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Sample records for activated b-cell abc

  1. CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas.

    PubMed

    Juilland, Mélanie; Gonzalez, Montserrat; Erdmann, Tabea; Banz, Yara; Jevnikar, Zala; Hailfinger, Stephan; Tzankov, Alexandar; Grau, Michael; Lenz, Georg; Novak, Urban; Thome, Margot

    2016-04-07

    A hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor-κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL.

  2. CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas

    PubMed Central

    Juilland, Mélanie; Gonzalez, Montserrat; Erdmann, Tabea; Banz, Yara; Jevnikar, Zala; Hailfinger, Stephan; Tzankov, Alexandar; Grau, Michael; Lenz, Georg; Novak, Urban

    2016-01-01

    A hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor–κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL. PMID:26747248

  3. Exploiting Synthetic Lethality for the Therapy of ABC Diffuse Large B Cell Lymphoma

    PubMed Central

    Yang, Yibin; Shaffer, Arthur L.; Emre, N.C. Tolga; Ceribelli, Michele; Zhang, Meili; Wright, George; Xiao, Wenming; Powell, John; Platig, John; Kohlhammer, Holger; Young, Ryan M.; Zhao, Hong; Yang, Yandan; Xu, Weihong; Buggy, Joseph J.; Balasubramanian, Sriram; Mathews, Lesley A.; Shinn, Paul; Guha, Rajarshi; Ferrer, Marc; Thomas, Craig; Waldmann, Thomas A.; Staudt, Louis M.

    2014-01-01

    Summary Knowledge of oncogenic mutations can inspire therapeutic strategies that are synthetically lethal, affecting cancer cells while sparing normal cells. Lenalidomide is an active agent in the activated B-cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), but its mechanism of action is unknown. Lenalidomide kills ABC DLBCL cells by augmenting interferon β (IFNβ) production, owing to the oncogenic MYD88 mutations in these lymphomas. In a cereblon-dependent fashion, lenalidomide downregulates IRF4 and SPIB, transcription factors that together prevent IFNβ production by repressing IRF7 and also amplify pro-survival NF-κB signaling by transactivating CARD11. Blockade of B cell receptor (BCR) signaling using the BTK inhibitor ibrutinib also downregulates IRF4 and consequently synergizes with lenalidomide in killing ABC DLBCLs, suggesting attractive therapeutic strategies. PMID:22698399

  4. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

    PubMed

    Davis, R Eric; Ngo, Vu N; Lenz, Georg; Tolar, Pavel; Young, Ryan M; Romesser, Paul B; Kohlhammer, Holger; Lamy, Laurence; Zhao, Hong; Yang, Yandan; Xu, Weihong; Shaffer, Arthur L; Wright, George; Xiao, Wenming; Powell, John; Jiang, Jian-Kang; Thomas, Craig J; Rosenwald, Andreas; Ott, German; Muller-Hermelink, Hans Konrad; Gascoyne, Randy D; Connors, Joseph M; Johnson, Nathalie A; Rimsza, Lisa M; Campo, Elias; Jaffe, Elaine S; Wilson, Wyndham H; Delabie, Jan; Smeland, Erlend B; Fisher, Richard I; Braziel, Rita M; Tubbs, Raymond R; Cook, J R; Weisenburger, Dennis D; Chan, Wing C; Pierce, Susan K; Staudt, Louis M

    2010-01-07

    A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

  5. STAT3 inhibition is a therapeutic strategy for ABC-like diffuse large B-cell lymphoma.

    PubMed

    Scuto, Anna; Kujawski, Maciej; Kowolik, Claudia; Krymskaya, Ludmila; Wang, Lin; Weiss, Lawrence M; Digiusto, David; Yu, Hua; Forman, Stephen; Jove, Richard

    2011-05-01

    Persistent STAT3 signaling contributes to malignant progression in many diverse types of human cancer. STAT3 is constitutively active in activated B-cell (ABC)-like diffuse large B-cell lymphomas (DLBCL), a class of nongerminal center derived DLBCL cells for which existing therapy is weakly effective. In this report, we provide a preclinical proof of concept that STAT3 is an effective molecular target for ABC-like DLBCL therapy. Direct inhibition of STAT3 with short hairpin RNA suppressed the growth of human ABC-like DLBCL in mouse models in a manner associated with apoptosis, repression of STAT3 target genes, and inhibition of a tumor-promoting microenvironment. Together, these results suggest that STAT3 is essential to maintain the pathophysiology of ABC-like DLBCL and therefore that STAT3 inhibition may offer a promising approach in its therapy.

  6. Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma

    PubMed Central

    Cardenas, Mariano G.; Yu, Wenbo; Beguelin, Wendy; Teater, Matthew R.; Geng, Huimin; Goldstein, Rebecca L.; Oswald, Erin; Hatzi, Katerina; Yang, Shao-Ning; Cohen, Joanna; Shaknovich, Rita; Vanommeslaeghe, Kenno; Cheng, Huimin; Liang, Dongdong; Cho, Hyo Je; Tam, Wayne; Du, Wei; Leonard, John P.; Elemento, Olivier; Cierpicki, Tomasz; Xue, Fengtian; MacKerell, Alexander D.; Melnick, Ari M.

    2016-01-01

    Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands. PMID:27482887

  7. Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma.

    PubMed

    Cardenas, Mariano G; Yu, Wenbo; Beguelin, Wendy; Teater, Matthew R; Geng, Huimin; Goldstein, Rebecca L; Oswald, Erin; Hatzi, Katerina; Yang, Shao-Ning; Cohen, Joanna; Shaknovich, Rita; Vanommeslaeghe, Kenno; Cheng, Huimin; Liang, Dongdong; Cho, Hyo Je; Abbott, Joshua; Tam, Wayne; Du, Wei; Leonard, John P; Elemento, Olivier; Cerchietti, Leandro; Cierpicki, Tomasz; Xue, Fengtian; MacKerell, Alexander D; Melnick, Ari M

    2016-09-01

    Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands.

  8. Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma.

    PubMed

    Hailfinger, Stephan; Lenz, Georg; Ngo, Vu; Posvitz-Fejfar, Anita; Rebeaud, Fabien; Guzzardi, Montserrat; Penas, Eva-Maria Murga; Dierlamm, Judith; Chan, Wing C; Staudt, Louis M; Thome, Margot

    2009-11-24

    A key element for the development of suitable anti-cancer drugs is the identification of cancer-specific enzymatic activities that can be therapeutically targeted. Mucosa-associated lymphoid tissue transformation protein 1 (MALT1) is a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, the least curable subtype of DLBCL. Recent data suggest that MALT1 has proteolytic activity, but it is unknown whether this activity is relevant for tumor growth. Here we report that MALT1 is constitutively active in DLBCL lines of the ABC but not the GCB subtype. Inhibition of the MALT1 proteolytic activity led to reduced expression of growth factors and apoptosis inhibitors, and specifically affected the growth and survival of ABC DLBCL lines. These results demonstrate a key role for the proteolytic activity of MALT1 in DLBCL of the ABC subtype, and provide a rationale for the development of pharmacological inhibitors of MALT1 in DLBCL therapy.

  9. Inhibition of MALT1 protease activity is selectively toxic for activated B cell-like diffuse large B cell lymphoma cells.

    PubMed

    Ferch, Uta; Kloo, Bernhard; Gewies, Andreas; Pfänder, Vera; Düwel, Michael; Peschel, Christian; Krappmann, Daniel; Ruland, Jürgen

    2009-10-26

    Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell-like (ABC) subtype of DLBCL is characterized by constitutive NF-kappaB activity and requires signals from CARD11, BCL10, and the paracaspase MALT1 for survival. CARD11, BCL10, and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11-BCL10-MALT1 (CBM) complexes couple upstream events to IkappaB kinase (IKK)/NF-kappaB activation. MALT1 also possesses a recently recognized proteolytic activity that cleaves and inactivates the negative NF-kappaB regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here, we demonstrate preassembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL, but not in germinal center B cell-like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-kappaB activity, and decreases the expression of NF-kappaB targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment.

  10. B cell activating factor (BAFF) selects IL-10(-)B cells over IL-10(+)B cells during inflammatory responses.

    PubMed

    Ma, Ning; Zhang, Yu; Liu, Qilin; Wang, Zhiding; Liu, Xiaoling; Zhu, Gaizhi; Yu, Dandan; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Wang, Tianxiao; Ma, Yuanfang; Shen, Beifen; Li, Yan; Xiao, He; Wang, Renxi

    2017-05-01

    B cell activating factor (BAFF) regulates B cell maturation, survival, function, and plays a critical pathogenic role in autoimmune diseases. It remains unclear how BAFF affects IL-10(-)B cells versus regulatory B cells (Bregs) in inflammatory responses. In this study, we found that IL-10-expressing Bregs decreased in lupus-prone MRL/lpr mice and experimental allergic encephalomyelitis (EAE) mice. On blockade of the effects of BAFF with TACI-IgG, IL-10(+) Bregs were upregulated in MRL/lpr and EAE mice. In addition, BAFF expanded IL-10(+)B cells over IL-10(-)B cells under noninflammatory conditions in vitro, whereas it expanded IL-10(-)B cells over IL-10(+)B cells during inflammatory responses, such as stimulation with autoantigen and LPS. Finally, the selection of IL-10(-)B cells over IL-10(+)B cells by BAFF was dependent on BAFF receptors (BAFFR, TACI, and BCMA) that were upregulated by inflammatory responses. This study suggests that BAFF selects IL-10(-)B cells over IL-10(+) regulatory B cells via BAFF receptors in inflammatory responses.

  11. Receptor Dissociation and B-Cell Activation.

    PubMed

    Yang, Jianying; Reth, Michael

    2016-01-01

    The B-cell antigen receptor (BCR) is one of the most abundant receptors on the surface of B cells with roughly 100,000-200,000 copies per cell. Signaling through the BCR is crucial for the activation and differentiation of B cells. Unlike other receptors, the BCR can be activated by a large set of structurally different ligands, but the molecular mechanism of BCR activation is still a matter of controversy. Although dominant for a long time, the cross-link model (CLM) of BCR activation is not supported by recent studies of the nanoscale organization of the BCR on the surface of resting B cells. In contrast to the prediction of CLM, the numerous BCR complexes on these cells are not randomly distributed monomers but rather form oligomers which reside within membrane confinements. This finding is more in line with the dissociation activation model (DAM), wherein B-cell activation is accompanied by an opening of the auto-inhibited BCR oligomers instead of a cross-linking of the BCR monomers. In this review, we discuss in detail the new findings and their implications for BCR signaling.

  12. Complement activation by a B cell superantigen.

    PubMed

    Kozlowski, L M; Soulika, A M; Silverman, G J; Lambris, J D; Levinson, A I

    1996-08-01

    Staphylococcal protein A (SpA), acting as a B cell superantigen, binds to the Fab region of human VH3+ Igs. Using SpA abrogated of its IgG Fc binding activity (Mod SpA) as a model B cell superantigen, we determined whether such an interaction causes complement activation. Addition of Mod SpA to human serum led to complement consumption and the generation of C3a. To determine whether this complement activation 1) was due to an interaction between VH3+ Igs and the Fab binding site of SpA and 2) proceeded via the classical complement pathway, we tested a panel of monoclonal IgM proteins for the ability to hind C1q following interaction with SpA. C1q binding was restricted to SpA-reactive, VH3+ IgM proteins. To formally determine whether the binding of SpA to the reactive VH3+ IgM proteins led to complement activation, we reconstituted the serum from a hypogammaglobulinemic patient with monoclonal IgM proteins and measured complement consumption and C3a generation following the addition of Mod SpA. We observed complement consumption and C3a production only in Mod SpA-treated serum reconstituted with a VH3+, SpA-binding, IgM protein. Taken together, these results provide compelling evidence that the interaction of the Fab binding site of SpA and VH3+ Igs can lead to complement activation via the classical pathway. This novel interaction may have significant implications for the in vivo properties of a B cell superantigen.

  13. Alloantigen presentation by B cells: analysis of the requirement for B-cell activation.

    PubMed Central

    Wilson, J L; Cunningham, A C; Kirby, J A

    1995-01-01

    This paper describes a model for investigation of the functional implications of B-cell activation for antigen presentation. Mixed lymphocyte cultures were used to assess the ability of freshly isolated B cells, mitogen-activated B cells and Epstein-Barr virus (EBV)-transformed B-cell lines to stimulate the activation and proliferation of allogeneic T cells under a variety of experimental conditions. It was found that resting B cells presented antigen poorly, while activated cells were highly immunogenic. Paraformaldehyde fixation completely eliminated antigen presentation by resting B cells, despite constitutive expression of class II MHC antigens. However, fixation had little effect on antigen presentation by activated B cells that expressed B7-1 and B7-2 in addition to class II major histocompatibility complex (MHC) molecules. Arrest of B-cell activation by serial fixation after treatment with F(ab')2 fragments of goat anti-human IgM produced cells with variable antigen-presenting capacity. Optimal antigen presentation was observed for cells fixed 72 hr after the initiation of B-cell activation. Although both B7-1 and B7-2 antigen expression increased after B-cell activation, it was found that the rate of T-cell proliferation correlated most closely with B7-2 expression. Stimulation of T cells by fixed activated B lymphocytes could be blocked by antibodies directed at class II MHC molecules, indicating involvement of the T-cell antigen receptor. In addition, T-cell proliferation was inhibited by antibodies specific for B7-1 and B7-2 and by the fusion protein CTLA4-Ig, demonstrating a requirement for CD28 signal transduction. The sole requirement of B7 family expression for antigen presentation by B lymphocytes was shown by demonstration of T-cell stimulation by fixed resting B cells in the presence of CD28 antibody as a source of artificial costimulation. PMID:8550066

  14. Control of translational activation by PIM kinase in activated B-cell diffuse large B-cell lymphoma confers sensitivity to inhibition by PIM447

    PubMed Central

    Peters, Tara L.; Li, Lingxiao; Tula-Sanchez, Ana A.; Pongtornpipat, Praechompoo; Schatz, Jonathan H.

    2016-01-01

    The PIM family kinases promote growth and survival of tumor cells and are expressed in a wide variety of human cancers. Their potential as therapeutic targets, however, is complicated by overlapping activities with multiple other pathways and remains poorly defined in most clinical scenarios. Here we explore activity of the new pan-PIM inhibitor PIM447 in a variety of lymphoid-derived tumors. We find strong activity in cell lines derived from the activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). Sensitive lines show lost activation of the mTORC1 signaling complex and subsequent lost activation of cap-dependent protein translation. In addition, we characterize recurrent PIM1 protein-coding mutations found in DLBCL clinical samples and find most preserve the wild-type protein's ability to protect cells from apoptosis but do not bypass activity of PIM447. Pan-PIM inhibition therefore may have an important role to play in the therapy of selected ABC-DLBCL cases. PMID:27556513

  15. Differential expression of viral agents in lymphoma tissues of patients with ABC diffuse large B-cell lymphoma from high and low endemic infectious disease regions

    PubMed Central

    Högfeldt, Therese; Jaing, Crystal; Loughlin, Kevin Mc; Thissen, James; Gardner, Shea; Bahnassy, Abeer A.; Gharizadeh, Baback; Lundahl, Joachim; Österborg, Anders; Porwit, Anna; Zekri, Abdel-Rahman N.; Khaled, Hussein M.; Mellstedt, Håkan; Moshfegh, Ali

    2016-01-01

    Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma (NHL) in adults, accounts for approximately 30–40% of newly diagnosed lymphomas worldwide. Environmental factors, such as viruses and bacteria, may contribute to cancer development through chronic inflammation and the integration of oncogenes, and have previously been indicated in cervical cancer, hepatocellular carcinoma, gastric cancer and lymphoproliferative disorders. In the present study, the presence of microbial agents was analyzed in the lymphoma tissue of patients with activated B-cell like (ABC) DLBCL. The present study compared two groups of patients from geographically varied regions that possess a difference in the prevalence of viral and other microbial agents. The patient populations were from Sweden (a low endemic infectious disease region) and Egypt (a high endemic infectious disease region). A differential expression of several viruses in lymphoma tissues was noted when comparing Swedish and Egyptian patients. JC polyomavirus (JCV) was detected in Swedish and Egyptian patients and, uniquely, the complete hepatitis B virus (HBV) genome was detected only in Egyptian lymphoma patients. None of these viruses were detected in control lymph tissues from Sweden or Egypt. In total, 38% of the Egyptian patients were found to have HBV surface antigens (HBsAgs) in their serum; however, HBsAgs were not found in any of the Swedish patients. The percentage of serum HBsAgs in Egyptian patients with ABC DLBCL was significantly increased compared with the general Egyptian population (P<0.05). The present study may support a notion that viral agents, including JCV and HBV, may be involved in the tumorigenesis of DLBCL in regions of high infectious disease. PMID:27698858

  16. Critical role of PI3K signaling for NF-kappaB-dependent survival in a subset of activated B-cell-like diffuse large B-cell lymphoma cells.

    PubMed

    Kloo, Bernhard; Nagel, Daniel; Pfeifer, Matthias; Grau, Michael; Düwel, Michael; Vincendeau, Michelle; Dörken, Bernd; Lenz, Peter; Lenz, Georg; Krappmann, Daniel

    2011-01-04

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) represents a very aggressive human lymphoma entity. Constitutive NF-κB activation caused by chronic active B-cell receptor (BCR) signaling is common feature of many ABC DLBCL cells; however, the pathways linking BCR signaling to the NF-κB prosurvival network are largely unknown. Here we report that constitutive activity of PI3K and the downstream kinase PDK1 are essential for the viability of two ABC DLBCL cell lines that carry mutations in the BCR proximal signaling adaptor CD79B. In these cells, PI3K inhibition reduces NF-κB activity and decreases the expression of NF-κB target genes. Furthermore, PI3K and PDK1 are required for maintaining MALT1 protease activity, which promotes survival of the affected ABC DLBCL cells. These results demonstrate a critical function of PI3K-PDK1 signaling upstream of MALT1 protease and NF-κB in distinct ABC DLBCL cells and provide a rationale for the pharmacologic use of PI3K inhibitors in DLBCL therapy.

  17. Regulation of B cell fate by chronic activity of the IgE B cell receptor

    PubMed Central

    Yang, Zhiyong; Robinson, Marcus J; Chen, Xiangjun; Smith, Geoffrey A; Taunton, Jack; Liu, Wanli; Allen, Christopher D C

    2016-01-01

    IgE can trigger potent allergic responses, yet the mechanisms regulating IgE production are poorly understood. Here we reveal that IgE+ B cells are constrained by chronic activity of the IgE B cell receptor (BCR). In the absence of cognate antigen, the IgE BCR promoted terminal differentiation of B cells into plasma cells (PCs) under cell culture conditions mimicking T cell help. This antigen-independent PC differentiation involved multiple IgE domains and Syk, CD19, BLNK, Btk, and IRF4. Disruption of BCR signaling in mice led to consistently exaggerated IgE+ germinal center (GC) B cell but variably increased PC responses. We were unable to confirm reports that the IgE BCR directly promoted intrinsic apoptosis. Instead, IgE+ GC B cells exhibited poor antigen presentation and prolonged cell cycles, suggesting reduced competition for T cell help. We propose that chronic BCR activity and access to T cell help play critical roles in regulating IgE responses. DOI: http://dx.doi.org/10.7554/eLife.21238.001 PMID:27935477

  18. Regulation of B cell fate by chronic activity of the IgE B cell receptor.

    PubMed

    Yang, Zhiyong; Robinson, Marcus J; Chen, Xiangjun; Smith, Geoffrey A; Taunton, Jack; Liu, Wanli; Allen, Christopher D C

    2016-12-09

    IgE can trigger potent allergic responses, yet the mechanisms regulating IgE production are poorly understood. Here we reveal that IgE(+) B cells are constrained by chronic activity of the IgE B cell receptor (BCR). In the absence of cognate antigen, the IgE BCR promoted terminal differentiation of B cells into plasma cells (PCs) under cell culture conditions mimicking T cell help. This antigen-independent PC differentiation involved multiple IgE domains and Syk, CD19, BLNK, Btk, and IRF4. Disruption of BCR signaling in mice led to consistently exaggerated IgE(+) germinal center (GC) B cell but variably increased PC responses. We were unable to confirm reports that the IgE BCR directly promoted intrinsic apoptosis. Instead, IgE(+) GC B cells exhibited poor antigen presentation and prolonged cell cycles, suggesting reduced competition for T cell help. We propose that chronic BCR activity and access to T cell help play critical roles in regulating IgE responses.

  19. Innate response activator B cells: origins and functions

    PubMed Central

    Swirski, Filip K.

    2015-01-01

    Innate response activator (IRA) B cells are a subset of B-1a derived B cells that produce the growth factors granulocyte macrophage colony stimulating factor and IL-3. In mouse models of sepsis and pneumonia, B-1a B cells residing in serosal sites recognize bacteria, migrate to the spleen or lung, and differentiate to IRA B cells that then contribute to the host response by amplifying inflammation and producing polyreactive IgM. In atherosclerosis, IRA B cells accumulate in the spleen, where they promote extramedullary hematopoiesis and activate classical dendritic cells. In this review, we focus on the ontogeny and function of IRA B cells in acute and chronic inflammation. PMID:25957266

  20. B Cell-Activating Factor Regulates Different Aspects of B Cell Functionality and Is Produced by a Subset of Splenic B Cells in Teleost Fish

    PubMed Central

    Tafalla, Carolina; González, Lucia; Castro, Rosario; Granja, Aitor G.

