the observed crosstalk between BCR and TLR signaling components may contribute to the discrimination of signals that emanate from single and dual receptor engagement to control adaptive B cell responses.

Stereotypical Chronic Lymphocytic Leukemia B-Cell Receptors Recognize Survival Promoting Antigens on Stromal Cells

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as “subset 1”) recognize antigens highly expressed in stromal cells – vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20–45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease.

Dasatinib-Induced Apoptosis in Chronic Lymphocytic Leukaemia Is Abrogated by Selective Micro-Environmental Signals.

Abstract 1264Poster Board I-286 BackgroundRecent advances in chronic lymphocytic leukaemia (CLL) biology indicate that a plethora of micro-environmental factors are important in promoting the pathogenesis of the disease. As B cell antigen receptor (BCR) signalling plays a critical role in CLL progression, we chose to study the effect of dasatinib, a dual src/abl tyrosine kinase inhibitor, on CLL cells, thus targeting the key, BCR-proximal tyrosine kinases Lyn and c-Abl that are over-expressed in CLL. We were interested to determine the impact of BCR-signalling directed therapy on CLL cell viability in the context of the leukaemic micro-environment. ResultsClinically relevant concentrations of dasatinib (100 nM) both inhibited ERK-MAPK and Akt phosphorylation, and prevented calcium mobilisation following BCR crosslinking. Furthermore, dasatinib prevented up-regulation of Mcl-1 observed on prolonged BCR stimulation, abrogating the BCR-driven increase in CLL cell survival. Dasatinib induced apoptosis of CLL cells, with a mean reduction in viability of 35.2% ± 3.8% following 48 hr exposure (n=26). Neither ZAP-70 expression nor cytogenetic abnormalities were predictive of response. In addition, dasatinib exhibited synergy with both fludarabine and chlorambucil, with mean ED50 combination indices of 0.29 and 0.62 respectively. Additional micro-environmental elements responsible for the maintenance and progression of CLL include stromal or ‘nurse-like' cells and activated T lymphocytes, the latter of which express CD40 ligand and secrete IL-4. CLL cell co-culture with the murine bone marrow stromal cell line NT-L significantly inhibited both spontaneous and dasatinib induced apoptosis. As we observed that stromal co-culture significantly increased phosphorylation of both ERK-MAPK and the Akt/mTOR target p70 S6 kinase in CLL cells, we investigated whether pharmacological inhibition these signalling pathways may re-sensitise CLL cells to dasatinib in co-culture. Both the MEK inhibitor PD98059 and PI3K inhibitor LY294002 significantly increased the level of apoptosis observed on dasatinib treatment of CLL cells in stromal co-culture. Dasatinib also retained the ability to potentiate the effects of fludarabine and chlorambucil in stromal co-culture, however, CLL cells co-cultured with NT-L cells stably transfected with CD40 ligand (CD154L cells) were resistant to all drug combinations. Furthermore, on CD154L/IL-4 co-culture, dasatinib failed to prevent up-regulation of Bcl-xL and Mcl-1, or inhibit CLL cell proliferation. Conclusions:While dasatinib was capable of reversing BCR-mediated survival signals, it was unable to overcome stromal-derived survival signals, indicating that combination of dasatinib with agents targeting antigen-independent micro-environmental pathways are required to effectively target CLL cells in tissue niches. DisclosuresNo relevant conflicts of interest to declare.

Grb2, a simple adapter with complex roles in lymphocyte development, function, and signaling

Lymphocyte development, activation, and tolerance depend on antigen receptor signaling transduced via multiple intracellular signalosomes. These signalosomes are assembled by different adapters. Given that signaling molecules can be either positive or negative regulators for a biochemical target, the complex of a target with different regulator may dictate the final signaling outcome. Grb2 is a simple adapter known to be involved in a variety of growth factor receptor signaling. However, its role in antigen receptor signaling as well as lymphocyte development and function has emerged only recently. Despite its simple molecular structure, recent experiments show that Grb2 may play a complex role in T and B-cell antigen receptor signaling. In this article, we review recent findings about the physiological role of Grb2 in T and B-cell development and activation and summarize the current mechanistic understanding of how Grb2 exerts its function following T and B-cell antigen receptor stimulation.