    2017-01-01

    In mammals, B cell functionality is greatly influenced by cytokines released by innate cells, such as macrophages or dendritic cells, upon the early recognition of common pathogen patterns through invariant receptors. B cell-activating factor (BAFF) is one of these innate B cell-helper signals and plays a key role in the survival and differentiation of B cells. Although, evolutionarily, teleost fish constitute the first animal group in which adaptive immunity based on Ig receptors is present, fish still rely greatly on innate responses. In this context, we hypothesized that BAFF would play a key role in the control of B cell responses in fish. Supporting this, our results show that teleost BAFF recapitulates mammalian BAFF stimulating actions on B cells, upregulating the expression of membrane MHC II, improving the survival of fish naïve B cells and antibody-secreting cells, and increasing the secretion of IgM. Surprisingly, we also demonstrate that BAFF is not only produced in fish by myeloid cells but is also produced by a subset of splenic B cells. Thus, if this B cell-produced BAFF proves to be actively regulating this same B cell subset, our findings point to an ancient mechanism to control B cell differentiation and survival in lower vertebrates, which has been silenced in mammals in physiological conditions, but reemerges under pathological conditions, such as B cell lymphomas and autoimmune diseases. PMID:28360916

  1. Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance.

    PubMed

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E; Notarangelo, Luigi D; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2015-11-17

    Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.

  2. Phenotypic Approaches to Identify Inhibitors of B Cell Activation

    PubMed Central

    Kim, Suzie; Wiener, Jake; Rao, Navin L.; Milla, Marcos E.; DiSepio, Daniel

    2015-01-01

    An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin’s lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton’s tyrosine kinase (BTK) inhibitors. LFA-1/ICAM-1 association was further increased on coapplication of anti-IgM and mega CD40L when compared to individual application of either. Anti-IgM, mega CD40L, or the combination of both displayed distinct kinetic profiles that were inhibited by treatment with a BTK inhibitor. We also established a FLIPR-based assay to measure B cell activation in Ramos Burkitt’s lymphoma B cells and an RL cell line. Anti-IgM-mediated BCR activation elicited a robust calcium response that was inhibited by a panel of BTK inhibitors. Conversely, CD40R activation did not elicit a calcium response in the FLIPR assay. Compared to the FLIPR, the EPIC assay has the propensity to identify inhibitors of both BCR and CD40R-mediated B cell activation and may provide more pharmacological depth or novel mechanisms of action for inhibition of B cell activation. PMID:25948491

  3. The regulation and activation of lupus-associated B cells.

    PubMed

    Fields, Michele L; Hondowicz, Brian D; Wharton, Gina N; Adair, Brigette S; Metzgar, Michele H; Alexander, Shawn T; Caton, Andrew J; Erikson, Jan

    2005-04-01

    Anti-double-stranded DNA (anti-dsDNA) B cells are regulated in non-autoimmune mice. While some are deleted or undergo receptor editing, a population of anti-dsDNA (VH3H9/V lambda 1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B-cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T-cell help. When provided with T-cell help, the anti-dsDNA B cells differentiate into antibody-forming cells. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, the VH3H9/V lambda 1 anti-dsDNA B cells populate the B-cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti-dsDNA B-cell follicular entry, in the absence of autoantibody production, is dependent upon CD4(+) T cells. We hypothesize that control of autoantibody production in young autoimmune-prone mice may be regulated by the counterbalancing effect of T-regulatory (T(reg)) cells. Consistent with this model, we have demonstrated that T(reg) cells are able to prevent autoantibody production induced by T-cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T-cell-dependent and -independent activation on anti-dsDNA B cells.

  4. FcR-Like 2 Inhibition of B Cell Receptor-Mediated Activation of B Cells

    PubMed Central

    Jackson, Tanisha A.; Haga, Christopher L.; Ehrhardt, Götz R. A.; Davis, Randall S.; Cooper, Max D.

    2017-01-01

    FcR-like (FCRL) 2 is a transmembrane protein with immunomodulatory potential that is preferentially expressed by memory B cells in humans. It has two consensus ITIMs in addition to a putative ITAM sequence in its cytoplasmic domain. We have confirmed the cellular distribution of FCRL2 and analyzed its functional potential to show that coligation with the BCR leads to tyrosine phosphorylation of its ITIM motifs and subsequent Src homology region 2 domain-containing phosphatase-1 recruitment to facilitate inhibition of BCR signaling. Mutational analysis indicates that the tyrosine residues in both inhibitory motifs of FCRL2 are required for complete inhibition of BCR signaling, whereas tyrosines in the putative activation motif are dispensable for signal modulation. These findings suggest a negative immunomodulatory function for FCRL2 in the regulation of memory B cells. PMID:21068405

  5. Krüppel-Like Factor 4 Regulates B Cell Number and Activation-Induced B Cell Proliferation1

    PubMed Central

    Klaewsongkram, Jettanong; Yang, Yinhua; Golech, Susanne; Katz, Jonathan; Kaestner, Klaus H.; Weng, Nan-ping

    2008-01-01

    Krüppel-like factor 4 (Klf4) is a transcription factor and functions in regulating cell differentiation, cell growth, and cell cycle. Although Klf4 is expressed in lymphocytes, its function in lymphocytes is unknown. In this study, we report that the levels of Klf4 expression were low in pro-B cells and continuously increased in pre-B and in mature B cells. Upon activation, Klf4 was rapidly decreased in mature B cells after 2 h of activation. A modest decrease in numbers of pre-B cells in bone marrow and mature B cells in spleen was observed in Klf4-deficient mice. In the absence of Klf4, fewer B cells entered the S phase of the cell cycle and completed cell division in response to the engagement of BCR and/or CD40 in vitro. Furthermore, the delay in entering the cell cycle is associated with decreased expression of cyclin D2 in B cells that lack Klf4 expression. We then demonstrated that Klf4 directly bound to the promoter of cyclin D2 and regulated its expression. These findings demonstrate that Klf4 regulates B cell number and activation-induced B cell proliferation through directly acting on the promoter of cyclin D2. PMID:17878366

  6. Lymphomagenic CARD11/BCL10/MALT1 signaling drives malignant B-cell proliferation via cooperative NF-κB and JNK activation.

    PubMed

    Knies, Nathalie; Alankus, Begüm; Weilemann, Andre; Tzankov, Alexandar; Brunner, Kristina; Ruff, Tanja; Kremer, Marcus; Keller, Ulrich B; Lenz, Georg; Ruland, Jürgen

    2015-12-29

    The aggressive activated B cell-like subtype of diffuse large B-cell lymphoma is characterized by aberrant B-cell receptor (BCR) signaling and constitutive nuclear factor kappa-B (NF-κB) activation, which is required for tumor cell survival. BCR-induced NF-κB activation requires caspase recruitment domain-containing protein 11 (CARD11), and CARD11 gain-of-function mutations are recurrently detected in human diffuse large B-cell lymphoma (DLBCL). To investigate the consequences of dysregulated CARD11 signaling in vivo, we generated mice that conditionally express the human DLBCL-derived CARD11(L225LI) mutant. Surprisingly, CARD11(L225LI) was sufficient to trigger aggressive B-cell lymphoproliferation, leading to early postnatal lethality. CARD11(L225LI) constitutively associated with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) to simultaneously activate the NF-κB and c-Jun N-terminal kinase (JNK) signaling cascades. Genetic deficiencies of either BCL10 or MALT1 completely rescued the phenotype, and pharmacological inhibition of JNK was, similar to NF-κB blockage, toxic to autonomously proliferating CARD11(L225LI)-expressing B cells. Moreover, constitutive JNK activity was observed in primary human activated B cell-like (ABC)-DLBCL specimens, and human ABC-DLBCL cells were also sensitive to JNK inhibitors. Thus, our results demonstrate that enforced activation of CARD11/BCL10/MALT1 signaling is sufficient to drive transformed B-cell expansion in vivo and identify the JNK pathway as a therapeutic target for ABC-DLBCL.

  7. MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-{kappa}B-dependent lymphoma.

    PubMed

    Milhollen, Michael A; Traore, Tary; Adams-Duffy, Jennifer; Thomas, Michael P; Berger, Allison J; Dang, Lenny; Dick, Lawrence R; Garnsey, James J; Koenig, Erik; Langston, Steven P; Manfredi, Mark; Narayanan, Usha; Rolfe, Mark; Staudt, Louis M; Soucy, Teresa A; Yu, Jie; Zhang, Julie; Bolen, Joseph B; Smith, Peter G

    2010-09-02

    MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition. Treatment of germinal-center B cell-like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-kappaB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-kappaB-dependent lymphomas.

  8. FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas

    PubMed Central

    Brown, P J; Wong, K K; Felce, S L; Lyne, L; Spearman, H; Soilleux, E J; Pedersen, L M; Møller, M B; Green, T M; Gascoyne, D M; Banham, A H

    2016-01-01

    The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P<0.05). FOXP1 knockdown in ABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL patients (n=150), reduced HLA-DRA (<90% frequency) expression correlated with inferior overall survival (P=0.0003) and progression-free survival (P=0.0012) and with non-GCB subtype stratified by the Hans, Choi or Visco–Young algorithms (all P<0.01). In non-GCB DLBCL cases with <90% HLA-DRA, there was an inverse correlation with the frequency (P=0.0456) and intensity (P=0.0349) of FOXP1 expression. We propose that FOXP1 represents a novel regulator of genes targeted by the class II MHC transactivator CIITA (MHC II and CD74) and therapeutically targeting the FOXP1 pathway may improve antigen presentation and immune surveillance in high-risk DLBCL patients. PMID:26500140

  9. Activation of B cells by non-canonical helper signals.

    PubMed

    Cerutti, Andrea; Cols, Montserrat; Puga, Irene

    2012-09-01

    Cognate interaction between T and B lymphocytes of the adaptive immune system is essential for the production of high-affinity antibodies against microbes, and for the establishment of long-term immunological memory. Growing evidence shows that--in addition to presenting antigens to T and B cells--macrophages, dendritic cells and other cells of the innate immune system provide activating signals to B cells, as well as survival signals to antibody-secreting plasma cells. Here, we discuss how these innate immune cells contribute to the induction of highly diversified and temporally sustained antibody responses, both systemically and at mucosal sites of antigen entry.

  10. Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation.

    PubMed

    Wang, Jing; Tang, Shan; Wan, Zhengpeng; Gao, Yiren; Cao, Yiyun; Yi, Junyang; Si, Yanyan; Zhang, Haowen; Liu, Lei; Liu, Wanli

    2016-02-02

    Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.

  11. Coupled ATPase-adenylate kinase activity in ABC transporters

    PubMed Central

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  12. Transgelin-2 in B-Cells Controls T-Cell Activation by Stabilizing T Cell - B Cell Conjugates

    PubMed Central

    Chae, Myoung-Won; Kim, Hye-Ran; Kim, Chang-Hyun; Jun, Chang-Duk; Park, Zee-Yong

    2016-01-01

    The immunological synapse (IS), a dynamic and organized junction between T-cells and antigen presenting cells (APCs), is critical for initiating adaptive immunity. The actin cytoskeleton plays a major role in T-cell reorganization during IS formation, and we previously reported that transgelin-2, an actin-binding protein expressed in T-cells, stabilizes cortical F-actin, promoting T-cell activation in response to antigen stimulation. Transgelin-2 is also highly expressed in B-cells, although no specific function has been reported. In this study, we found that deficiency in transgelin-2 (TAGLN2-/-) in B-cells had little effect on B-cell development and activation, as measured by the expression of CD69, MHC class II molecules, and CD80/86. Nevertheless, in B-cells, transgelin-2 accumulated in the IS during the interaction with T-cells. These results led us to hypothesize that transgelin-2 may also be involved in IS stability in B-cells, thereby influencing T-cell function. Notably, we found that transgelin-2 deficiency in B-cells reduced T-cell activation, as determined by the release of IL-2 and interferon-γ and the expression of CD69. Furthermore, the reduced T-cell activation was correlated with reduced B-cell–T-cell conjugate formation. Collectively, these results suggest that actin stability in B-cells during IS formation is critical for the initiation of adaptive T-cell immunity. PMID:27232882

  13. Treatment of ongoing autoimmune encephalomyelitis with activated B-cell progenitors maturing into regulatory B cells

    PubMed Central

    Korniotis, Sarantis; Gras, Christophe; Letscher, Hélène; Montandon, Ruddy; Mégret, Jérôme; Siegert, Stefanie; Ezine, Sophie; Fallon, Padraic G.; Luther, Sanjiv A.; Fillatreau, Simon; Zavala, Flora

    2016-01-01

    The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here, we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes, where they trap T cells by releasing the CCR7 ligand, CCL19, and to inflamed central nervous system, where they locally limit immunopathogenesis through interleukin-10 production, thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment, where proB cells develop, is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition, these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases. PMID:27396388

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

    PubMed Central

    Fiala, Gina J.; Janowska, Iga; Prutek, Fabiola; Hobeika, Elias; Satapathy, Annyesha; Sprenger, Adrian; Plum, Thomas; Seidl, Maximilian; Dengjel, Jörn; Reth, Michael; Cesca, Fabrizia; Brummer, Tilman

    2015-01-01

    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, Ca2+, 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. PMID:26324445

  15. Akt and mTOR in B Cell Activation and Differentiation.

    PubMed

    Limon, Jose J; Fruman, David A

    2012-01-01

    Activation of phosphoinositide 3-kinase (PI3K) is required for B cell proliferation and survival. PI3K signaling also controls key aspects of B cell differentiation. Upon engagement of the B cell receptor (BCR), PI3K activation promotes Ca(2+) mobilization and activation of NFκB-dependent transcription, events which are essential for B cell proliferation. PI3K also initiates a distinct signaling pathway involving the Akt and mTOR serine/threonine kinases. It has been generally assumed that activation of Akt and mTOR downstream of PI3K is essential for B cell function. However, Akt and mTOR have complex roles in B cell fate decisions and suppression of this pathway can enhance certain B cell responses while repressing others. In this review we will discuss genetic and pharmacological studies of Akt and mTOR function in normal B cells, and in malignancies of B cell origin.

  16. Activation-induced cytidine deaminase (AID) expression in human B-cell precursors is essential for central B-cell tolerance

    PubMed Central

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M.; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E.; Notarangelo, Luigi D.; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D.; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2015-01-01

    SUMMARY Activation-induced cytidine deaminase (AID), the enzyme mediating class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B-cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B-cell intrinsic AID expression mediates central B-cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. PMID:26546282

  17. MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program.

    PubMed

    Hu, Shimin; Xu-Monette, Zijun Y; Tzankov, Alexander; Green, Tina; Wu, Lin; Balasubramanyam, Aarthi; Liu, Wei-min; Visco, Carlo; Li, Yong; Miranda, Roberto N; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L; Hsi, Eric D; Choi, William W L; Zhao, Xiaoying; van Krieken, J Han; Huang, Qin; Huh, Jooryung; Ai, Weiyun; Ponzoni, Maurilio; Ferreri, Andrés J M; Zhou, Fan; Slack, Graham W; Gascoyne, Randy D; Tu, Meifeng; Variakojis, Daina; Chen, Weina; Go, Ronald S; Piris, Miguel A; Møller, Michael B; Medeiros, L Jeffrey; Young, Ken H

    2013-05-16

    Diffuse large B-cell lymphoma (DLBCL) is stratified into prognostically favorable germinal center B-cell (GCB)-like and unfavorable activated B-cell (ABC)-like subtypes based on gene expression signatures. In this study, we analyzed 893 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We show that MYC/BCL2 protein coexpression occurred significantly more commonly in the ABC subtype. Patients with the ABC or GCB subtype of DLBCL had similar prognoses with MYC/BCL2 coexpression and without MYC/BCL2 coexpression. Consistent with the notion that the prognostic difference between the 2 subtypes is attributable to MYC/BCL2 coexpression, there is no difference in gene expression signatures between the 2 subtypes in the absence of MYC/BCL2 coexpression. DLBCL with MYC/BCL2 coexpression demonstrated a signature of marked downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. We conclude that MYC/BCL2 coexpression in DLBCL is associated with an aggressive clinical course, is more common in the ABC subtype, and contributes to the overall inferior prognosis of patients with ABC-DLBCL. In conclusion, the data suggest that MYC/BCL2 coexpression, rather than cell-of-origin classification, is a better predictor of prognosis in patients with DLBCL treated with R-CHOP.

  18. A Role for TLR Signaling During B Cell Activation in Antiretroviral-Treated HIV Individuals

    PubMed Central

    Keshavarzian, Ali; French, Audrey; Demarais, Patricia; Landay, Alan

    2013-01-01

    Abstract The mechanisms underlying B cell activation that persists during antiretroviral therapy (ART) are unknown. Toll-like receptor (TLR) signaling is a critical mediator of innate cell activation and though B cells express TLRs, few studies have investigated a role for TLR signaling in B cell activation during HIV infection. We addressed this question by assessing the activated phenotype and TLR expression/responsiveness of B cells from ART-treated HIV-infected subjects (HIVART+). We evaluated activation markers implicated in B cell-mediated T cell trans infection during HIV pathogenesis. We found no significant difference in TLR expression between B cells of HIVART+ and HIV− subjects. However, B cells of HIVART+ subjects exhibited heightened endogenous expression levels of IL-6 (p=0.0051), T cell cognate ligands CD40 (p=0.0475), CD54 (p=0.0229), and phosphorylated p38 (p<0.0001), a marker of TLR signaling. In vitro, B cells of HIVART+ individuals were less responsive to TLR stimulation compared to B cells of HIV− subjects. The activated phenotype of in vitro TLR-stimulated B cells of HIV− subjects was similar to ex vivo B cells from HIVART+ individuals. TLR2 stimulation was a potent mediator of B cell activation, whereas B cells were least responsive to TLR4 stimulation. Compared to HIV− subjects, the serum level of lipoteichoic acid (TLR2 ligand) in HIVART+ subjects was significantly higher (p=0.0207), correlating positively with viral load (p=0.0127, r=0.6453). Our data suggest that during HIV infection TLR-activated B cells may exert a pathogenic role and B cells from HIVART+ subjects respond to in vitro TLR stimulation, yet exhibit a TLR tolerant phenotype suggesting prior in vivo TLR stimulation. PMID:23763346

  19. Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection

    PubMed Central

    Sciaranghella, Gaia; Tong, Neath; Mahan, Alison E.; Suscovich, Todd J.; Alter, Galit

    2013-01-01

    Objective To define the impact of chronic viremia and associated immune activation on B-cell exhaustion in HIV infection. Design Progressive HIV infection is marked by B-cell anergy and exhaustion coupled with dramatic hypergammaglobulinemia. Although both upregulation of CD95 and loss of CD21 have been used as markers of infection-associated B-cell dysfunction, little is known regarding the specific profiles of dysfunctional B cells and whether persistent viral replication and its associated immune activation play a central role in driving B-cell dysfunction. Methods Multiparameter flow cytometry was used to define the profile of dysfunctional B cells. The changes in the expression of CD21 and CD95 were tracked on B-cell subpopulations in patients with differential control of viral replication. Results Although the emergence of exhausted, CD21low tissue-like memory B cells followed similar patterns in both progressors and controllers, the frequency of CD21low activated memory B cells was lower in spontaneous controllers. Conclusion Our results suggest that the loss of CD21 and the upregulation of CD95 occur as separate events during the development of B-cell dysfunction. The loss of CD21 is a marker of B-cell exhaustion induced in the absence of appreciable viral replication, whereas the upregulation of CD95 is tightly linked to persistent viral replication and its associated immune activation. Thus, these dysfunctional profiles potentially represent two functionally distinct states within the B-cell compartment. PMID:23135171

  20. Control of Viremia Enables Acquisition of Resting Memory B Cells with Age and Normalization of Activated B Cell Phenotypes in HIV-Infected Children

    PubMed Central

    Muema, Daniel M.; Macharia, Gladys N.; Hassan, Amin S.; Mwaringa, Shalton M.; Fegan, Greg W.; Berkley, James A.; Urban, Britta C.

    2015-01-01

    HIV affects the function of all lymphocyte populations, including B cells. Phenotypic and functional defects of B cells in HIV-infected adults have been well characterized, but defects in children have not been studied to the same extent. We determined the proportion of B cell subsets and frequencies of Ag-specific memory B cells in peripheral blood from HIV-infected children and healthy controls, using flow cytometry and B cell ELISPOT, respectively. In addition, we measured the quantities and avidities of plasma Abs against various Ags by ELISA. We also determined plasma levels of BAFF and expression of BAFF receptors on B cells. Children with high HIV viremia had increased proportions of activated mature B cells, tissue-like memory B cells and plasmablasts, and low proportions of naive B cells when compared with community controls and children with low HIV viremia, similar to adults infected with HIV. HIV-infected groups had lower proportions of resting memory B cells than did community controls. Notably, high HIV viremia prevented the age-dependent accumulation of class-switched resting memory B cells. HIV-infected children, regardless of the level of viremia, showed lower quantities and avidities of IgG and lower frequencies of memory B cells against Expanded Program on Immunization vaccines. The HIV-infected children had an altered BAFF profile that could have affected their B cell compartment. Therefore, B cell defects in HIV-infected children are similar to those seen in HIV-infected adults. However, control of HIV viremia is associated with normalization of activated B cell subsets and allows age-dependent accumulation of resting memory B cells. PMID:26116511

  1. Metabolic activity is necessary for activation of T suppressor cells by B cells

    SciTech Connect

    Elkins, K.L.; Stashak, P.W.; Baker, P.J. )

    1990-04-15

    Ag-primed B cells must express cell-surface IgM, but not IgD or Ia Ag, and must remain metabolically active, in order to activate suppressor T cells (Ts) specific for type III pneumococcal polysaccharide. Ag-primed B cells that were gamma-irradiated with 1000r, or less, retained the ability to activate Ts; however, Ag-primed B cells exposed to UV light were not able to do so. gamma-Irradiated and UV-treated Ag-primed B cells both expressed comparable levels of cell-surface IgM, and both localized to the spleen after in vivo transfer; neither could proliferate in vitro in response to mitogens. By contrast, gamma-irradiated primed B cells were still able to synthesize proteins, whereas UV-treated primed B cells could not. These findings suggest that in order for Ag-primed B cells to activate Ts, they must (a) express cell-associated IgM (sIgM) antibody bearing the idiotypic determinants of antibody specific for type III pneumococcal polysaccharide, and (b) be able to synthesize protein for either the continued expression of sIgM after cell transfer, or for the elaboration of another protein molecule that is also required for the activation of Ts; this molecule does not appear to be Ia Ag.

  2. ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans

    PubMed Central

    Hathcock, Karen S.; Padilla-Nash, Hesed M.; Camps, Jordi; Shin, Dong-Mi; Triner, Daniel; Shaffer, Arthur L.; Maul, Robert W.; Steinberg, Seth M.; Gearhart, Patricia J.; Staudt, Louis M.; Morse, Herbert C.; Ried, Thomas

    2015-01-01

    The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M+ B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell–like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors. PMID:26400962

  3. Clonal B cells in Waldenström's macroglobulinemia exhibit functional features of chronic active B-cell receptor signaling

    PubMed Central

    Argyropoulos, K V; Vogel, R; Ziegler, C; Altan-Bonnet, G; Velardi, E; Calafiore, M; Dogan, A; Arcila, M; Patel, M; Knapp, K; Mallek, C; Hunter, Z R; Treon, S P; van den Brink, M R M; Palomba, M L

    2016-01-01

    Waldenström's macroglobulinemia (WM) is a B-cell non-Hodgkin's lymphoma (B-NHL) characterized by immunoglobulin M (IgM) monoclonal gammopathy and the medullary expansion of clonal lymphoplasmacytic cells. Neoplastic transformation has been partially attributed to hyperactive MYD88 signaling, secondary to the MYD88 L265P mutation, occurring in the majority of WM patients. Nevertheless, the presence of chronic active B-cell receptor (BCR) signaling, a feature of multiple IgM+ B-NHL, remains a subject of speculation in WM. Here, we interrogated the BCR signaling capacity of primary WM cells by utilizing multiparametric phosphoflow cytometry and found heightened basal phosphorylation of BCR-related signaling proteins, and augmented phosphoresponses on surface IgM (sIgM) crosslinking, compared with normal B cells. In support of those findings we observed high sIgM expression and loss of phosphatase activity in WM cells, which could both lead to signaling potentiation in clonal cells. Finally, led by the high-signaling heterogeneity among WM samples, we generated patient-specific phosphosignatures, which subclassified patients into a ‘high' and a ‘healthy-like' signaling group, with the second corresponding to patients with a more indolent clinical phenotype. These findings support the presence of chronic active BCR signaling in WM while providing a link between differential BCR signaling utilization and distinct clinical WM subgroups. PMID:26867669

  4. Activation of the STAT3 Signaling Pathway Is Associated With Poor Survival in Diffuse Large B-Cell Lymphoma Treated With R-CHOP

    PubMed Central

    Huang, Xin; Meng, Bin; Iqbal, Javeed; Ding, B. Belinda; Perry, Anamarija M.; Cao, Wenfeng; Smith, Lynette M.; Bi, Chengfeng; Jiang, Chunsun; Greiner, Timothy C.; Weisenburger, Dennis D.; Rimsza, Lisa; Rosenwald, Andreas; Ott, German; Delabie, Jan; Campo, Elias; Braziel, Rita M.; Gascoyne, Randy D.; Cook, James R.; Tubbs, Raymond R.; Jaffe, Elaine S.; Armitage, James O.; Vose, Julie M.; Staudt, Louis M.; McKeithan, Timothy W.; Chan, Wing C.; Ye, B. Hilda; Fu, Kai

    2013-01-01

    Purpose We previously reported that constitutive STAT3 activation is a prominent feature of the activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL). In this study, we investigated whether STAT3 activation can risk stratify patients with DLBCL. Patients and Methods By an immunohistochemical method, we investigated phosphotyrosine STAT3 (PY-STAT3) expression from 185 patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Cell line-based siRNA experiments were also performed to generate an 11-gene, PY-STAT3 activation signature, which was used to study a previously published cohort of 222 patients with DLBCL. The STAT3 activation status determined by these two methods and by STAT3 mRNA levels were then correlated with survival. Results PY-STAT3 was detected in 37% of DLBCL and enriched in ABC-DLBCL cases (P = .03). PY-STAT3 positivity significantly correlated with poor overall survival (OS; P = .01) and event-free survival (EFS; P = .006). Similar observations were made for high levels of STAT3 mRNA. In multivariable analysis, PY-STAT3 status (P = .02), International Prognostic Index (P = .02), and BCL2 expression (P = .046) were independent prognosticators of OS in this cohort. Among the cell-of-origin subgroups, PY-STAT3 was associated with poor EFS among non–germinal center B-cell DLBCL cases only (P = .027). Similarly, the 11-gene STAT3 activation signature correlated with poor survival in the entire DLBCL cohort (OS, P < .001; EFS, P < .001) as well as the ABC-DLBCL subgroup (OS, P = .029; EFS, P = .025). Conclusion STAT3 activation correlated with poor survival in patients with DLBCL treated with R-CHOP, especially those with tumors of the ABC-DLBCL subtype. PMID:24220563

  5. Induction of polyclonal B cell activation and differentiation by the AIDS retrovirus (HTLV-III/LAV)

    SciTech Connect

    Higgins, S.E.; Schnittman, S.M.; Lane, H.C.; Folks, T.; Koenig, S.; Fauci, A.S.

    1986-03-05

    The immune systems of individuals infected with HTLV-III/LAV are characterized by a profound defect in cellular immunity together with paradoxical polyclonal B cell activation. The present study examined the direct effects of HTLV-III/LAV on B lymphocytes. Peripheral blood B cells from healthy donors were incubated with a variety of HTLV-III/LAV isolates for 1 h and /sup 3/H-thymidine incorporation was measured at multiple time points. Responses ranged from 9000-28,000 cpm and peaked on day 4. This B cell activation was not enhanced by the addition of interleukin-2 to culture, was not synergistic with Staphylococcus aureus Cowan I, was not modulated by the addition of T lymphocytes to culture, and was not associated with B cell transformation. Supernatant Ig could first be detected in virus-activated cultures at day 4, plateaued by day 8, and yielded a mean of 12,500 ng IgG+IgM/ml/50,000 B cells. Thus, HTLV-III/LAV is a potent T cell independent B cell mitogen capable of inducing B cell activation, proliferation, and differentiation comparable in magnitude to that of the most potent B cell activators. This biological property of HTLV-III/LAV may help explain the profound polyclonal B cell activation observed in patients with AIDS and may provide investigators with another probe for investigating the mechanisms of B cell activation.

  6. Addition of an Indoleamine-2,3,-dioxygenase Inhibitor to B cell Depletion Therapy Blocks Autoreactive B Cell Activation and Recurrence of Arthritis

    PubMed Central

    Pigott, Elizabeth; Mandik-Nayak, Laura

    2012-01-01

    Objective Define the role indoleamine-2,3-dioxygenase (IDO) plays in driving pathogenic B cells responses leading to arthritis and determine if inhibitors of the IDO pathway can be used in conjunction with B cell depletion therapy to prevent the re-emergence of autoantibodies and arthritis following reconstitution of the B cell repertoire. Methods Immunoglobulin transgenic mice were treated with the IDO inhibitor 1-methyl-tryptophan (1MT) and followed for the extent of autoreactive B cell activation. Arthritic mice (K/BxN) were treated with B cell depletion therapy alone or in combination with 1MT. Mice were followed for the presence of autoantibody secreting cells, inflammatory cytokines, and joint inflammation. Results 1MT did not affect the initial activation or survival of autoreactive B cells, but did inhibit their ability to differentiate into autoantibody secreting cells. Treatment with anti-CD20 depleted the B cell repertoire and attenuated arthritis symptoms; however, arthritis symptoms rapidly returned as B cells repopulated the repertoire. Administration of 1MT prior to B cell repopulation prevented the production of autoantibodies, inflammatory cytokines, and flare in arthritis symptoms. Conclusion IDO activity is essential for the differentiation of autoreactive B cells into antibody secreting cells, but is not necessary for their initial stages of activation. Addition of 1MT to B cell depletion therapy prevents the differentiation of autoantibody secreting cells and recurrence of autoimmune arthritis following reconstitution of the B cell repertoire. These data suggest that IDO inhibitors could be used in conjunction with B cell depletion as an effective co-therapeutic strategy in the treatment of rheumatoid arthritis. PMID:22294267

  7. Quorum Sensing Contributes to Activated IgM-Secreting B Cell Homeostasis

    PubMed Central

    Montaudouin, Caroline; Anson, Marie; Hao, Yi; Duncker, Susanne V.; Fernandez, Tahia; Gaudin, Emmanuelle; Ehrenstein, Michael; Kerr, William G.; Colle, Jean-Hervé; Bruhns, Pierre; Daëron, Marc; Freitas, António A.

    2013-01-01

    Maintenance of plasma IgM levels is critical for immune system function and homeostasis in humans and mice. However, the mechanisms that control homeostasis of the activated IgM-secreting B cells are unknown. After adoptive transfer into immune-deficient hosts, B lymphocytes expand poorly, but fully reconstitute the pool of natural IgM-secreting cells and circulating IgM levels. By using sequential cell transfers and B cell populations from several mutant mice, we were able to identify novel mechanisms regulating the size of the IgM-secreting B cell pool. Contrary to previous mechanisms described regulating homeostasis, which involve competition for the same niche by cells having overlapping survival requirements, homeostasis of the innate IgM-secreting B cell pool is also achieved when B cell populations are able to monitor the number of activated B cells by detecting their secreted products. Notably, B cell populations are able to assess the density of activated B cells by sensing their secreted IgG. This process involves the FcγRIIB, a low-affinity IgG receptor that is expressed on B cells and acts as a negative regulator of B cell activation, and its intracellular effector the inositol phosphatase SHIP. As a result of the engagement of this inhibitory pathway, the number of activated IgM-secreting B cells is kept under control. We hypothesize that malfunction of this quorum-sensing mechanism may lead to uncontrolled B cell activation and autoimmunity. PMID:23209322

  8. Activation of resting human B cells by helper T-cell clone supernatant: characterization of a human B-cell-activating factor.

    PubMed Central

    Diu, A; Gougeon, M L; Moreau, J L; Reinherz, E L; Thèze, J

    1987-01-01

    The effects of helper T-cell clone supernatants on resting human B cells were investigated. Four different helper T-cell clones (two T4+ and two T8+) were stimulated by anti-T3 monoclonal antibodies on Sepharose beads or anti-T11(2) plus anti-T11(3) monoclonal antibodies. The supernatants from these activated clones induced the proliferation of highly purified resting B lymphocytes from the peripheral blood. The B cells exhibited a cell size and a surface-antigen pattern (4F2 antigen and transferrin receptor) of phase G0 B cells, and they were functionally resting. In response to T-cell supernatants a large fraction of the B cells enlarged and expressed 4F2 antigens and transferrin receptors. In gel filtration, the corresponding activity migrated with an apparent Mr of 12,000-15,000. Our findings strongly support the existence of a human B-cell-activating factor acting on resting B cells and causing them to enter phase G1 of the cell cycle. PMID:2962196

  9. B-cell activation in cats with feline infectious peritonitis (FIP) by FIP-virus-induced B-cell differentiation/survival factors.

    PubMed

    Takano, Tomomi; Azuma, Natsuko; Hashida, Yoshikiyo; Satoh, Ryoichi; Hohdatsu, Tsutomu

    2009-01-01

    It has been suggested that antibody overproduction plays a role in the pathogenesis of feline infectious peritonitis (FIP). However, only a few studies on the B-cell activation mechanism after FIP virus (FIPV) infection have been reported. The present study shows that: (1) the ratio of peripheral blood sIg(+) CD21(-) B-cells was higher in cats with FIP than in SPF cats, (2) the albumin-to-globulin ratio has negative correlation with the ratio of peripheral blood sIg(+) CD21(-) B-cell, (3) cells strongly expressing mRNA of the plasma cell master gene, B-lymphocyte-induced maturation protein 1 (Blimp-1), were increased in peripheral blood in cats with FIP, (4) mRNA expression of B-cell differentiation/survival factors, IL-6, CD40 ligand, and B-cell-activating factor belonging to the tumor necrosis factor family (BAFF), was enhanced in macrophages in cats with FIP, and (5) mRNAs of these B-cell differentiation/survival factors were overexpressed in antibody-dependent enhancement (ADE)-induced macrophages. These data suggest that virus-infected macrophages overproduce B-cell differentiation/survival factors, and these factors act on B-cells and promote B-cell differentiation into plasma cells in FIPV-infected cats.

  10. Synergistic activation of cells by Epstein-Barr virus and B-cell growth factor.

    PubMed Central

    Hutt-Fletcher, L M

    1987-01-01

    Infection with Epstein-Barr virus (EBV) is initiated by virus binding to the C3dg-C3d receptor CR2. Several workers have implicated this receptor in the control of B-cell activation by examining the effects of antibodies to CR2 and isolated C3d on B-cell proliferation and differentiation. We report here on the activating effects of irradiated EBV, which retains its capacity to bind to CR2 but loses its ability to function as a T-independent B-cell activator. EBV synergized with B-cell growth factor in the induction of uptake of tritiated thymidine by T cell-depleted leukocytes from seronegative donors but did not induce secretion of immunoglobulin. Synergism could be inhibited with an anti-viral antibody that inhibited binding of EBV to CR2. No similar synergism was found between EBV and recombinant interleukin 2, interleukin 1 alpha, or gamma interferon or with the lipid A fraction of bacterial lipopolysaccharide. EBV may thus initiate B-cell activation as it binds to CR2. Infectious virus may, under normal circumstances, induce the cell to make those growth factors necessary to support B-cell proliferation; the difficulty of transforming cells with transfected EBV DNA may in part reflect the absence of an activation event provided by intact virus as it attaches to CR2. The synergism of EBV and B-cell growth factor more clearly distinguishes the effects of B-cell growth factor from those of interleukin 1 and interleukin 2 in other models of B-cell activation. Thus, this may be a useful model for further delineation of unique effects of B-cell growth factor on B-cell function. PMID:3027404

  11. Regulation of Cell Surface CB2 Receptor during Human B Cell Activation and Differentiation.

    PubMed

    Castaneda, Julie T; Harui, Airi; Roth, Michael D

    2017-03-31

    Cannabinoid receptor type 2 (CB2) is the primary receptor pathway mediating the immunologic consequences of cannabinoids. We recently reported that human peripheral blood B cells express CB2 on both the extracellular membrane and at intracellular sites, where-as monocytes and T cells only express intracellular CB2. To better understand the pattern of CB2 expression by human B cells, we examined CD20(+) B cells from three tissue sources. Both surface and intracellular expression were present and uniform in cord blood B cells, where all cells exhibited a naïve mature phenotype (IgD(+)/CD38(Dim)). While naïve mature and quiescent memory B cells (IgD(-)/CD38(-)) from tonsils and peripheral blood exhibited a similar pattern, tonsillar activated B cells (IgD(-)/CD38(+)) expressed little to no surface CB2. We hypothesized that regulation of the surface CB2 receptor may occur during B cell activation. Consistent with this, a B cell lymphoma cell line known to exhibit an activated phenotype (SUDHL-4) was found to lack cell surface CB2 but express intracellular CB2. Furthermore, in vitro activation of human cord blood resulted in a down-regulation of surface CB2 on those B cells acquiring the activated phenotype but not on those retaining IgD expression. Using a CB2 expressing cell line (293 T/CB2-GFP), confocal microscopy confirmed the presence of both cell surface expression and multifocal intracellular expression, the latter of which co-localized with endoplasmic reticulum but not with mitochondria, lysosomes, or nucleus. Our findings suggest a dynamic multi-compartment expression pattern for CB2 in B cells that is specifically modulated during the course of B cell activation.

  12. The novel kinesin spindle protein (KSP) inhibitor SB-743921 exhibits marked activity in in vivo and in vitro models of aggressive large B-cell lymphoma.

    PubMed

    Bongero, Danielle; Paoluzzi, Luca; Marchi, Enrica; Zullo, Kelly M; Neisa, Roberto; Mao, Yinghui; Escandon, Rafael; Wood, Ken; O'Connor, Owen A

    2015-01-01

    The kinesin spindle protein (KSP) is a mitotic protein essential for cell cycle control and motility. SB-743921 (hereafter SB-921) is an inhibitor that selectively targets the ATP-binding domain of the KSP. The preclinical activity of SB-921 was evaluated in models of diffuse large B-cell lymphoma (DLBCL). The cytotoxicity of SB-921 was evaluated in a series of germinal center (GC-DLBCL) and post-germinal center (ABC-DLBCL) DLBCL cell lines and a murine lymphoma xenograft model. GC-DLBCL lines generally demonstrated greater sensitivity to SB-921. IC50 values ranged between 1 nM and 900 nM for GC-DLBCL compared to 1 nM to 10 μM for ABC lines. SB-921 demonstrated marked activity in a xenograft model of Ly-1 (GC-DLBCL). While SB-921 was relatively more active in GC derived cell lines, ABC-derived lines still underwent apoptosis at higher concentrations. These results demonstrate that SB-921 inhibits proliferation and induces apoptosis in both GC-DLBCL and ABC-DLBCL.

  13. B cells from African American lupus patients exhibit an activated phenotype

    PubMed Central

    Menard, Laurence C.; Habte, Sium; Gonsiorek, Waldemar; Lee, Deborah; Banas, Dana; Holloway, Deborah A.; Cunningham, Mark; Stetsko, Dawn; Casano, Francesca; Kansal, Selena; Davis, Patricia M.; Carman, Julie; Zhang, Clarence K.; Abidi, Ferva; Furie, Richard; Nadler, Steven G.; Suchard, Suzanne J.

    2016-01-01

    Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease driven by both innate and adaptive immune cells. African Americans tend to present with more severe disease at an earlier age compared with patients of European ancestry. In order to better understand the immunological differences between African American and European American patients, we analyzed the frequencies of B cell subsets and the expression of B cell activation markers from a total of 68 SLE patients and 69 normal healthy volunteers. We found that B cells expressing the activation markers CD86, CD80, PD1, and CD40L, as well as CD19+CD27–IgD– double-negative B cells, were enriched in African American patients vs. patients of European ancestry. In addition to increased expression of CD40L, surface levels of CD40 on B cells were lower, suggesting the engagement of the CD40 pathway. In vitro experiments confirmed that CD40L expressed by B cells could lead to CD40 activation and internalization on adjacent B cells. To conclude, these results indicate that, compared with European American patients, African American SLE patients present with a particularly active B cell component, possibly via the activation of the CD40/CD40L pathway. These data may help guide the development of novel therapies. PMID:27699274

  14. B cell receptor-mediated internalization of salmonella: a novel pathway for autonomous B cell activation and antibody production.

    PubMed

    Souwer, Yuri; Griekspoor, Alexander; Jorritsma, Tineke; de Wit, Jelle; Janssen, Hans; Neefjes, Jacques; van Ham, S Marieke

    2009-06-15

    The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion of anti-Salmonella Ab by the Salmonella-specific B cells. In addition, BCR-mediated internalization leads to efficient Ag delivery to the MHC class II Ag-loading compartments, even though Salmonella remains vital intracellularly in primary B cells. Consequently, BCR-mediated bacterial uptake induces efficient CD4(+) T cell help, which boosts Salmonella-specific Ab production. BCR-mediated internalization of Salmonella by B cells is superior over extracellular Ag extraction to induce rapid and specific humoral immune responses and efficiently combat infection.

  15. Evidence for B cell activation in patients with active rheumatoid arthritis.

    PubMed Central

    Youinou, P Y; Irving, W L; Shipley, M; Hayes, J; Lydyard, P M

    1984-01-01

    Peripheral blood lymphocytes and in some cases synovial eluate cells from 51 patients with rheumatoid arthritis (RA), were analysed for the percentages of cells bearing surface light chains (total B cells), IgM and IgD. In addition, their capacity to form rosettes with mouse erythrocytes (mRFC)--a property of a B cell subpopulation--was determined. Activity of the disease was assessed by clinical and laboratory criteria and classified as very active, moderately active and inactive. Normal, age and sex matched individuals and a group of patients with a variety of other rheumatological disorders, were used as control populations. Although there was no significant difference in percentages of total B cells in any of the groups compared with normal controls, there was a small but significant increase in the ratio of cells bearing IgM to those bearing IgD in patients with very active disease. This was paralleled by a significant decrease in the mRFC in this disease activity group. Patients with inactive disease showed no change in their proportions of IgM:IgD, but did show a significant increase in mRFC. These results are discussed in terms of the presence of activated B cells in patients with very active RA. PMID:6607144

  16. TSPAN33 is a novel marker of activated and malignant B cells

    PubMed Central

    Luu, Van Phi; Hevezi, Peter; Vences-Catalan, Felipe; Maravillas-Montero, Jose Luis; White, Clayton Alexander; Casali, Paolo; Llorente, Luis; Jakez-Ocampo, Juan; Lima, Guadalupe; Vilches-Cisneros, Natalia; Flores-Gutiérrez, Juan Pablo; Santos-Argumedo, Leopoldo; Zlotnik, Albert

    2014-01-01

    We have identified Tspan33 as a gene encoding a transmembrane protein exhibiting a restricted expression pattern including expression in activated B cells. TSPAN33 is a member of the tetraspanin family. TSPAN33 is not expressed in resting B cells, but is strongly induced in primary human B cells following activation. Human 2E2 cells, a Burkitt’s lymphoma-derived B cell model of activation and differentiation, also upregulate TSPAN33 upon activation. TSPAN33 is expressed in several lymphomas including Hodgkin’s and Diffuse large B Cell Lymphoma. TSPAN33 is also expressed in some autoimmune diseases where B cells participate in the pathology, including rheumatoid arthritis patients, systemic lupus erythematosus (SLE), and in spleen B cells from MRL/Faslpr/lpr mice (a mouse model of SLE). We conclude that TSPAN33 may be used as a diagnostic biomarker or as a target for therapeutic antibodies for treatment of certain B cell lymphomas or autoimmune diseases. PMID:24211713

  17. The ABCs of Activity-Based Costing: A Cost Containment and Reallocation Tool.

    ERIC Educational Resources Information Center

    Turk, Frederick J.

    1992-01-01

    This article describes activity-based costing (ABC) and how this tool may help management understand the costs of major activities and identify possible alternatives. Also discussed are the traditional costing systems used by higher education and ways of applying ABC to higher education. (GLR)

  18. Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro.

    PubMed

    Chiappini, Nico; Cantisani, Rocco; Pancotto, Laura; Ruggiero, Paolo; Rosa, Domenico; Manetti, Andrea; Romano, Antonio; Montagnani, Francesca; Bertholet, Sylvie; Castellino, Flora; Del Giudice, Giuseppe

    2015-01-01

    Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19⁺CD20⁺GM-CSF⁺ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19⁺CD20⁺GM-CSF⁻ B cells. These cells reside within the B cell follicles, are mostly IgM⁺IgD⁺, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.

  19. Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro

    PubMed Central

    Pancotto, Laura; Ruggiero, Paolo; Rosa, Domenico; Manetti, Andrea; Romano, Antonio; Montagnani, Francesca; Bertholet, Sylvie; Castellino, Flora; Del Giudice, Giuseppe

    2015-01-01

    Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19+CD20+GM-CSF+ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19+CD20+GM-CSF- B cells. These cells reside within the B cell follicles, are mostly IgM+IgD+, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils. PMID:26066485

  20. Signals from activation of B-cell receptor with anti-IgD can override the stimulatory effects of excess BAFF on mature B cells in vivo.

    PubMed

    Nguyen, Tue G; Morris, Jonathan M

    2014-09-01

    The selection and maturation of B-cell clones are critically determined by tonic signals from activated B cell receptors (BCR) and survival signals from BAFF cytokine. These finely tuned and coordinated signals provide a net positive signal that can promote the selection, maturation, proliferation and differentiation of a developing B cell. Stimulation with an anti-IgD antibody can also activate BCR but can lead to depletion and an arrest of mature B-cell development in vivo. It is not known whether survival signals from excess BAFF can override the suppressive effects of treatment with anti-IgD on mature B cells in vivo. Herein, we examined the effects of co-treatment of BAFF and anti-IgD on the mature B-cell compartment and antibody production in vivo by treating mice with either 1mg/kg BAFF or anti-IgD alone or in combination for 3 consecutive days. We found that co-treatment with anti-IgD significantly abrogated these stimulatory effects of BAFF treatment on splenic CD19+ B cells as well as mature CD19+IgD(hi)IgM+ B cells in vivo. Anti-IgD down-regulated the expression of the BCR complex (mIgM, mIgD and CD19) and the BAFF receptor TACI without regard to the presence of BAFF. Anti-IgD treatment also significantly negated BAFF-induced IgM production in vivo. Both BAFF and anti-IgD could individually stimulate IL-10 synthesis in B cells but did not affect one another. Taken together, our data suggest that activation of BCR with an anti-IgD antibody can override the stimulatory effects from excess BAFF on B cell proliferation and antibody production by down-regulating the expression of BCR complex and BAFF receptors.

  1. The Role of Activity Based Costing (ABC) in Educational Support Services: A White Paper.

    ERIC Educational Resources Information Center

    Edds, Daniel B.

    Many front-line managers who are assuming more financial responsibility for their organizations find traditional cost accounting inadequate for their needs and are turning to Activity Based Costing (ABC). ABC is not a financial reporting system to serve the needs of regulatory agencies, but a tool that tracks costs from the general ledger…

  2. Follicular B cell trafficking within the spleen actively restricts humoral immune responses

    PubMed Central

    Hoek, Kristen L.; Gordy, Laura E.; Collins, Patrick L.; Parekh, Vrajesh V.; Aune, Thomas M.; Joyce, Sebastian; Thomas, James W.; Van Kaer, Luc; Sebzda, Eric

    2010-01-01

    Summary Follicular (FO) and marginal zone (MZ) B cells are maintained in distinct locations within the spleen but the genetic basis for this separation is still enigmatic. We now report that B cell sequestration requires lineage-specific regulation of migratory receptors by the transcription factor, Klf2. Moreover, using gene-targeted mice we show that altered splenic B cell migration confers a significant in vivo gain-of-function phenotype to FO B cells, including the ability to quickly respond to MZ-associated antigens and pathogens in a T cell-dependent manner. This work demonstrates that in wild-type animals, naïve FO B cells are actively removed from the MZ, thus restricting their capacity to respond to blood-borne pathogens. PMID:20691614

  3. CD4 T cell activation by B cells in human Leishmania (Viannia) infection

    PubMed Central

    2014-01-01

    Background An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated. Methods CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC. Results Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells. Conclusions The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to

  4. Suppression of human B cell activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin involves altered regulation of B cell lymphoma-6.

    PubMed

    Phadnis-Moghe, Ashwini S; Crawford, Robert B; Kaminski, Norbert E

    2015-03-01

    The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces marked suppression of the primary humoral immune response in virtually every animal species evaluated thus far. In addition, epidemiological studies performed in areas of dioxin contamination have identified an association between TCDD exposure and an increased incidence of non-Hodgkin's lymphoma (NHL). Recent studies using an in vitro CD40 ligand model of human B cell differentiation have shown that TCDD impairs both B cell activation and differentiation. The present study extends these findings by identifying B cell lymphoma-6 [BCL-6] as a putative cellular target for deregulation by TCDD, which may contribute to suppression of B cell function as well as NHL. BCL-6 is a multifunctional transcriptional repressor frequently mutated in NHLs and known to regulate critical events of B cell activation and differentiation. In the presence of TCDD, BCL-6 protein levels were elevated and concurrently the same population of cells with high BCL-6 levels showed decreased CD80 and CD69 expression indicative of impaired cellular activation. The elevated BCL-6 levels resulted in a concomitant increase in BCL-6 DNA binding activity at its cognate binding site within an enhancer region for CD80. Furthermore, a small molecule inhibitor of BCL-6 activity reversed TCDD-mediated suppression of CD80 expression in human B cells. In the presence of a low-affinity ligand of the aryl hydrocarbon receptor (AHR), suppression of B cell activation and altered BCL-6 regulation were not observed. These results provide new mechanistic insights into the role of BCL-6 in the suppression of human B cell activation by TCDD.

  5. Targeting Non-proteolytic Protein Ubiquitination for the Treatment of Diffuse Large B Cell Lymphoma.

    PubMed

    Yang, Yibin; Kelly, Priscilla; Shaffer, Arthur L; Schmitz, Roland; Yoo, Hee Min; Liu, Xinyue; Huang, Da Wei; Webster, Daniel; Young, Ryan M; Nakagawa, Masao; Ceribelli, Michele; Wright, George W; Yang, Yandan; Zhao, Hong; Yu, Xin; Xu, Weihong; Chan, Wing C; Jaffe, Elaine S; Gascoyne, Randy D; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa; Staudt, Louis M

    2016-04-11

    Chronic active B cell receptor (BCR) signaling, a hallmark of the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), engages the CARD11-MALT1-BCL10 (CBM) adapter complex to activate IκB kinase (IKK) and the classical NF-κB pathway. Here we show that the CBM complex includes the E3 ubiquitin ligases cIAP1 and cIAP2, which are essential mediators of BCR-dependent NF-κB activity in ABC DLBCL. cIAP1/2 attach K63-linked polyubiquitin chains on themselves and on BCL10, resulting in the recruitment of IKK and the linear ubiquitin chain ligase LUBAC, which is essential for IKK activation. SMAC mimetics target cIAP1/2 for destruction, and consequently suppress NF-κB and selectively kill BCR-dependent ABC DLBCL lines, supporting their clinical evaluation in patients with ABC DLBCL.

  6. Exogenous and endogenous TLR ligands activate anti-chromatin and polyreactive B cells.

    PubMed

    Fields, Michele L; Metzgar, Michele H; Hondowicz, Brian D; Kang, Sun-Ah; Alexander, Shawn T; Hazard, Kristin D; Hsu, Alice C; Du, Yang-Zhu; Prak, Eline Luning; Monestier, Marc; Erikson, Jan

    2006-06-01

    Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.

  7. Kaposi's Sarcoma-Associated Herpesvirus Latency Locus Compensates for Interleukin-6 in Initial B Cell Activation.

    PubMed

    Sin, Sang-Hoon; Kang, Sun Ah; Kim, Yongbaek; Eason, Anthony; Tan, Kelly; An, Hyowon; Dittmer, Dirk P

    2015-12-09

    Interleukin 6 (IL-6) is considered a proliferation and survival factor for B cells. To assess the role of IL-6 in Kaposi sarcoma-associated herpesvirus (KSHV) latency, KSHV latency locus-transgenic mice (referred to as latency mice) lacking IL-6 were evaluated. IL-6(-/-) latency mice had the same phenotypes as the latency mice, i.e., increased frequency of marginal zone B cells, hyperplasia, and hyperglobulinemia, indicating that the KSHV latency locus, which includes all viral microRNAs (miRNAs), can compensate for lack of IL-6 in premalignant B cell activation.

  8. Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells

    PubMed Central

    Avalos, Ana M.; Bilate, Angelina M.; Witte, Martin D.; Tai, Albert K.; He, Jiang; Frushicheva, Maria P.; Thill, Peter D.; Meyer-Wentrup, Friederike; Theile, Christopher S.; Chakraborty, Arup K.; Zhuang, Xiaowei

    2014-01-01

    Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR. By altering length and valency of epitope-bearing synthetic peptides, we examined the properties of ligands required for optimal OB1 B cell activation. Monovalent engagement of the BCR with an epitope-bearing 17-mer synthetic peptide readily activated OB1 B cells. Dimers of the minimal peptide epitope oriented in an N to N configuration were more stimulatory than their C to C counterparts. Although shorter length correlated with less activation, a monomeric 8-mer peptide epitope behaved as a weak agonist that blocked responses to cell-bound peptide antigen, a blockade which could not be reversed by CD40 ligation. The 8-mer not only delivered a suboptimal signal, which blocked subsequent responses to OVA, anti-IgG, and anti-kappa, but also competed for binding with OVA. Our results show that fine-tuning of BCR-ligand recognition can lead to B cell nonresponsiveness, activation, or inhibition. PMID:24493799

  9. The impact of inflammation and immune activation on B cell differentiation during HIV-1 infection.

    PubMed

    Ruffin, Nicolas; Thang, Pham Hong; Rethi, Bence; Nilsson, Anna; Chiodi, Francesca

    2011-01-01

    One important pathogenic feature of human immunodeficiency virus (HIV)-1 infection is chronic immune activation and impaired survival of T and B cells. A decline of resting memory B cells was reported to occur in both children and adults infected with HIV-1; these cells are responsible for maintaining an adequate serological response to antigens previously encountered in life through natural infection or vaccination. Further understanding of the mechanisms leading to impaired B cell differentiation and germinal center reaction might be essential to design new HIV vaccines and therapies that could improve humoral immune responses in HIV-1 infected individuals. In the present article we summarize the literature and present our view on critical mechanisms of B cell development impaired during HIV-1 infection. We also discuss the impact of microbial translocation, a driving force for persistent inflammation during HIV-1 infection, on survival of terminally differentiated B cells and how the altered expression of cytokines/chemokines pivotal for communication between T and B cells in lymphoid tissues may impair formation of memory B cells.

  10. The Impact of Inflammation and Immune Activation on B Cell Differentiation during HIV-1 Infection

    PubMed Central

    Ruffin, Nicolas; Thang, Pham Hong; Rethi, Bence; Nilsson, Anna; Chiodi, Francesca

    2012-01-01

    One important pathogenic feature of human immunodeficiency virus (HIV)-1 infection is chronic immune activation and impaired survival of T and B cells. A decline of resting memory B cells was reported to occur in both children and adults infected with HIV-1; these cells are responsible for maintaining an adequate serological response to antigens previously encountered in life through natural infection or vaccination. Further understanding of the mechanisms leading to impaired B cell differentiation and germinal center reaction might be essential to design new HIV vaccines and therapies that could improve humoral immune responses in HIV-1 infected individuals. In the present article we summarize the literature and present our view on critical mechanisms of B cell development impaired during HIV-1 infection. We also discuss the impact of microbial translocation, a driving force for persistent inflammation during HIV-1 infection, on survival of terminally differentiated B cells and how the altered expression of cytokines/chemokines pivotal for communication between T and B cells in lymphoid tissues may impair formation of memory B cells. PMID:22566879

  11. Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

    PubMed Central

    Villar, Rina F.; Patel, Jinal; Weaver, Grant C.; Kanekiyo, Masaru; Wheatley, Adam K.; Yassine, Hadi M.; Costello, Catherine E.; Chandler, Kevin B.; McTamney, Patrick. M.; Nabel, Gary J.; McDermott, Adrian B.; Mascola, John R.; Carr, Steven A.; Lingwood, Daniel

    2016-01-01

    Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed. PMID:27796362

  12. Osteoclast activity modulates B-cell development in the bone marrow.

    PubMed

    Mansour, Anna; Anginot, Adrienne; Mancini, Stéphane J C; Schiff, Claudine; Carle, Georges F; Wakkach, Abdelilah; Blin-Wakkach, Claudine

    2011-07-01

    B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.

  13. Homocysteine Activates B Cells via Regulating PKM2-Dependent Metabolic Reprogramming

    PubMed Central

    Deng, Jiacheng; Lü, Silin; Liu, Huiying; Liu, Bo; Jiang, Changtao; Xu, Qingbo

    2017-01-01

    The overactivation of immune cells plays an important role in the pathogenesis of hyperhomocysteinemia (HHcy)-accelerated atherosclerosis. Homocysteine (Hcy) activates B cell proliferation and Ab secretion; however, the underlying mechanisms for these effects remain largely unknown. Metabolic reprogramming is critical for lymphocyte activation and effector function. In this study, we showed that Hcy-activated B cells displayed an increase in both oxidative phosphorylation and glycolysis, with a tendency to shift toward the latter, as well as an accumulation of intermediates in the pentose phosphate pathway, to provide energy and biosynthetic substrates for cell growth and function. Mechanistically, Hcy increased both the protein expression and glycolytic enzyme activity of the pyruvate kinase muscle isozyme 2 (PKM2) in B cells, whereas the PKM2 inhibitor shikonin restored Hcy-induced metabolic changes, as well as B cell proliferation and Ab secretion both in vivo and in vitro, indicating that PKM2 plays a critical role in metabolic reprogramming in Hcy-activated B cells. Further investigation revealed that the Akt–mechanistic target of rapamycin signaling pathway was involved in this process, as the mechanistic target of rapamycin inhibitor rapamycin inhibited Hcy-induced changes in PKM2 enzyme activity and B cell activation. Notably, shikonin treatment effectively attenuated HHcy-accelerated atherosclerotic lesion formation in apolipoprotein E–deficient mice. In conclusion, our results demonstrate that PKM2 is required to support metabolic reprogramming for Hcy-induced B cell activation and function, and it might serve as a critical regulator in HHcy-accelerated initiation of atherosclerosis. PMID:27903739

  14. T cell-mediated activation and regulation of anti-chromatin B cells.

    PubMed

    Pagán, Antonio J; Ramón, Hilda E; Hondowicz, Brian D; Erikson, Jan

    2006-07-01

    We have taken an immunoglobulin transgenic approach to study how self-reactive B cells are held in check in healthy mice and what parameters contribute to their activation in autoimmunity. Using this strategy, we have documented that a population of anti-chromatin B cells migrate to the periphery. In a healthy background, these cells have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B cell interface in the spleen. Importantly, they are capable of differentiating into antibody-forming cells when provided with T cell help. T(H)1 and T(H)2 cells induce IgG2a and IgG1 autoantibodies, respectively. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, these autoreactive B cells populate the B cell follicle, and this is dependent upon CD4 T cells. However, after 10 weeks of age serum autoantibodies are produced. We hypothesize that control of autoantibody production in young autoimmune-prone mice is regulated by the counterbalancing influence of regulatory T cells. We show that while autoantibody production is blocked in the context of regulatory T cells, early events characterizing a productive T cell-B cell interaction are not disturbed, with the notable exceptions of T(H) ICOS levels and IFN-gamma and IL-10 production.

  15. IL-27 Directly Enhances Germinal Center B Cell Activity and Potentiates Lupus in Sanroque Mice.

    PubMed

    Vijayan, Dipti; Mohd Redzwan, Norhanani; Avery, Danielle T; Wirasinha, Rushika C; Brink, Robert; Walters, Giles; Adelstein, Stephen; Kobayashi, Masao; Gray, Paul; Elliott, Michael; Wong, Melanie; King, Cecile; Vinuesa, Carola G; Ghilardi, Nico; Ma, Cindy S; Tangye, Stuart G; Batten, Marcel

    2016-10-15

    Germinal centers (GC) give rise to high-affinity and long-lived Abs and are critical in immunity and autoimmunity. IL-27 supports GCs by promoting survival and function of T follicular helper cells. We demonstrate that IL-27 also directly enhances GC B cell function. Exposure of naive human B cells to rIL-27 during in vitro activation enhanced their differentiation into CD20(+)CD38(+)CD27(low)CD95(+)CD10(+) cells, consistent with the surface marker phenotype of GC B cells. This effect was inhibited by loss-of-function mutations in STAT1 but not STAT3 To extend these findings, we studied the in vivo effects of IL-27 signals to B cells in the GC-driven Roquin(san/san) lupus mouse model. Il27ra(-/-)Roquin(san/san) mice exhibited significantly reduced GCs, IgG2a(c)(+) autoantibodies, and nephritis. Mixed bone marrow chimeras confirmed that IL-27 acts through B cell- and CD4(+) T cell-intrinsic mechanisms to support GCs and alter the production of pathogenic Ig isotypes. To our knowledge, our data provide the first evidence that IL-27 signals directly to B cells promote GCs and support the role of IL-27 in lupus.

  16. B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation.

    PubMed

    Maity, Palash Chandra; Blount, Amy; Jumaa, Hassan; Ronneberger, Olaf; Lillemeier, Björn F; Reth, Michael

    2015-09-15

    The B cell antigen receptors (BCRs) play an important role in the clonal selection of B cells and their differentiation into antibody-secreting plasma cells. Mature B cells have both immunoglobulin M (IgM) and IgD types of BCRs, which have identical antigen-binding sites and are both associated with the signaling subunits Igα and Igβ, but differ in their membrane-bound heavy chain isoforms. By two-color direct stochastic optical reconstruction microscopy (dSTORM), we showed that IgM-BCRs and IgD-BCRs reside in the plasma membrane in different protein islands with average sizes of 150 and 240 nm, respectively. Upon B cell activation, the BCR protein islands became smaller and more dispersed such that the IgM-BCRs and IgD-BCRs were found in close proximity to each other. Moreover, specific stimulation of one class of BCR had minimal effects on the organization of the other. These conclusions were supported by the findings from two-marker transmission electron microscopy and proximity ligation assays. Together, these data provide evidence for a preformed multimeric organization of BCRs on the plasma membrane that is remodeled after B cell activation.

  17. Implication of calpain in caspase activation during B cell clonal deletion.

    PubMed Central

    Ruiz-Vela, A; González de Buitrago, G; Martínez-A, C

    1999-01-01

    In the absence of costimulating signals, B cell receptor (BCR) crosslinking on immature B cells triggers the apoptotic cell death program. In the WEHI-231 B cell lymphoma model, anti-IgM crosslinking triggers activation of caspase-7 independently of caspase-8, followed by apoptosis. Two main mechanisms for caspase-7 activation have been proposed: (i) caspase-8 recruitment to death receptors (Fas or tumour necrosis factor); and (ii) changes in mitochondrial membrane permeability and cytochrome c release, which activate caspase-9. Here we report that caspase-7 activation induced by BCR crosslinking is independent of caspase-8 and cytochrome c translocation from mitochondria to the cytosol, as well as of mitochondrial depolarization. In addition, in a cell-free system, the S-100 fraction of anti-IgM-treated WEHI-231 cells induces a caspase activation pattern different from that activated by cytochrome c and dATP. We demonstrate that calpain specifically triggers activation and processing of caspase-7 both in vitro and in vivo, and that both processes are inhibited by calpain inhibitors. Furthermore, calpain activation is associated with decreased expression levels of calpastatin, which is upregulated by CD40 ligation. These data confirm a role for calpain during BCR crosslinking, which may be critical for cell deletion by apoptosis during B cell development and activation. PMID:10487751

  18. MALT1 small molecule inhibitors specifically suppress ABC-DLBCL in vitro and in vivo.

    PubMed

    Fontan, Lorena; Yang, Chenghua; Kabaleeswaran, Venkataraman; Volpon, Laurent; Osborne, Michael J; Beltran, Elena; Garcia, Monica; Cerchietti, Leandro; Shaknovich, Rita; Yang, Shao Ning; Fang, Fang; Gascoyne, Randy D; Martinez-Climent, Jose Angel; Glickman, J Fraser; Borden, Katherine; Wu, Hao; Melnick, Ari

    2012-12-11

    MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo.

  19. Dynamics of Major Histocompatibility Complex Class II Compartments during B Cell Receptor–mediated Cell Activation

    PubMed Central

    Lankar, Danielle; Vincent-Schneider, Hélène; Briken, Volker; Yokozeki, Takeaki; Raposo, Graça; Bonnerot, Christian

    2002-01-01

    Antigen recognition by clonotypic B cell receptor (BcR) is the first step of B lymphocytes differentiation into plasmocytes. This B cell function is dependent on efficient major histocompatibility complex (MHC) class II–restricted presentation of BcR-bound antigens. In this work, we analyzed the subcellular mechanisms underlying antigen presentation after BcR engagement on B cells. In quiescent B cells, we found that MHC class II molecules mostly accumulated at the cell surface and in an intracellular pool of tubulovesicular structures, whereas H2-M molecules were mostly detected in distinct lysosomal compartments devoid of MHC class II. BcR stimulation induced the transient intracellular accumulation of MHC class II molecules in newly formed multivesicular bodies (MVBs), to which H2-M was recruited. The reversible downregulation of cathepsin S activity led to the transient accumulation of invariant chain–MHC class II complexes in MVBs. A few hours after BcR engagement, cathepsin S activity increased, the p10 invariant chain disappeared, and MHC class II–peptide complexes arrived at the plasma membrane. Thus, BcR engagement induced the transient formation of antigen-processing compartments, enabling antigen-specific B cells to become effective antigen-presenting cells. PMID:11854359

  20. IL-7 modulates B cells survival and activation by inducing BAFF and CD70 expression in T cells.

    PubMed

    Sammicheli, Stefano; Ruffin, Nicolas; Lantto, Rebecka; Vivar, Nancy; Chiodi, Francesca; Rethi, Bence

    2012-06-01

    Interleukin-7 (IL-7) promotes the maintenance and activation of peripheral T cells, whereas it does not act directly on mature B cells due to the lack of IL-7Rα expression on these. We report here that, in spite of the insensitivity of B cells to IL-7, high concentration of IL-7 can lead to increased B cell survival and antibody production in the presence of T cells, without the use of any further B cell stimulatory signal. IL-7 promoted B cell activation through inducing CD70 expression on resting T cells, particularly on CD4+ memory cells. The interaction of CD70 molecules with the B cell costimulatory receptor CD27 led to B cell proliferation, the accumulation of CD38 + CD20- plasmablasts and antibody production. In addition, IL-7 treatment induced BAFF secretion from resting peripheral T cells thereby promoting B cell survival. IL-7 levels can increase in lymphopenic conditions, in autoimmune diseases or in patients receiving T cell regenerative IL-7 therapy. Based on our findings high IL-7 levels can lead to increased B cell activation by inducing the B cell regulatory proteins CD70 and BAFF in resting T cells. Such activity might be beneficial in short term immune-stimulatory IL-7 therapies; permanently increased IL-7 levels, on the other hand, can contribute to impaired B cell tolerance.

  1. Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification.

    PubMed

    Prabhakar, Vikas; Capila, Ishan; Bosques, Carlos J; Pojasek, Kevin; Sasisekharan, Ram

    2005-02-15

    GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC I's active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.

  2. Role of the Bp35 cell surface polypeptide in human B-cell activation.

    PubMed Central

    Clark, E A; Shu, G; Ledbetter, J A

    1985-01-01

    A 35-kDa polypeptide, Bp35, expressed on the surface of all B cells, plays a role in B-cell activation. Monoclonal antibodies to Bp35 stimulate human tonsillar B cells to proliferate. The activation induced by anti-Bp35 is similar to anti-Ig-mediated in several ways: the activation does not require T cells but is augmented by T-cell-derived allogeneic factors; monovalent Fab fragments to Bp35 do not trigger proliferation but instead block activation by whole antibody, indicating that cross-linking is required; and induction by anti-Bp35, like the induction by anti-Ig, is inhibited by monoclonal anti-IgM via an Fc domain-dependent mechanism. However, several features of anti-Bp35-mediated proliferation are clearly different from activation by anti-Ig: anti-Bp35 monoclonal antibodies do not require attachment to beads to function, the proliferation induced by anti-Bp35 and anti-Ig is additive, and Fab fragments of anti-Bp35 augment proliferation induced by anti-Ig. Models for the possible function of the Bp35 polypeptide as either a "bridge" or a "second signal" with surface Ig in B-cell activation are discussed. PMID:3872456

  3. Suboptimal B-cell antigen receptor signaling activity in vivo elicits germinal center counterselection mechanisms.

    PubMed

    Königsberger, Sebastian; Weis, Vanessa; Prodöhl, Jan; Stehling, Martin; Hobeika, Elias; Reth, Michael; Kiefer, Friedemann

    2015-02-01

    Syk and Zap-70 constitute a closely related nonreceptor protein tyrosine kinase family, of which both members are functionally indispensable for conferring their respective antigen receptors with enzymatic activity. In this study, we analyze the impact of altering BCR signaling output on B-cell germinal center (GC) fate selection by constitutive, as well as inducible, monoallelic Syk kinase loss in the presence of a Zap-70 knock-in rescue allele. Cre-mediated Syk deletion in Syk(flox/Zap-70) B cells lowers pErk, but not pAkt-mediated signaling. Surprisingly, the use of a B-cell-specific constitutive mb1-cre deleter mouse model showed that a small cohort of peripheral Syk(flox/Zap-70);mb1-cre B cells efficiently circumvents deletion, which ultimately favors these Syk-sufficient cells to contribute to the GC reaction. Using a developmentally unbiased Syk(flox/Zap-70);mb1-creER(T2) approach in combination with an inducible tdRFP allele, we further demonstrate that this monoallelic deletion escape is not fully explained by leakiness of Cre expression, but is possibly the result of differential Syk locus accessibility in maturing B cells. Altogether, this underscores the importance of proper Syk kinase function not only during central and peripheral selection processes, but also during GC formation and maintenance.

  4. The role of B cell-activating factor in autoimmune myasthenia gravis.

    PubMed

    Lisak, Robert P; Ragheb, Samia

    2012-12-01

    B cell-activating factor (BAFF) is important in the development and maturation of B cells and their progeny-plasma blasts and plasma cells. There is increasing evidence that BAFF is involved in the pathogenesis of several autoimmune diseases including myasthenia gravis. Increased expression of BAFF and receptors for BAFF have been demonstrated in thymus of patients with myasthenia gravis, and an increase in serum levels of BAFF have been reported in patients with myasthenia gravis. While the exact role of BAFF in the pathogenesis of myasthenia gravis is not clear, BAFF and its receptors may provide potential targets for therapy in patients with myasthenia gravis.

  5. Brucella fractions behave as nonspecific mitogens and polyclonal B-cell activators for human lymphocytes.

    PubMed Central

    Vendrell, J P; Rabesandratana, H; Huguet, M F; Cannat, A; Serre, A

    1985-01-01

    Two lipid-A-free fractions which were extracted from Brucella melitensis and were designated PI and SF stimulated human unsensitized mononuclear cells to proliferate and to secrete immunoglobulins. Both of these effects were observed in cultures of peripheral blood, tonsils, and cord blood lymphocytes. Neither B cells nor T cells alone proliferated in the presence of these fractions, whereas the proliferative response of T cells plus B cells was largely independent of accessory cells. Polyclonal activation was estimated by counting the cells which secreted immunoglobulins of different isotypes into culture supernatants. This phenomenon was strongly T dependent. PMID:3876286

  6. Induction and activation of the aryl hydrocarbon receptor by IL-4 in B cells.

    PubMed

    Tanaka, Go; Kanaji, Sachiko; Hirano, Ayumi; Arima, Kazuhiko; Shinagawa, Akira; Goda, Chiho; Yasunaga, Shin'ichiro; Ikizawa, Koichi; Yanagihara, Yukiyoshi; Kubo, Masato; Kuriyama-Fujii, Yoshiaki; Sugita, Yuji; Inokuchi, Akira; Izuhara, Kenji

    2005-06-01

    It is widely known that IL-4 and IL-13 act on various kinds of cells, including B cells, resulting in enhancement of proliferation, class switching to IgE and expression of several surface proteins. These functions are important for the recognition of the various antigens in B cells and are known to be involved in the pathogenesis of allergic diseases. However, it has not been known whether IL-4/IL-13 is involved in the metabolism of various kinds of xenobiotics including 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), and it remains undetermined whether TCDD, an environmental pollutant, influences IgE production in B cells, exaggerating allergic reactions. We identified IL-4- or IL-13-inducible genes in a human Burkitt lymphoma cell line, DND-39, using microarray technology, in which the AHR gene was included. The AHR gene product, the aryl hydrocarbon receptor (AhR), was induced by IL-4 in both mouse and human B cells in a STAT6-dependent manner. IL-4 alone had the ability to translocate the induced AhR to the nuclei. TCDD, a ligand for AhR, rapidly degraded the induced AhR by the proteasomal pathway, although IL-4-activated AhR sustained its expression. AhR activated by IL-4 caused expression of a xenobiotic-metabolizing gene, CYP1A1, and TCDD synergistically acted on the induction of this gene by IL-4. However, the induction of AhR had no effect on IgE synthesis or CD23 expression. These results indicate that the metabolism of xenobiotics would be a novel biological function of IL-4 and IL-13 in B cells, whereas TCDD is not involved in IgE synthesis in B cells.

  7. LIME acts as a transmembrane adapter mediating BCR-dependent B-cell activation.

    PubMed

    Ahn, Eunseon; Lee, Hyunsook; Yun, Yungdae

    2006-02-15

    Assembly of a signaling complex around the transmembrane adapter LAT is essential for the transmission of T-cell receptor (TCR)-mediated signaling. However, a LAT-like molecule responsible for the initial activation events in B-cell receptor (BCR) signaling has not yet been identified. Here, we show that LIME is a transmembrane adaptor required for BCR-mediated B-cell activation. LIME was found to be expressed in mouse splenic B cells. Upon BCR cross-linking, LIME was tyrosine phosphorylated by Lyn and associated with Lyn, Grb2, PLC-gamma2, and PI3K. Reduction of LIME expression by the introduction of siRNA resulted in the disruption of BCR-mediated activation of MAPK, calcium flux, NF-AT, PI3K, and NF-kappaB. Taken together, these results establish that LIME is an essential transmembrane adaptor linking BCR ligation to the downstream signaling events that lead to B-cell activation.

  8. miR-181b negatively regulates activation-induced cytidine deaminase in B cells.

    PubMed

    de Yébenes, Virginia G; Belver, Laura; Pisano, David G; González, Susana; Villasante, Aranzazu; Croce, Carlo; He, Lin; Ramiro, Almudena R

    2008-09-29

    Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3' untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.

  9. Activated GL7(+) B cells are maintained within the inflamed CNS in the absence of follicle formation during viral encephalomyelitis.

    PubMed

    DiSano, Krista D; Stohlman, Stephen A; Bergmann, Cornelia C

    2017-02-01

    Central nervous system (CNS) inflammation associated with viral infection and autoimmune disease results in the accumulation of B cells in various differentiation stages. However, the contribution between peripheral and CNS activation remains unclear. During gliatropic coronavirus induced encephalomyelitis, accumulation of protective antibody secreting cells is preceded by infiltration of B cells with a naïve and early differentiation phenotype (Phares et al., 2014). Investigation of the temporal dynamics of B cell activation in draining cervical lymph nodes (CLN) and the CNS revealed that peak CNS infiltration of early activated, unswitched IgD(+) and IgM(+) B cells coincided with polyclonal activation in CLN. By contrast, isotype-switched IgG(+) B cells did not accumulate until peripheral germinal center formation. In the CNS, unswitched B cells were confined to the perivascular space and meninges, with only rare B cell clusters, while isotype-switched B cells localized to parenchymal areas. Although ectopic follicle formation was not observed, more differentiated B cell subsets within the CNS expressed the germinal center marker GL7, albeit at lower levels than CLN counterparts. During chronic infection, CNS IgD(int) and IgD(-) B cell subsets further displayed sustained markers of proliferation and CD4 T cell help, which were only transiently expressed in the CLN. A contribution of local CD4 T cell help to sustain B cell activation was supported by occasional B cells adjacent to T cells. The results suggest that accumulation of differentiated B cell subsets within the CNS is largely dictated by peripheral activation, but that local events contribute to their sustained activation independent of ectopic follicle formation.

  10. Enhanced Follicular Dendritic Cell-B Cell Interaction in HIV and SIV Infections and its Potential Role in Polyclonal B Cell Activation

    PubMed Central

    Lewis, Mark. G.; Kosco-Vilbois, Marie H.

    1998-01-01

    Human immunodeficiency virus (HIV) infections have been characterized by both polyclonal Bcell activation and enhanced responsiveness to B-cell growth factors on one hand and the loss of specific antibody (Ab) responses and refractoriness to the normal signals for B-cell activation on the other. Histopathological studies of lymph node from HIV- and simian immunodeficiency virus (SIV)-infected individuals have indicated initial follicular hyperplasia and the appearance of large irregular germinal centers that undergo progressive involution concomitant with follicular dendritic-cell (FDC) disruption. During this process, follicular dendritic-cell -enriched lymph-node-cell cultures exhibit increased ability to induce cluster formation (“in vitro germinal centers”), lymphocyte proliferation and antibody production compared to uninfected controls. This paper discusses how enhanced FDC-B-cell interaction within SIV-infected germinal centers may result in a reduced ability to select high-affinity B cells and alter the dynamics of antibodyproducing- cell and memory-cell generation resulting in the observed hyperactivity. PMID:9716906

  11. Single molecule analysis of B cell receptor motion during signaling activation

    NASA Astrophysics Data System (ADS)

    Rey Suarez, Ivan; Koo, Peter; Mochrie, Simon; Song, Wenxia; Upadhyaya, Arpita

    B cells are an essential part of the adaptive immune system. They patrol the body looking for signs of infection in the form of antigen on the surface of antigen presenting cells. The binding of the B cell receptor (BCR) to antigen induces signaling cascades that lead to B cell activation and eventual production of high affinity antibodies. During activation, BCR organize into signaling microclusters, which are platforms for signal amplification. The physical processes underlying receptor movement and aggregation are not well understood. Here we study the dynamics of single BCRs on activated murine primary B cells using TIRF imaging and single particle tracking. The tracks obtained are analyzed using perturbation expectation-maximization (pEM) a systems-level analysis that allows the identification of different short-time diffusive states from a set of single particle tracks. We identified five different diffusive states on wild type cells, which correspond to different molecular states of the BCR. By using actin polymerization inhibitors and mutant cells lacking important actin regulators we were able to identify the BCR molecule configuration associated with each diffusive state.

  12. B-cell activation in HIV infection: relationship of spontaneous immunoglobulin secretion to various immunological parameters.

    PubMed Central

    Mizuma, H; Litwin, S; Zolla-Pazner, S

    1988-01-01

    Peripheral blood mononuclear cells from HIV-infected individuals spontaneously secrete elevated levels of IgG, IgM and IgD. This increased level of synthesis and secretion is similar in HIV-infected subjects with no or few symptoms, in ARC patients and in AIDS patients. Thus, abnormal B-cell activation is characteristic of patients with mild as well as severe manifestations of HIV infection. The level of spontaneous cellular secretion of IgG, IgM and IgD correlates with serum levels of these isotypes. Levels of spontaneous cellular secretion of IgG and IgM correlate negatively with the percentage but not with the absolute number of T4-positive cells and correlate positively with the percentage but not with the absolute number of T8-positive cells. The data suggest that the proportional distribution of these T-cell subsets is a critical factor in the B-cell dysregulation leading to overproduction of IgG and IgM. On the other hand, spontaneous IgD secretion correlates with neither the percent nor the absolute number of T4 or T8 cells suggesting that the increase of IgD-secretion by B cells is independent of the T-cell defects. The data imply that more than one mechanism underlies the B-cell activation in HIV-infected individuals. PMID:3260155

  13. MARCH1 down-regulation in IL-10-activated B cells increases MHC class II expression.

    PubMed

    Galbas, Tristan; Steimle, Viktor; Lapointe, Réjean; Ishido, Satoshi; Thibodeau, Jacques

    2012-07-01

    IL-10 is vastly studied for its anti-inflammatory properties on most immune cells. However, it has been reported that IL-10 activates B cells, up-regulates their MHC class II molecules and prevents apoptosis. As MARCH1 was shown to be responsible for the intracellular sequestration of MHC class II molecules in dendritic cells and monocytes in response to IL-10, we set out to clarify the role of this ubiquitin ligase in B cells. Here, we demonstrate in mice that splenic follicular B cells represent the major cell population that up-regulate MHC II molecules in the presence of IL-10. Activation of these cells through TLR4, CD40 or the IL-10 receptor caused the down-regulation of MARCH1 mRNA. Accordingly, B cells from MARCH1-deficient mice do not up-regulate I-A(b) in response to IL-10. In all, our results demonstrate that IL-10 can have opposite effects on MARCH1 regulation in different cell types.

  14. Effect of Antiretroviral Therapy on the Memory and Activation Profiles of B Cells in HIV-Infected African Women.

    PubMed

    Tanko, Ramla F; Soares, Andreia P; Müller, Tracey L; Garrett, Nigel J; Samsunder, Natasha; Abdool Karim, Quarraisha; Abdool Karim, Salim S; Riou, Catherine; Burgers, Wendy A

    2017-02-01

    Human immunodeficiency virus infection induces a wide range of effects in B cells, including skewed memory cell differentiation, compromised B cell function, and hypergammaglobulinemia. However, data on the extent to which these B cell abnormalities can be reversed by antiretroviral therapy (ART) are limited. To investigate the effect of ART on B cells, the activation (CD86) and differentiation (IgD, CD27, and CD38) profiles of B cells were measured longitudinally in 19 HIV-infected individuals before (median, 2 mo) and after ART initiation (median, 12 mo) and compared with 19 age-matched HIV-uninfected individuals using flow cytometry. Twelve months of ART restored the typical distribution of B cell subsets, increasing the proportion of naive B cells (CD27(-)IgD(+)CD38(-)) and concomitantly decreasing the immature transitional (CD27(-)IgD(+)CD38(+)), unswitched memory (CD27(+)IgD(+)CD38(-)), switched memory (CD27(+)IgD(-)CD38(-) or CD27(-)IgD(-)CD38(-)), and plasmablast (CD27(+)IgD(-)CD38(high)) subsets. However, B cell activation was only partially normalized post-ART, with the frequency of activated B cells (CD86(+)CD40(+)) reduced compared with pre-ART levels (p = 0.0001), but remaining significantly higher compared with HIV-uninfected individuals (p = 0.0001). Interestingly, unlike for T cell activation profiles, the extent of B cell activation prior to ART did not correlate with HIV plasma viral load, but positively associated with plasma sCD14 levels (p = 0.01, r = 0.58). Overall, ART partially normalizes the skewed B cell profiles induced by HIV, with some activation persisting. Understanding the effects of HIV on B cell dysfunction and restoration following ART may provide important insights into the mechanisms of HIV pathogenesis.

  15. Murine Visceral Leishmaniasis: IgM and Polyclonal B-Cell Activation Lead to Disease Exacerbation

    PubMed Central

    Deak, Eszter; Jayakumar, Asha; Wing Cho, Ka; Goldsmith-Pestana, Karen; Dondji, Blaise; Lambris, John D.; McMahon-Pratt, Diane

    2010-01-01

    In visceral leishmaniasis, the draining lymph node (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with L. infantum. Although not unexpected, at early times post-infection there is a marked B cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JHD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JHD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JHD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to wild type BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5aR (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-γ). Overall these studies indicate that polyclonal B cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention. PMID:20213734

  16. Reduced Levels of Hspa9 Attenuates Stat5 Activation in Mouse B-cells

    PubMed Central

    Krysiak, Kilannin; Tibbitts, Justin F.; Shao, Jin; Liu, Tuoen; Ndonwi, Matthew; Walter, Matthew J.

    2014-01-01

    HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndromes (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. While homozygous knockout of Hspa9 is embryonic lethal, mice with heterozygous deletion of Hspa9 (Hspa9+/−) are viable and have a 50% reduction in Hspa9 expression. Hspa9+/− mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9+/− mice have a cell- intrinsic reduction in bone marrow CFU-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9+/− B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNAi and observe reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B- lymphopoiesis in vivo. Knockdown of Hspa9 in an IL-7 dependent mouse B-cell line reduced Stat5 phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B-cells. Collectively, these data implicate a role for Hspa9 in B-lymphopoiesis and Stat5 activation downstream of IL-7 signaling. PMID:25550197

  17. Global gene regulation during activation of immunoglobulin class switching in human B cells

    PubMed Central

    Zhang, Youming; Fear, David J.; Willis-Owen, Saffron A. G.; Cookson, William O.; Moffatt, Miriam F.

    2016-01-01

    Immunoglobulin class switch recombination (CSR) to IgE is a tightly regulated process central to atopic disease. To profile the B-cell transcriptional responses underlying the activation of the germinal centre activities leading to the generation of IgE, naïve human B-cells were stimulated with IL-4 and anti-CD40. Gene expression and alternative splicing were profiled over 12 days using the Affymetrix Human Exon 1.0 ST Array. A total of 1,399 genes, forming 13 temporal profiles were differentially expressed. CCL22 and CCL17 were dramatically induced but followed a temporal trajectory distinct from classical mediators of isotype switching. AICDA, NFIL3, IRF4, XBP1 and BATF3 shared a profile with several genes involved in innate immunity, but with no recognised role in CSR. A transcription factor BHLHE40 was identified at the core of this profile. B-cell activation was also accompanied by variation in exon retention affecting >200 genes including CCL17. The data indicate a circadian component and central roles for the Th2 chemokines CCL22 and CCL17 in the activation of CSR. PMID:27897229

  18. CXCL13/CXCR5 signaling enhances BCR-triggered B-cell activation by shaping cell dynamics.

    PubMed

    Sáez de Guinoa, Julia; Barrio, Laura; Mellado, Mario; Carrasco, Yolanda R

    2011-08-11

    Continuous migration of B cells at the follicle contrasts with their stable arrest after encounter with antigen. Two main ligand/receptor pairs are involved in these cell behaviors: the chemokine CXCL13/chemokine receptor CXCR5 and antigen/BCR. Little is known regarding the interplay between CXCR5 and BCR signaling in the modulation of B-cell dynamics and its effect on B-cell activation. We used a 2-dimensional model to study B-cell migration and antigen recognition in real time, and found that BCR signaling strength alters CXCL13-mediated migration, leading to a heterogeneous B-cell behavior pattern. In addition, we demonstrate that CXCL13/CXCR5 signaling does not impair BCR-triggered immune synapse formation and that CXCR5 is excluded from the central antigen cluster. CXCL13/CXCR5 signaling enhances BCR-mediated B-cell activation in at least 2 ways: (1) it assists antigen gathering at the synapse by promoting membrane ruffling and lymphocyte function-associated antigen 1 (LFA-1)-supported adhesion, and (2) it allows BCR signaling integration in motile B cells through establishment of LFA-1-supported migratory junctions. Both processes require functional actin cytoskeleton and non-muscle myosin II motor protein. Therefore, the CXCL13/CXCR5 signaling effect on shaping B-cell dynamics is an effective mechanism that enhances antigen encounter and BCR-triggered B-cell activation.

  19. Lipoxin A4 protects against lipopolysaccharide-induced sepsis by promoting innate response activator B cells generation.

    PubMed

    Cheng, Qiong; Wang, Zheng; Ma, Ruihua; Chen, Yongtao; Yan, Yan; Miao, Shuo; Jiao, Jingyu; Cheng, Xue; Kong, Lingfei; Ye, Duyun

    2016-10-01

    Sepsis is a serious disease that leads to severe inflammation, dysregulation of immune system, multi-organ failure and death. Innate response activator (IRA) B cells, which produce granulocyte-macrophage colony-stimulating factor (GM-CSF), protect against microbial sepsis. Lipid mediator lipoxin A4 (LXA4) exerts anti-inflammatory and immunoregulatory effects, and it has been reported that LXA4 receptor ALX/FPR2 is expressed on B cells. Here, we investigated the potential role of LXA4 on IRA B cells in lipopolysaccharide (LPS)-induced sepsis. We found that LXA4 significantly promoted the expansion of splenic IRA B cells and increased GM-CSF expression in splenic B cells with LPS stimulation. After splenectomy, LXA4 treatment did not change the serum or peritoneal IL-1β, IL-6 and TNF-α levels in LPS-induced sepsis. LXA4 accelerated the migration of peritoneal B cells to spleen for their differentiation into IRA B cells, whereas this effect was independent of peritoneal macrophage. Furthermore, LXA4 enhanced the phosphorylation level of signal transducer and activator of transcription 5 (STAT5) in splenic B cells. These results suggest that LXA4 protects against LPS-induced sepsis by promoting the generation and migration of splenic IRA B cells, and the underlying molecular mechanism may be related to STAT5 activation. It might provide new insights and therapeutic approaches for treating sepsis.

  20. Deregulated lncRNAs in B Cells from Patients with Active Tuberculosis

    PubMed Central

    Fu, Yurong; Xu, Xianqin; Xue, Junfang; Duan, Wenping; Yi, Zhengjun

    2017-01-01

    Role of lncRNAs in human adaptive immune response to TB infection is largely unexplored. To address this issue, here we characterized lncRNA expression profile in primary human B cell response to TB infection using microarray assay. Several lncRNAs and mRNAs were chosen for RT-qPCR validation. Bioinformatics prediction was applied to delineate function of the deregulated mRNAs. We found that 844 lncRNAs and 597 mRNAs were differentially expressed between B cell samples from individuals with or without TB. KEGG pathway analysis for the deregulated mRNAs indicated a number of pathways, such as TB, TLR signaling pathway and antigen processing and presentation. Moreover, corresponding to the dysregulation of many lncRNAs, we also found that their adjacent protein-coding genes were also deregulated. Functional annotation for the corresponding mRNAs showed that these lncRNAs were mainly associated with TLR signaling, TGF-β signaling. Interestingly, SOCS3, which is a critical negative regulator of cytokine response to TB infection and its nearby lncRNA XLOC_012582, were highly expressed in active TB B cells. Subsequent RT-qPCR results confirmed the changes. Whether upregulated XLOC_012582 causes SOCS3 overexpression and is eventually involved in the context of exacerbations of active TB represents an interesting issue that deserves to be further explored. Taken together, for the first time, we identified a set of deregulated lncRNAs in active TB B cells and their functions were predicted. Such findings provided novel insight into the pathogenesis of TB and further studies should focus on the function and pathogenic mechanisms of the lncRNAs involved in active TB. PMID:28125665

  1. RelA NF-κB subunit activation as a therapeutic target in diffuse large B-cell lymphoma

    PubMed Central

    Manyam, Ganiraju C.; Visco, Carlo; Tzankov, Alexandar; Wang, Jing; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W.L.; Han van Krieken, J.; Huh, Jooryung; Ponzoni, Maurilio; Ferreri, Andrés J.M.; Møller, Michael B.; Parsons, Ben M.; Winter, Jane N.; Piris, Miguel A.; Jeffrey Medeiros, L.; Pham, Lan V.; Young, Ken H.

    2016-01-01

    It has been well established that nuclear factor kappa-B (NF-κB) activation is important for tumor cell growth and survival. RelA/p65 and p50 are the most common NF-κB subunits and involved in the classical NF-κB pathway. However, the prognostic and biological significance of RelA/p65 is equivocal in the field. In this study, we assessed RelA/p65 nuclear expression by immunohistochemistry in 487 patients with de novo diffuse large B-cell lymphoma (DLBCL), and studied the effects of molecular and pharmacological inhibition of NF-κB on cell viability. We found RelA/p65 nuclear expression, without associations with other apparent genetic or phenotypic abnormalities, had unfavorable prognostic impact in patients with stage I/II DLBCL. Gene expressionprofiling analysis suggested immune dysregulation and antiapoptosis may be relevant for the poorer prognosis associated with p65 hyperactivation in germinal center B-cell–like (GCB) DLBCL and in activated B-cell–like (ABC) DLBCL, respectively. We knocked down individual NF-κB subunits in representative DLBCL cells in vitro, and found targeting p65 was more effective than targeting other NF-κB subunits in inhibiting cell growth and survival. In summary, RelA/p65 nuclear overexpression correlates with significant poor survival in early-stage DLBCL patients, and therapeutic targeting RelA/p65 is effective in inhibiting proliferation and survival of DLBCL with NF-κB hyperactivation. PMID:27941215

  2. Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity

    PubMed Central

    Siggs, Owen M.; Stockenhuber, Alexander; Deobagkar-Lele, Mukta; Bull, Katherine R.; Crockford, Tanya L.; Kingston, Bethany L.; Crawford, Greg; Anzilotti, Consuelo; Steeples, Violetta; Ghaffari, Sahar; Czibik, Gabor; Bellahcene, Mohamed; Watkins, Hugh; Ashrafian, Houman; Davies, Benjamin; Woods, Angela; Carling, David; Yavari, Arash; Beutler, Bruce; Cornall, Richard J.

    2016-01-01

    Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt–Hogg–Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1. Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51–like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK. PMID:27303042

  3. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    PubMed Central

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2013-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT’s reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT’s reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease. PMID:23012103

  4. Deposition of idiotype-anti-idiotype immune complexes in renal glomeruli after polyclonal B cell activation

    SciTech Connect

    Goldman, M.; Rose, L.M.; Hochmann, A.; Lambert, P.H.

    1982-05-01

    We investigated the possible role of idiotypic interactions in the pathogenesis of the glomerular lesions observed in mice undergoing polyclonal B cell activation. BALB/c mice were studied for the presence of renal deposits of T15 idiotype-anti-T15 idiotype-immune complexes (IC) after injection of bacterial lipopolysaccharides (LPS). The T15 idiotype is the major idiotype of BALB/c mice anti-phosphorylcholine (PC) antibodies, which are cross-reactive with the idiotype of the TEPC-15 myeloma protein. This model was used because T15 idiotype-anti-T15 idiotype IC have been detected in the circulation of BALB/c mice after polyclonal B cell activation. First, an idiotype-specific immunofluorescence technique allowed us to detect T15 idiotype-bearing immunoglobulins in glomeruli from day 6 to day 28 after LPS injection. Second, fluorescein isothiocyanate-conjugated TEPC-15 myeloma protein was found to localize in the glomeruli after in vivo injection 18 d after LPS administration. This renal localization was shown to be idiotype-specific and could be quantified in a trace-labeling experiment. Third, kidney-deposited immunoglobulins of mice injected with LPS were eluted, radiolabeled, and analyzed by radioimmunoassay. Both T15 idiotype-bearing immunoglobulins and anti-T15 idiotype antibodies were detected in the eluates, providing further evidence for a renal deposition of T15 idiotype-anti-T15 idiotype IC. Polyclonal B cell activation is likely to result in a simultaneous triggering of many idiotypic clones and of corresponding anti-idiotypic clones represented in the B cell repertoire. This could lead to the formation of a variety of idiotype-anti-idiotype IC that could participate in the development of glomerular lesions.

  5. A 24,000 MW Trypanosoma cruzi antigen is a B-cell activator.

    PubMed Central

    Da Silva, A C; Espinoza, A G; Taibi, A; Ouaissi, A; Minoprio, P

    1998-01-01

    Trypanosoma cruzi, the causative agent of Chagas' disease, is a protozoan parasite that infects humans and other mammals in Central and Latin America. Several alterations of the immune response after infection have been described, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal B- and T-cell activation, including the expansion of self-reactive clones. We have investigated the effects of the intraperitoneal injection of a recombinant 24,000 MW T. cruzi-specific antigen (rTc24) on the immune response of normal and deficient strains of mice. We analysed the in vivo and ex vivo levels of lymphocyte activation and the proliferative responses to rTc24 by determining the expression of CD69 activation marker and the levels of thymidine incorporation by spleen cells. The numbers of antibody-producing cells were determined by ELISPOT and the levels of immunoglobulin in the sera by isotype-specific enzyme-linked immunosorbent assay. We observed an increased [3H]thymidine ([3H]TdR) incorporation by spleen cells after rTc24 stimulation in vivo and in vitro. This proliferative activity induced by rTc24 was independent of the mouse strain used in the experiments (including C3H/HeJ mice) and ruled out the possibility that rTc24 preparations were contaminated by lipopolysaccharide. The injection of rTc24 protein induced preferentially the activation of B cells, as determined by the increased expression of CD69 molecules on IgM+ spleen cells. Considerable increases of IgM-secreting B cells were determined in both athymic and euthymic BALB/c mice. Mice that are deficient in B cells (BALB.Xid) responded to rTc24 but to a lesser extent. These increases in IgM B-cell numbers were accompanied by elevated levels of IgM immunoglobulins in the sera of injected animals. Our results suggest a role for rTc24 in B-cell activation. PMID:9741340

  6. Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

    PubMed

    Di Niro, Roberto; Lee, Seung-Joo; Vander Heiden, Jason A; Elsner, Rebecca A; Trivedi, Nikita; Bannock, Jason M; Gupta, Namita T; Kleinstein, Steven H; Vigneault, Francois; Gilbert, Tamara J; Meffre, Eric; McSorley, Stephen J; Shlomchik, Mark J

    2015-07-21

    The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

  7. Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation.

    PubMed

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-21

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux.

  8. Vav3 Modulates B Cell Receptor Responses by Regulating Phosphoinositide 3-Kinase Activation

    PubMed Central

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M.; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-01

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux. PMID:11805146

  9. Role of type I interferons in the activation of autoreactive B cells

    PubMed Central

    Kiefer, Kerstin; Oropallo, Michael A; Cancro, Michael P; Marshak-Rothstein, Ann

    2013-01-01

    Type I interferons (IFNs) are a family of cytokines involved in the defense against viral infections that play a key role in the activation of both the innate and adaptive immune system. IFNs both directly and indirectly enhance the capacity of B lymphocytes to respond to viral challenge and produce cytotoxic and neutralizing antibodies. However, prolonged type I IFN exposure is not always beneficial to the host. If not regulated properly IFN can drive autoantibody production as well as other parameters of systemic autoimmune disease. Type I IFNs impact B-cell function through a variety of mechanisms, including effects on receptor engagement, Toll-like receptor expression, cell migration, antigen presentation, cytokine responsiveness, cytokine production, survival, differentiation and class-switch recombination. Type I IFNs are also cytotoxic for a variety of cell types and thereby contribute to the accumulation of cell debris that serves as a potential source for autoantigens. Type I IFN engagement of a variety of accessory cells further promotes B-cell survival and activation, as exemplified by the capacity of type I IFNs to increase the level of B-cell survival factors, such as B lymphocyte stimulator, produced by dendritic cells. Therefore, it is not surprising that the loss of expression of the type I IFN receptor can have dramatic effects on the production of autoantibodies and on the clinical features of systemic autoimmune diseases such as systemic lupus erythematosus. PMID:22430248

  10. Dendritic cell-elicited B-cell activation fosters immune privilege via IL-10 signals in hepatocellular carcinoma

    PubMed Central

    Ouyang, Fang-Zhu; Wu, Rui-Qi; Wei, Yuan; Liu, Rui-Xian; Yang, Dong; Xiao, Xiao; Zheng, Limin; Li, Bo; Lao, Xiang-Ming; Kuang, Dong-Ming

    2016-01-01

    B cells are prominent components of human solid tumours, but activation status and functions of these cells in human cancers remain elusive. Here we establish that over 50% B cells in hepatocellular carcinoma (HCC) exhibit an FcγRIIlow/− activated phenotype, and high infiltration of these cells positively correlates with cancer progression. Environmental semimature dendritic cells, but not macrophages, can operate in a CD95L-dependent pathway to generate FcγRIIlow/− activated B cells. Early activation of monocytes in cancer environments is critical for the generation of semimature dendritic cells and subsequent FcγRIIlow/− activated B cells. More importantly, the activated FcγRIIlow/− B cells from HCC tumours, but not the resting FcγRIIhigh B cells, without external stimulation suppress autologous tumour-specific cytotoxic T-cell immunity via IL-10 signals. Collectively, generation of FcγRIIlow/− activated B cells may represent a mechanism by which the immune activation is linked to immune tolerance in the tumour milieu. PMID:27853178

  11. Gambogic acid induces apoptosis in diffuse large B-cell lymphoma cells via inducing proteasome inhibition.

    PubMed

    Shi, Xianping; Lan, Xiaoying; Chen, Xin; Zhao, Chong; Li, Xiaofen; Liu, Shouting; Huang, Hongbiao; Liu, Ningning; Zang, Dan; Liao, Yuning; Zhang, Peiquan; Wang, Xuejun; Liu, Jinbao

    2015-04-08

    Resistance to chemotherapy is a great challenge to improving the survival of patients with diffuse large B-cell lymphoma (DLBCL), especially those with activated B-cell-like DLBCL (ABC-DLBCL). Therefore it is urgent to search for novel agents for the treatment of DLBCL. Gambogic acid (GA), a small molecule derived from Chinese herb gamboges, has been approved for Phase II clinical trial for cancer therapy by Chinese FDA. In the present study, we investigated the effect of GA on cell survival and apoptosis in DLBCL cells including both GCB- and ABC-DLBCL cells. We found that GA induced growth inhibition and apoptosis of both GCB- and ABC-DLBCL cells in vitro and in vivo, which is associated with proteasome malfunction. These findings provide significant pre-clinical evidence for potential usage of GA in DLBCL therapy particularly in ABC-DLBCL treatment.

  12. Doxorubicin enhances the capacity of B cells to activate T cells in urothelial urinary bladder cancer.

    PubMed

    Zirakzadeh, A Ali; Kinn, Johan; Krantz, David; Rosenblatt, Robert; Winerdal, Malin E; Hu, Jin; Hartana, Ciputra Adijaya; Lundgren, Christian; Bergman, Emma Ahlén; Johansson, Markus; Holmström, Benny; Hansson, Johan; Sidikii, Alexander; Vasko, Janos; Marits, Per; Sherif, Amir; Winqvist, Ola

    2016-12-24

    Cancer is currently treated by a combination of therapies, including chemotherapy which is believed to suppress the immune system. Combination of immunotherapy and chemotherapy correlates with improved survival but needs careful planning in order to achieve a synergistic effect. In this study, we have demonstrated that doxorubicin treatment of B cells resulted in increased expression of CD86 and concordantly increased CD4(+) T cell activation in the presence of superantigen, an effect that was inhibited by the addition of a CD86 blocking antibody. Furthermore, doxorubicin resulted in decreased expression of the anti-inflammatory cytokines IL-10 and TNF-α. Finally, B cells from urinary bladder cancer patients, treated with a neoadjuvant regiment containing doxorubicin, displayed increased CD86-expression. We conclude that doxorubicin induces CD86 expression on B cells and hence enhances their antigen-presenting ability in vitro, a finding verified in patients. Development of tailored time and dose schedules may increase the effectiveness of combining chemotherapy and immunotherapy.

  13. DOCK2 regulates cell proliferation through Rac and ERK activation in B cell lymphoma

    SciTech Connect

    Wang, Lei; Nishihara, Hiroshi; Kimura, Taichi; Kato, Yasutaka; Tanino, Mishie; Nishio, Mitsufumi; Obara, Masato; Endo, Tomoyuki; Koike, Takao; Tanaka, Shinya

    2010-04-23

    DOCK2; a member of the CDM protein family, regulates cell motility and cytokine production through the activation of Rac in mammalian hematopoietic cells and plays a pivotal role in the modulation of the immune system. Here we demonstrated the alternative function of DOCK2 in hematopoietic tumor cells, especially in terms of its association with the tumor progression. Immunostaining for DOCK2 in 20 cases of human B cell lymphoma tissue specimens including diffuse large B cell lymphoma and follicular lymphoma revealed the prominent expression of DOCK2 in all of the lymphoma cells. DOCK2-knockdown (KD) of the B cell lymphoma cell lines, Ramos and Raji, using the lentiviral shRNA system presented decreased cell proliferation compared to the control cells. Furthermore, the tumor formation of DOCK2-KD Ramos cell in nude mice was significantly abrogated. Western blotting analysis and pull-down assay using GST-PAK-RBD kimeric protein suggested the presence of DOCK2-Rac-ERK pathway regulating the cell proliferation of these lymphoma cells. This is the first report to clarify the prominent role of DOCK2 in hematopoietic malignancy.

  14. Diffuse large B-cell lymphoma with concurrent high MYC and BCL2 expression shows evidence of active B-cell receptor signaling by quantitative immunofluorescence

    PubMed Central

    Kovach, Alexandra E.; Le, Long P.; Feng, Derek; Baxter, Richard H. G.; Sohani, Aliyah R.

    2017-01-01

    B-cell receptor (BCR)-mediated signaling plays an important role in the pathogenesis of a subset of diffuse large B-cell lymphoma (DLBCL), and novel agents targeting this pathway are now in clinical use. We have previously identified a signature of active BCR signaling on formalin-fixed paraffin-embedded specimens using quantitative immunofluorescence, allowing for identification of patients who might benefit from anti-BCR therapies. We sought to characterize the clinicopathologic significance of active BCR signaling in DLBCL by correlating measures of signaling intensity with clinical features and various tumor cell characteristics. High MYC and concurrent high MYC and BCL2 double-expression was positively correlated with individual markers of active BCR signaling and cases with MYC/BCL2 double-expression showed overall greater BCR activation compared to cases lacking double-expression. Our findings suggest that the BCR signaling pathway may be more active in MYC/BCL2 double-expressor DLBCL and may represent a rational therapeutic target in this aggressive DLBCL subgroup. PMID:28212447

  15. C-Myc dysregulation is a co-transforming event for NF-κB activated B-cells.

    PubMed

    David, Amandine; Arnaud, Nicolas; Fradet, Magali; Lascaux, Hélène; Ouk-Martin, Catherine; Gachard, Nathalie; Zimber-Strobl, Ursula; Feuillard, Jean; Faumont, Nathalie

    2017-02-23

    While c-Myc dysregulation is constantly associated with highly proliferating B-cell tumors, NF-κB addiction is found in indolent lymphomas as well as diffuse large B-cell lymphomas either with an activated B-cell like phenotype or associated with Epstein-Barr virus. We raised the question of the effect of c-Myc in B-cells with NF-κB activated by three different inducers: Epstein-Barr Virus-latency III program, TLR-9 and CD40. Induction of c-Myc overexpression increased proliferation of Epstein-Barr Virus-latency III immortalized B-cells, an effect that was dependent on NF-κB. Results from transcriptomic signatures and functional studies showed that c-Myc over-expression increased Epstein-Barr Virus-latency III driven proliferation depending on NF-κB. In vitro, induction of c-Myc increased proliferation of B-cell with TLR9 dependant activation of Myd88, with decreased apoptosis. In the transgenic λc-Myc mouse model with c-Myc over expression in B-cells, in vivo activation of Myd88 by TLR9 induced splenomegaly related to increased S-phase entry of B-cells. Transgenic mice with both continuous CD40 signaling in B-cells and the λc-Myc transgene developed very aggressive lymphomas with characteristics of activated diffuse large B-cell lymphomas. The main characteristic gene expression profile signatures of these tumors were those of proliferation and energetic metabolism. These results suggest that c-Myc is an NF-κB co-transforming event in aggressive lymphomas with an activated phenotype, activated B-cell like diffuse large B-cell lymphomas. This would explain why NF-κB is associated with both indolent and aggressive lymphomas and opens new perspectives on the possibility of combinatory therapies targeting both c-Myc proliferating program and NF-κB activation pathways in diffuse large B-cell lymphomas.

  16. Neuropilin-1 expression characterizes T follicular helper (Tfh) cells activated during B cell differentiation in human secondary lymphoid organs.

    PubMed

    Renand, Amédée; Milpied, Pierre; Rossignol, Julien; Bruneau, Julie; Lemonnier, François; Dussiot, Michael; Coulon, Séverine; Hermine, Olivier

    2013-01-01

    T follicular helper (Tfh) cells play an essential role in the development of antigen-specific B cell immunity. Tfh cells regulate the differentiation and survival of activated B cells outside and inside germinal centers (GC) of secondary lymphoid organs. They act through cognate contacts with antigen-presenting B cells, but there is no current marker to specifically identify those Tfh cells which productively interact with B cells. Here we show that neuropilin 1 (Nrp1), a cell surface receptor, is selectively expressed by a subset of Tfh cells in human secondary lymphoid organs. Nrp1 expression on Tfh cells correlates with B cell differentiation in vivo and in vitro, is transient, and can be induced upon co-culture with autologous memory B cells in a cell contact-dependent manner. Comparative analysis of ex vivo Nrp1(+) and Nrp1(-) Tfh cells reveals gene expression modulation during activation. Finally, Nrp1 is expressed by malignant Tfh-like cells in a severe case of angioimmunoblastic T-cell lymphoma (AITL) associated with elevated terminal B cell differentiation. Thus, Nrp1 is a specific marker of Tfh cells cognate activation in humans, which may prove useful as a prognostic factor and a therapeutic target in neoplastic diseases associated with Tfh cells activity.

  17. Early Activation of Teleost B Cells in Response to Rhabdovirus Infection

    PubMed Central

    Abós, Beatriz; Castro, Rosario; González Granja, Aitor; Havixbeck, Jeffrey J.; Barreda, Daniel R.

    2014-01-01

    ABSTRACT To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM+) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM+ cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM+ cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM+ cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM+ cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM+ cell proliferation, a consistent effect on IgM+ cell survival was detected. IMPORTANCE Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we

  18. RNA exosome regulates AID DNA mutator activity in the B cell genome

    PubMed Central

    Pefanis, Evangelos; Basu, Uttiya

    2015-01-01

    The immunoglobulin diversification processes of somatic hypermutation and class switch recombination critically rely on transcription coupled targeting of AID to Ig loci in activated B lymphocytes. AID catalyzes deamination of cytidine deoxynucleotides on exposed single stranded DNA. In addition to driving immunoglobulin diversity, promiscuous targeting of AID mutagenic activity poses a deleterious threat to genomic stability. Recent genome-wide studies have uncovered pervasive AID activity throughout the B cell genome. It is increasingly apparent that AID activity is frequently targeted to genomic loci undergoing early transcription termination where RNA exosome promotes the resolution of stalled transcription complexes via co-transcriptional RNA degradation mechanisms. Here we review aspects and consequences of eukaryotic transcription that lead to early termination, RNA exosome recruitment, and ultimately targeting of AID mutagenic activity. PMID:26073986

  19. Characterization of two distinct antigens expressed on either resting or activated human B cells as defined by monoclonal antibodies.

    PubMed Central

    Kokai, Y; Ishii, Y; Kikuchi, K

    1986-01-01

    Two antigen systems (L29 & L30) expressed on two distinct human B cell subpopulations were identified by using BL1-4D6 and TB3-7D5 monoclonal antibodies, respectively. L29 was expressed on approximately one-third of B cells in human lymphoid tissues. These B cells associated with L29 were large activated B cells located in the germinal centres of lymphoid follicles. L30, on the other hand, existed on approximately two-thirds of B cells mainly located in the mantle zone of lymphoid follicles, most of which also expressed IgM and IgD on their cell membrane. In addition, L30 was shared on mature granulocytes. With the use of polyclonal activators such as pokeweek mitogen (PWM) and protein A-bearing staphylococci (SAC), L29 antigen was inducible on PWM- or SAC-stimulated B cells in correspondence with the emergence of Tac and T10 antigens of these B cells. In contrast, L30 antigen on the B cells stimulated by the polyclonal activators was decreased in its expression and was finally lost from these B cells. Although none of L29 and L30 was expressed on normal, non-activated human thymus and peripheral T cells, L29 but not L30 was expressed on concanavalin A-activated T cells. Immunochemical studies showed that L30 consist of a single polypeptide with mol. wt of 40,000. L29 antigen is presently under study. Images Fig. 2 Fig. 4 PMID:3527505

  20. Genetic and cellular dissection of the activation of AM14 rheumatoid factor B cells in a mouse model of lupus

    PubMed Central

    Sang, Allison; Zheng, Ying Yi; Choi, Seung-Chul; Zeumer, Leilani; Morel, Laurence

    2015-01-01

    The RF-specific AM14 tg BCR has been used as a model to dissect the mechanisms of B cell tolerance to ICs containing nucleic acids. We have shown previously that AM14 RF B cells break tolerance in the TC mouse model of lupus through the dual engagement of the AM14 BCR and TLR9. In this study, we showed that neither the expression of Sle1 or Sle2 susceptibility loci alone was sufficient to activate AM14 RF B cells, suggesting that the production of antichromatin IgG2aa autoAg mediated by Sle1 and an intrinsically higher B cell activation mediated by Sle2 were required. We also showed that the B6 genetic background enhanced the selection of AM14 RF B cells to the MZB cell compartment regardless of the expression of the Sle loci and therefore, of their activation into AFCs. Furthermore, some AM14 RF B cells were selected into the B-1a compartment, where they did not differentiate into AFCs. Therefore, it is unlikely that the selection of AM14 RF B cells to the MZB or B-1a cell compartments in TC.AM14a mice is responsible for their breach of tolerance. Finally, we showed that the presence of expression of Sle1 in non-tg cells, most likely T cells, is necessary for the activation of AM14 RF B cells into AFCs. Overall, these results suggest a threshold model of activation of AM14 RF B cells on the B6 background with additive genetic and cellular contribution of multiple sources. PMID:25957308

  1. Accurate Classification of Germinal Center B-Cell-Like/Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Using a Simple and Rapid Reverse Transcriptase-Multiplex Ligation-Dependent Probe Amplification Assay: A CALYM Study.

    PubMed

    Mareschal, Sylvain; Ruminy, Philippe; Bagacean, Cristina; Marchand, Vinciane; Cornic, Marie; Jais, Jean-Philippe; Figeac, Martin; Picquenot, Jean-Michel; Molina, Thierry Jo; Fest, Thierry; Salles, Gilles; Haioun, Corinne; Leroy, Karen; Tilly, Hervé; Jardin, Fabrice

    2015-04-09

    Diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is subdivided into germinal center B-cell-like and activated B-cell-like subtypes. Unfortunately, these lymphomas are difficult to differentiate in routine diagnosis, impeding the development of treatments. Patients with these lymphomas can benefit from specific therapies. We therefore developed a simple and rapid classifier based on a reverse transcriptase multiplex ligation-dependent probe amplification assay and 14 gene signatures. Compared with the Affymetrix U133+2 gold standard, all 46 samples (95% CI, 92%-100%) of a validation cohort classified by both techniques were attributed to the expected subtype. Similarly, 93% of the 55 samples (95% CI, 82%-98%) of a second independent series characterized with a mid-throughput gene expression profiling method were classified correctly. Unclassifiable sample proportions reached 13.2% and 13.8% in these cohorts, comparable with the frequency originally reported. The developed assay was also sensitive enough to obtain reliable results from formalin-fixed, paraffin-embedded samples and flexible enough to include prognostic factors such as MYC/BCL2 co-expression. Finally, in a series of 135 patients, both overall (P = 0.01) and progression-free (P = 0.004) survival differences between the two subtypes were confirmed. Because the multiplex ligation-dependent probe amplification method is already in use and requires only common instruments and reagents, it could easily be applied to clinical trial patient stratification to help in treatment decisions.

  2. B cell helper assays.

    PubMed

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  3. Corticosteroids inhibit anti-IgE activities of specialized proresolving mediators on B cells from asthma patients

    PubMed Central

    Kim, Nina; Thatcher, Thomas H.; Sime, Patricia J.; Phipps, Richard P.

    2017-01-01

    Specialized proresolving mediators (SPMs) promote the resolution of inflammation and exert beneficial effects in animal models of chronic inflammatory diseases, including asthma. Previously, we have shown that certain SPMs reduce IgE production in B cells from healthy individuals, which has a critical role in allergic asthma. Here, we investigated the effects of SPMs on B cell IgE production in asthma patients. Peripheral blood mononuclear cells from asthma patients were treated with 17-HDHA or RvD1, and IgE levels were measured. RvD1 and 17-HDHA dampened IgE production in B cells from most asthma patients, whereas B cells from a subset of patients taking oral steroids were refractory to SPM treatment. Molecular mechanisms underlying the interaction between corticosteroids and SPMs were investigated by treating B cells from nonasthmatic donors with corticosteroids in vitro. Corticosteroids blocked the inhibitory effects of 17-HDHA and RvD1 on B cell IgE production by abolishing the suppressive activity of these mediators on IgE class switching. Corticosteroids decreased the expression of transcriptional repressor Bcl-6 as well as its suppressive activity on epsilon germline transcription. We conclude that 17-HDHA and RvD1 can reduce IgE production in asthma patients not taking high doses of steroids but that corticosteroids interfere with the ability of B cells to respond to proresolving mediators. PMID:28194434

  4. Molecular cloning, expression and bioactivity of B cell activating factor (BAFF) in African ostrich.

    PubMed

    Yang, Keli; Xiao, Ke; Huang, Haibo; Lu, Shun; Zhong, Juming; Ansari, Abdur Rahman; Khaliq, Haseeb; Song, Hui; Liu, Huazhen; Peng, Kemei

    2015-09-01

    B cell activating factor (BAFF), which belongs to the tumor necrosis factor (TNF) family, is testified to play a critical role in B cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, the cDNA of open reading frame (ORF) in African ostrich (Struthio camelus) BAFF (designated OsBAFF) was cloned by reverse transcription-PCR (RT-PCR). The OsBAFF gene encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like BAFFs from chicken (cBAFF), quail (qBAFF), duck (dBAFF), goose (gBAFF) and dove (doBAFF). RT-PCR analysis showed that the OsBAFF gene is strongly expressed in the bursa of Fabricius, thymus, spleen, and bone marrow. The soluble OsBAFF had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in Escherichia coli Rosset (DE3). In vitro, purified OsBAFF was not only able to promote the survival of African ostrich bursal lymphocytes, but also able to co-stimulate proliferation of mouse splenic B cells. The expression of OsBAFF in lymphocyte cells was higher than the control after LPS stimulation. These findings indicated that OsBAFF plays an important role in survival and proliferation of African ostrich bursal lymphocytes, which may provide valuable information for research into the immune system of African ostrich and OsBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and any other birds.

  5. Polyclonal B cell activation, circulating immune complexes and autoimmunity in human american visceral leishmaniasis.

    PubMed Central

    Galvão-Castro, B; Sá Ferreira, J A; Marzochi, K F; Marzochi, M C; Coutinho, S G; Lambert, P H

    1984-01-01

    In a prospective study, serum and plasma samples from 17 patients with kala-azar were collected in Rio de Janeiro. High levels of immune complexes (IC) were detected in serum by means of 125I-Clq and conglutinin binding assays. The Clq binding material had a sedimentation coefficient of 19-25S, as determined by ultracentrifugation on sucrose gradient. Plasma levels of C3 and C3 breakdown products were measured and the C3d levels were increased in six out of 11 patients. The occurrence of polyclonal B cell activation was suggested by (a) a marked increase of serum IgG and IgM levels and (b) of the presence of antibodies against various proteins and haptens (SRBC, DNP-BSA, FITC-BSA,KLH). There was a close association between the presence of IC and anti-immunoglobulin antibodies. Anti-smooth muscle antibodies were also observed. These data are consistent with a major role of polyclonal B cell activation in the induction of IC during visceral leishmaniasis. PMID:6424987

  6. 1,25-dihydroxyvitamin D{sub 3} impairs NF-{kappa}B activation in human naive B cells

    SciTech Connect

    Geldmeyer-Hilt, Kerstin; Heine, Guido; Hartmann, Bjoern; Baumgrass, Ria; Radbruch, Andreas; Worm, Margitta

    2011-04-22

    Highlights: {yields} In naive B cells, VDR activation by calcitriol results in reduced NF-{kappa}B p105 and p50 protein expression. {yields} Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-{kappa}B p65. {yields} Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. {yields} Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1{alpha},25-dihydroxyvitamin D{sub 3} (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-{kappa}B p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-{kappa}B mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-{kappa}B activation by interference with NF-{kappa}B p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.

  7. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    NASA Astrophysics Data System (ADS)

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-01

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  8. B-cell activation with CD40L or CpG measures the function of B-cell subsets and identifies specific defects in immunodeficient patients.

    PubMed

    Marasco, Emiliano; Farroni, Chiara; Cascioli, Simona; Marcellini, Valentina; Scarsella, Marco; Giorda, Ezio; Piano Mortari, Eva; Leonardi, Lucia; Scarselli, Alessia; Valentini, Diletta; Cancrini, Caterina; Duse, Marzia; Grimsholm, Ola; Carsetti, Rita

    2017-01-01

    Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system.

  9. NFAM1, an immunoreceptor tyrosine-based activation motif-bearing molecule that regulates B cell development and signaling.

    PubMed

    Ohtsuka, Makoto; Arase, Hisashi; Takeuchi, Arata; Yamasaki, Sho; Shiina, Ritsuko; Suenaga, Tadahiro; Sakurai, Daiju; Yokosuka, Tadashi; Arase, Noriko; Iwashima, Makio; Kitamura, Toshio; Moriya, Hideshige; Saito, Takashi

    2004-05-25

    A functional cDNA cloning system was developed by using a retrovirus library encoding CD8-chimeric proteins and a nuclear factor of activated T cells (NFAT)-GFP reporter cell line to identify molecules inducing NFAT activation. By using this strategy, NFAT activating molecule 1 (NFAM1) was cloned as an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cell surface molecule belonging to the Ig superfamily and is predominantly expressed in spleen B and T cells. NFAM1 crosslinking induced ITAM phosphorylation, ZAP-70/Syk recruitment, NFAT activation, and cytokine production. In vivo overexpression of NFAM1 in bone marrow chimeras and transgenic mice induced severe impairment of early B cell development in an ITAM-dependent manner. In NFAM1-expressing B cells, B cell antigen receptor stimulation induced NFAM1 translocation to lipid raft, and NFAM1 co-crosslinking augmented B cell antigen receptor signaling. The results suggest that NFAM1 modulates B cell signaling through its ITAM, which regulates B cell development.

  10. Enhanced activation of B cells in a granulocyte colony-stimulating factor-mobilized peripheral blood stem cell graft.

    PubMed

    Tayebi, H; Lapierre, V; Saas, P; Lienard, A; Sutton, L; Milpied, N; Attal, M; Cahn, J Y; Kuentz, M; Blaise, D; Hervé, P; Tiberghien, P; Robinet, E

    2001-09-01

    In a randomized study that compared human leucocyte antigen-identical allogeneic granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cell (PBSC) versus bone marrow (BM) transplantation, the expression of activation markers, CD23, CD25 and CD45RO by B cells, was compared in blood before and after G-CSF mobilization and in PBSC versus BM grafts. The fractions of CD23+ and CD25+ B cells were higher in PBSC than in BM grafts. Moreover, we observed a G-CSF-induced increase in B-cell fractions in blood as well as in PBSC grafts when compared with BM grafts. Such an enhanced B-cell activation could contribute to the accelerated kinetics of immuno-haematological reconstitution, the occurrence of acute haemolysis in the ABO minor incompatibility setting, as well as the increased incidence of chronic graft-versus-host disease observed after PBSC transplantation.

  11. Defining antigen-specific plasmablast and memory B cell subsets in blood following viral infection and vaccination of humans

    PubMed Central

    Ellebedy, Ali H.; Jackson, Katherine J.L.; Kissick, Haydn T.; Nakaya, Helder I.; Davis, Carl W.; Roskin, Krishna M.; McElroy, Anita K.; Oshansky, Christine M.; Elbein, Rivka; Thomas, Shine; Lyon, George M.; Spiropoulou, Christina F.; Mehta, Aneesh K.; Thomas, Paul G.; Boyd, Scott D.; Ahmed, Rafi

    2016-01-01

    Antigen-specific B cells bifurcate into antibody secreting cells (ASC) and memory B cells after infection or vaccination. ASCs or plasmablasts have been extensively studied in humans but less is known about B cells that get activated but do not differentiate into early plasmablasts. Here, we define the phenotype and transcriptional program of an antigen-specific B cell subset, referred to as activated B cells (ABC), that is distinct from ASCs and is committed to the memory B cell lineage. ABCs were detected in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after influenza vaccination we interrogated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced HA-specific clones revealed minimal increase in SHM over time suggesting that repeated annual immunization may have limitations in enhancing the quality of influenza-specific antibody. PMID:27525369

  12. Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.

    PubMed

    McGuire, Andrew T; Gray, Matthew D; Dosenovic, Pia; Gitlin, Alexander D; Freund, Natalia T; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B; Glenn, Jolene; Seaman, Michael S; Schief, William R; Strong, Roland K; Nussenzweig, Michel C; Stamatatos, Leonidas

    2016-02-24

    VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

  13. A Recombinant Bispecific CD20×CD95 Antibody With Superior Activity Against Normal and Malignant B-cells

    PubMed Central

    Nalivaiko, Kristina; Hofmann, Martin; Kober, Karina; Teichweyde, Nadine; Krammer, Peter H; Rammensee, Hans-Georg; Grosse-Hovest, Ludger; Jung, Gundram

    2016-01-01

    Monoclonal antibodies directed to the B-cell-specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here, we compare the anti-B-cell activity of three different antibodies directed to CD20: (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof, and (iii) a bispecific CD20×CD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant, as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from peripheral blood mononuclear cell (PBMC) cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its nonoptimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20×CD95-antibody might become a new, prototypical reagent for the treatment of B-cell-mediated autoimmune disease. PMID:26581163

  14. Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice

    PubMed Central

    McGuire, Andrew T.; Gray, Matthew D.; Dosenovic, Pia; Gitlin, Alexander D.; Freund, Natalia T.; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B.; Glenn, Jolene; Seaman, Michael S.; Schief, William R.; Strong, Roland K.; Nussenzweig, Michel C.; Stamatatos, Leonidas

    2016-01-01

    VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype. PMID:26907590

  15. The frequency of early-activated hapten-specific B cell subsets predicts the efficacy of vaccines for nicotine dependence

    PubMed Central

    Laudenbach, M; Tucker, AM; Runyon, SP; Carroll, FI; Pravetoni, M

    2015-01-01

    Therapeutic vaccines for nicotine addiction show pre-clinical efficacy. Yet, clinical evaluation of the first-generation nicotine vaccines did not meet expectations because only a subset of immunized subjects achieved effective serum antibody levels. Recent studies suggest that vaccine design affects B cell activation, and that the frequency of the hapten-specific B cell subsets contributes to vaccine efficacy against drugs of abuse. To extend this hypothesis to nicotine immunogens, we synthesized a novel hapten containing a carboxymethylureido group at the 2-position of the nicotine structure (2CMUNic) and compared its efficacy to the previously characterized 6CMUNic hapten. Haptens were conjugated to the keyhole limpet hemocyanin (KLH) carrier protein, and evaluated for efficacy against nicotine in mice using the clinically approved alum adjuvant. Using a novel fluorescent antigen-based magnetic enrichment strategy paired with multicolor flow cytometry analysis, polyclonal hapten-specific B cell subsets were measured in mice immunized with either 6CMUNic-KLH or 2CMUNic-KLH. The 6CMUNic-KLH showed significantly greater efficacy than 2CMUNic-KLH on nicotine distribution to serum and to the brain. The 6CMUNic-KLH elicited higher anti-nicotine serum antibody titers, and a greater frequency of hapten-specific B cells than 2CMUNic-KLH. Within the splenic polyclonal B cell population, a higher number of hapten-specific IgMhigh and germinal center B cells predicted greater vaccine efficacy against nicotine distribution. These early pre-clinical findings suggest that hapten structure affects activation of B cells, and that variations in the frequency of early-activated hapten-specific B cell subsets underlie individual differences in vaccine efficacy. PMID:26409811

  16. Simultaneous Inhibition of PI3Kδ and PI3Kα Induces ABC-DLBCL Regression by Blocking BCR-Dependent and -Independent Activation of NF-κB and AKT.

    PubMed

    Paul, Juliane; Soujon, Maurice; Wengner, Antje M; Zitzmann-Kolbe, Sabine; Sturz, Andrea; Haike, Katja; Keng Magdalene, Koh Hui; Tan, Sze Huey; Lange, Martin; Tan, Soo Yong; Mumberg, Dominik; Lim, Soon Thye; Ziegelbauer, Karl; Liu, Ningshu

    2017-01-09

    Compared with follicular lymphoma, high PI3Kα expression was more prevalent in diffuse large B cell lymphoma (DLBCL), although both tumor types expressed substantial PI3Kδ. Simultaneous inhibition of PI3Kα and PI3Kδ dramatically enhanced the anti-tumor profile in ABC-DLBCL models compared with selective inhibition of PI3Kδ, PI3Kα, or BTK. The anti-tumor activity was associated with suppression of p-AKT and a mechanism of blocking nuclear factor-κB activation driven by CD79(mut), CARD11(mut), TNFAIP3(mut), or MYD88(mut). Inhibition of PI3Kα/δ resulted in tumor regression in an ibrutinib-resistant CD79B(WT)/MYD88(mut) patient-derived ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79B(mut)-ABC-DLBCL to show a robust response to PI3K and BTK inhibitor monotherapies. A combination of ibrutinib with the PI3Kα/δ inhibitor copanlisib produced a sustained complete response in vivo in CD79B(mut)/MYD88(mut) ABC-DLBCL models.

  17. Alternative splicing regulates activation-induced cytidine deaminase (AID): implications for suppression of AID mutagenic activity in normal and malignant B cells

    PubMed Central

    Wu, Xiaosheng; Darce, Jaime R.; Chang, Sook Kyung; Nowakowski, Grzegorz S.

    2008-01-01

    The mutagenic enzyme activation-induced cytidine deaminase (AID) is required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B cells. Deregulated expression of AID is associated with various B-cell malignancies and, currently, it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B cells, and there is limited evidence that this also occurs in normal blood B cells. The functional significance of these splice variants remains unknown. Here we show that normal GC human B cells and blood memory B cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B cells as well as malignant B cells from chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly, our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B cells. PMID:18684869

  18. Activation of ATR-Chk1 pathway facilitates EBV-mediated transformation of primary tonsillar B-cells

    PubMed Central

    Mordasini, Vanessa; Ueda, Seigo; Aslandogmus, Roberta; Berger, Christoph; Gysin, Claudine; Hühn, Daniela; Sartori, Alessandro A

    2017-01-01

    Primary infection of the immunocompromised host with the oncovirus Epstein-Barr virus (EBV) that targets mainly B-cells is associated with an increased risk for EBV-associated tumors. The early events subsequent to primary infection with potential for B-cell transformation are poorly studied. Here, we modeled in vitro the primary infection by using B-cells isolated from tonsils, the portal of entry of EBV, since species specificity of EBV hampers modeling in experimental animals. Increasing evidence indicates that the host DNA damage response (DDR) can influence and be influenced by EBV infection. Thus, we inoculated tonsillar B-cells (TBCs) with EBV-B95.8 and investigated cell proliferation and the DDR during the first 96 hours thereafter. We identified for the first time that EBV infection of TBCs induces a period of hyperproliferation 48-96 hours post infection characterized by the activation of ataxia telangiectasia and Rad3-releated (ATR) and checkpoint kinase-1 (Chk1). Whereas inhibition of Chk1 did not affect B-cell transformation, the specific inhibition of ATR robustly decreased the transformation efficiency of EBV. Our results suggest that activation of ATR is key for EBV-induced B-cell transformation. Thus, targeting the interaction between ATR/Chk1 and EBV could offer new options for the treatment of EBV-associated malignancies. PMID:28031537

  19. Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies.

    PubMed

    Rodgers, David T; Mazagova, Magdalena; Hampton, Eric N; Cao, Yu; Ramadoss, Nitya S; Hardy, Ian R; Schulman, Andrew; Du, Juanjuan; Wang, Feng; Singer, Oded; Ma, Jennifer; Nunez, Vanessa; Shen, Jiayin; Woods, Ashley K; Wright, Timothy M; Schultz, Peter G; Kim, Chan Hyuk; Young, Travis S

    2016-01-26

    Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR-T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.

  20. Mechanisms of polyclonal B-cell activation in autoimmune B6-lpr/lpr mice.

    PubMed

    Warren, R W; Roths, J B; Murphy, E D; Pisetsky, D S

    1984-03-01

    The influence of the lpr gene on spontaneous and lipopolysaccharide (LPS)-induced immunoglobulin production was studied in B6 mice homozygous for the mutant lpr gene (B6-lpr/lpr). Male and female mice of this congenic strain were followed for 1 year and sera serially tested by the enzyme-linked immunosorbent assay (ELISA) for the production of antibodies to single-stranded DNA (anti-sDNA), immunoglobulin (anti-IgG), and keyhole limpet hemocyanin (anti-KLH), models of autoantibody and non-autoantibody responses, respectively. Female B6-lpr/lpr mice demonstrated marked spontaneous responses to all three antigens; the responses of male B6-lpr/lpr mice were significantly lower but still exceeded those of the congenic B6-+/+ controls. These results demonstrate a generalized influence of sex on lpr associated responses. To determine whether this sex difference could be demonstrated with other forms of B-cell activation, young B6-+/+ and B6-lpr/lpr male and female mice were immunized with lipopolysaccharide and the induced responses determined. This immunization caused significant increases in the IgM response only. The levels of the induced responses produced after LPS treatment were comparable for +/+ and lpr/lpr mice. These results indicate that the enhanced responsiveness of female mice to lpr action is not reflected in the polyclonal response to LPS, which, furthermore, was unaffected by the presence of lpr. The differential influence of sex on lpr and LPS-induced responses and their apparent independence suggests that lpr and LPS promote B-cell activation by dissimilar mechanisms.

  1. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

    PubMed Central

    Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

    2016-01-01

    Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

  2. Presence of Rheumatoid Factor during Chronic HCV Infection Is Associated with Expansion of Mature Activated Memory B-Cells that Are Hypo-Responsive to B-Cell Receptor Stimulation and Persist during the Early Stage of IFN Free Therapy.

    PubMed

    Reyes-Avilés, Elane; Kostadinova, Lenche; Rusterholtz, Anne; Cruz-Lebrón, Angelica; Falck-Ytter, Yngve; Anthony, Donald D

    2015-01-01

    Approximately half of those with chronic hepatitis C virus (HCV) infection have circulating rheumatoid factor (RF), and a portion of these individuals develop cryoglobulinemic vasculitis. B cell phenotype/function in relation to RF in serum has been unclear. We examined B cell subset distribution, activation state (CD86), cell cycle state (Ki67), and ex-vivo response to BCR, TLR9 and TLR7/8 stimulation, in chronic HCV-infected donors with or without RF, and uninfected donors. Mature-activated B-cells of HCV-infected donors had lower CD86 expression compared to uninfected donors, and in the presence of RF they also showed reduced CD86 expression in response to BCR and TLR9 stimulation. Additionally, mature activated memory B cells of HCV RF+ donors less commonly expressed Ki67+ than HCV RF- donors, and did not proliferate as well in response to BCR stimulation. Proportions of mature-activated B cells were enhanced, while naïve B-cells were lower in the peripheral blood of HCV-RF+ compared to RF- and uninfected donors. None of these parameters normalize by week 8 of IFN free direct acting antiviral (DAA) therapy in HCV RF+ donors, while in RF- donors, mature activated B cell proportions did normalize. These data indicate that while chronic HCV infection alone results in a lower state of activation in mature activated memory B cells, the presence of RF in serum is associated with a more pronounced state of unresponsiveness and an overrepresentation of these B cells in the blood. This phenotype persists at least during the early time window after removal of HCV from the host.

  3. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model.

    PubMed

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay (®) (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  4. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model

    PubMed Central

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay ® (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  5. LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation

    PubMed Central

    Llibre, Alba; López-Macías, Constantino; Marafioti, Teresa; Mehta, Hema; Partridge, Amy; Kanzig, Carina; Rivellese, Felice; Galson, Jacob D.; Walker, Lucy J.; Milne, Paul; Phillips, Rodney E.; Kelly, Dominic F.; Freeman, Gordon J.; Klenerman, Paul

    2016-01-01

    Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones, light and dark, with coordinated roles, controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells, but its functional role in the GC reaction has not been explored. In this study, we report high expression of LLT1 on GC-associated B cells, early plasmablasts, and GC-derived lymphomas. LLT1 expression was readily induced via BCR, CD40, and CpG stimulation on B cells. Unexpectedly, we found high expression of the LLT1 ligand, CD161, on follicular dendritic cells. Triggering of LLT1 supported B cell activation, CD83 upregulation, and CXCR4 downregulation. Overall, these data suggest that LLT1–CD161 interactions play a novel and important role in B cell maturation within the GC in humans. PMID:26829983

  6. Differential development of progenitor activity for three B-cell lineages.

    PubMed Central

    Kantor, A B; Stall, A M; Adams, S; Herzenberg, L A; Herzenberg, L A

    1992-01-01

    Cell-transfer studies presented here distinguish three murine B cell lineages: conventional B cells, which develop late and are continually replenished from progenitors in adult bone marrow; Ly-1 B cells (B-1a), which develop early and maintain their numbers by self-replenishment; and Ly-1B "sister" (B-1b) cells, which share many of the properties of Ly-1 B cells, including self-replenishment and feedback regulation of development but can also readily develop from progenitors in adult bone marrow. The sequential emergence of these lineages, the time at which their progenitors function during ontogeny, and the distinctions among their repertoires and functions suggest that evolution has created a layered immune system in which the immune response potential of each successive lineage is adapted to its particular niche. Images PMID:1565622

  7. Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells

    PubMed Central

    Wu, Jung-Lin; Wu, Hsin-Yi; Tsai, Dong-Yan; Chiang, Ming-Feng; Chen, Yi-Ju; Gao, Shijay; Lin, Chun-Cheng; Lin, Chun-Hung; Khoo, Kay-Hooi; Chen, Yu-Ju; Lin, Kuo-I.

    2016-01-01

    Crosslinking of B-cell receptor (BCR) sets off an apoptosis programme, but the underlying pathways remain obscure. Here we decipher the molecular mechanisms bridging B-cell activation and apoptosis mediated by post-translational modification (PTM). We find that O-GlcNAcase inhibition enhances B-cell activation and apoptosis induced by BCR crosslinking. This proteome-scale analysis of the functional interplay between protein O-GlcNAcylation and phosphorylation in stimulated mouse primary B cells identifies 313 O-GlcNAcylation-dependent phosphosites on 224 phosphoproteins. Among these phosphoproteins, temporal regulation of the O-GlcNAcylation and phosphorylation of lymphocyte-specific protein-1 (Lsp1) is a key switch that triggers apoptosis in activated B cells. O-GlcNAcylation at S209 of Lsp1 is a prerequisite for the recruitment of its kinase, PKC-β1, to induce S243 phosphorylation, leading to ERK activation and downregulation of BCL-2 and BCL-xL. Thus, we demonstrate the critical PTM interplay of Lsp1 that transmits signals for initiating apoptosis after BCR ligation. PMID:27555448

  8. Differential requirements for the canonical NF-κB transcription factors c-REL and RELA during the generation and activation of mature B-cells

    PubMed Central

    De Silva, Nilushi S.; Anderson, Michael M.; Silva, Kathryn; Carette, Amanda; Orelli, Fabiano; Bhagat, Govind; Klein, Ulf

    2016-01-01

    Signaling through the canonical NF-κB pathway is critical for the generation and maintenance of mature B-cells and for antigen-dependent B-cell activation. c-REL (rel) and RELA (rela) are the downstream transcriptional activators of the canonical NF-κB pathway. Studies of B-cells derived from constitutional rel knockout mice and chimeric mice repopulated with rela−/− fetal liver cells provided evidence that the subunits can have distinct roles during B-cell development. However, the B-cell-intrinsic functions of c-REL and RELA during B-cell generation and antigen-dependent B-cell activation have not been determined in vivo. To clarify this issue, we crossed mice with conditional rel and rela alleles individually or in combination to mice that express Cre-recombinase in B-cells. We here report that, whereas single deletion of rel or rela did not impair mature B-cell generation and maintenance, their simultaneous deletion led to a dramatic reduction of follicular and marginal zone B-cells. Upon T-cell-dependent immunization, B-cell-specific deletion of the c-REL subunit alone abrogated the formation of germinal centers (GC), whereas rela deletion did not affect GC formation. T-independent responses were strongly impaired in mice with B-cell-specific deletion of rel, and only modestly in mice with RELA-deficient B-cells. Our findings identify differential requirements for the canonical NF-κB subunits c-REL and RELA at distinct stages of mature B-cell development. The subunits are jointly required for the generation of mature B-cells. During antigen-dependent B-cell activation, c-REL is the critical subunit required for the initiation of the GC-reaction and for optimal T-independent antibody responses, with RELA being largely dispensable at this stage. PMID:27649781

  9. Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils.

    PubMed

    Chen, Kang; Xu, Weifeng; Wilson, Melanie; He, Bing; Miller, Norman W; Bengtén, Eva; Edholm, Eva-Stina; Santini, Paul A; Rath, Poonam; Chiu, April; Cattalini, Marco; Litzman, Jiri; B Bussel, James; Huang, Bihui; Meini, Antonella; Riesbeck, Kristian; Cunningham-Rundles, Charlotte; Plebani, Alessandro; Cerutti, Andrea

    2009-08-01

    Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

  10. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

    PubMed

    Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

  11. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Sánchez-Martínez, Diego; Lanuza, Pilar M.; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A.; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments. PMID:27833611

  12. Antitumor activity of fucoidan against diffuse large B cell lymphoma in vitro and in vivo.

    PubMed

    Yang, Guang; Zhang, Qianqiao; Kong, Yuanyuan; Xie, Bingqian; Gao, Minjie; Tao, Yi; Xu, Hongwei; Zhan, Fenghuang; Dai, Bojie; Shi, Jumei; Wu, Xiaosong

    2015-11-01

    Fucoidan is one of the major sulfated polysaccharides isolated from brown seaweeds. In this study, we determined the anti-cancer activity of fucoidan on diffuse large B cell lymphoma (DLBCL) cells both in vitro and in vivo. Fucoidan inhibited the growth of DLBCL cells in a dose- and time-dependent manner, and fucoidan treatment provoked G0/G1 cell cycle arrest, which was accompanied by p21 up-regulation and cyclin D1, Cdk4, and Cdk6 down-regulation. Fucoidan also induced caspase-dependent cell apoptosis in DLBCL cell lines and primary DLBCL cell. In addition, fucoidan treatment caused the loss of mitochondrial membrane potential and the release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Fucoidan also potentiated the activities of carfilzomib in killing DLBCL cells. Oral administration of fucoidan effectively inhibited tumor growth in xenograft mouse models. Our findings reveal the novel function of fucoidan as an anti-DLBCL agent, which can be used in the clinical treatment of DLBCL.

  13. A Recently Established Murine Model of Nasal Polyps Demonstrates Activation of B Cells, as Occurs in Human Nasal Polyps.

    PubMed

    Kim, Dong-Young; Lee, Sun Hye; Carter, Roderick G; Kato, Atsushi; Schleimer, Robert P; Cho, Seong H

    2016-08-01

    Animal model systems are invaluable for examining human diseases. Our laboratory recently established a mouse model of nasal polyps (NPs) and investigated similarities and differences between this mouse model and human NPs. We especially focus on the hypothesis that B cell activation occurs during NP generation in the murine model. After induction of ovalbumin-induced allergic rhinosinusitis, 6% ovalbumin and Staphylococcus aureus enterotoxin B (10 ng) were instilled into the nasal cavity of mice three times per week for 8 weeks. The development of structures that somewhat resemble NPs (which we will refer to as NPs) was confirmed by hematoxylin and eosin staining. The mRNA and protein levels of various inflammatory cell markers and mediators were measured by real-time PCR in nasal tissue and by ELISA in nasal lavage fluid (NLF), respectively. Total Ig isotype levels in NLF were also quantitated using the Mouse Ig Isotyping Multiplex kit (EMD Millipore, Billerica, MA) on a Luminex 200 instrument (Life Technologies, Grand Island, NY). Similar to human NPs, there were significant increases in gene expression of inflammatory cell markers, such as CD19, CD138, CD11c, and mast cell protease-6 in nasal tissue samples of the NP group compared with those of the control group. In further investigations of B cell activation, mRNA expressions of B cell activating factor and a proliferation-inducing ligand were found to be significantly increased in mouse NP tissue. B cell-activating factor protein concentration and IgA and IgG1 levels in NLF were significantly higher in the NP group compared with the control group. In this study, the NP mouse model demonstrated enhanced B cell responses, which are reminiscent of B cell responses in human NPs.

  14. The bioflavonoid galangin blocks aryl hydrocarbon receptor activation and polycyclic aromatic hydrocarbon-induced pre-B cell apoptosis.

    PubMed

    Quadri, S A; Qadri, A N; Hahn, M E; Mann, K K; Sherr, D H

    2000-09-01

    Bioflavonoids are plant compounds touted for their potential to treat or prevent several diseases including cancers induced by common environmental chemicals. Much of the biologic activity of one such class of pollutants, polycyclic aromatic hydrocarbons (PAH), is mediated by the aryl hydrocarbon receptor/transcription factor (AhR). For example, the AhR regulates PAH immunotoxicity that manifests as pre-B cell apoptosis in models of B cell development. Because bioflavonoids block PAH-induced cell transformation and are structurally similar to AhR ligands, it was postulated that some of them would suppress PAH-induced, AhR-dependent immunotoxicity, possibly through a direct AhR blockade. This hypothesis was tested using a model of B cell development in which pre-B cells are cultured with and are dependent on bone marrow stromal or hepatic parenchymal cell monolayers. Of seven bioflavonoids screened, galangin (3,5,7-trihydroxyflavone) blocked PAH-induced but not C(2)-ceramide- or H(2)O(2)-induced pre-B cell apoptosis. Because galangin blocked AhR-dependent reporter gene expression, AhR complex-DNA binding, and AhR nuclear translocation, inhibition of a relatively early step in AhR signaling was implicated. This hypothesis was supported by the ability of galangin to bind the AhR and stabilize AhR-90-kDa heat shock protein complexes in the presence of AhR agonists. These studies demonstrate the utility of pre-B cell culture systems in identifying compounds capable of blocking PAH immunotoxicity, define at least one mechanism of galangin activity (i.e., repression of AhR activation), and motivate the use of this and similar dietary bioflavonoids as relatively nontoxic inhibitors of AhR agonist activity and as pharmacologic agents with which to dissect AhR signaling pathways.

  15. The immunoregulator soluble TACI is released by ADAM10 and reflects B-cell activation in autoimmunity

    PubMed Central

    Hoffmann, Franziska S.; Kuhn, Peer-Hendrik; Laurent, Sarah A.; Hauck, Stefanie M.; Berer, Kerstin; Wendlinger, Simone A.; Krumbholz, Markus; Khademi, Mohsen; Olsson, Tomas; Dreyling, Martin; Pfister, Hans-Walter; Alexander, Tobias; Hiepe, Falk; Kümpfel, Tania; Crawford, Howard C.; Wekerle, Hartmut; Hohlfeld, Reinhard; Lichtenthaler, Stefan F.; Meinl, Edgar

    2014-01-01

    BAFF and APRIL, which control B-cell homeostasis, are therapeutic targets in autoimmune diseases. TACI-Fc (atacicept), a soluble fusion protein containing the extracellular domain of the BAFF-APRIL-receptor TACI, was applied in clinical trials. However disease activity in multiple sclerosis (MS) unexpectedly increased, whereas in systemic lupus erythematosus (SLE) atacicept was beneficial. Here, we show that an endogenous soluble form of TACI exists in vivo. TACI proteolysis involved shedding by ADAM10 releasing sTACI from activated B cells. The membrane-bound stub was subsequently cleaved by γ-secretase reducing ligand-independent signaling of the remaining C-terminal fragment. The shed ectodomain assembled ligand-independently in a homotypic way. It functioned as a decoy receptor inhibiting BAFF- and APRIL-mediated B-cell survival and NFκB-activation. We determined sTACI levels in autoimmune diseases with established hyper-activation of the BAFF-APRIL system. sTACI levels were elevated both in the cerebrospinal fluid (CSF) of the brain-restricted autoimmune disease MS correlating with intrathecal IgG production, as well as in the serum of the systemic autoimmune disease SLE correlating with disease activity. Together, we show that TACI is sequentially processed by ADAM10 and γ-secretase. The released sTACI is an immunoregulator that shares decoy functions with atacicept. Itreflectssystemic and compartmentalized B-cell accumulation and activation. PMID:25505277

  16. Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

    SciTech Connect

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M.

    2008-03-19

    Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a {beta}-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: ({alpha}/{alpha}){sub 5} for CS (chondroitin lyases AC) and {beta}-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located {approx}12 {angstrom} from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

  17. Secretion of thioredoxin after in vitro activation of human B cells.

    PubMed

    Ericson, M L; Hörling, J; Wendel-Hansen, V; Holmgren, A; Rosén, A

    1992-10-01

    The redox-active enzyme thioredoxin (Trx) is secreted by various virus-transformed cell lines of B- and T-cell origin and has been considered to play an autoregulatory role as a cofactor during cellular growth processes. We show in this paper that exposure of B lymphocytes from normal, healthy donors and B cells from B-type chronic lymphocytic leukemia (B-CLL) to Staphylococcus aureus Cowan I (SAC) induced expression of Trx mRNA. By combining SAC, or the phorbol ester TPA, with IL-2 and the conditioned medium of a T-cell hybridoma (BSF-MP6), we could strongly enhance the Trx expression. After [35S]methionine labeling of stimulated B-CLL cells in vitro, Trx was immunoprecipitated both from cell extracts and from the medium with antibodies against human placenta Trx. Secretion of newly synthesized Trx was also confirmed by a quantitative radioimmunoassay for human Trx. During 24 h cultivation experiments, treatment with SAC induced a 5-fold increase of the Trx content of normal B lymphocytes as well as in B-CLL cells. Approximately two-thirds of the total amount of the enzyme was released into the medium.

  18. B cell activating factor is central to bleomycin- and IL-17-mediated experimental pulmonary fibrosis.

    PubMed

    François, Antoine; Gombault, Aurélie; Villeret, Bérengère; Alsaleh, Ghada; Fanny, Manoussa; Gasse, Paméla; Adam, Sylvain Marchand; Crestani, Bruno; Sibilia, Jean; Schneider, Pascal; Bahram, Seiamak; Quesniaux, Valérie; Ryffel, Bernhard; Wachsmann, Dominique; Gottenberg, Jacques-Eric; Couillin, Isabelle

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive devastating, yet untreatable fibrotic disease of unknown origin. We investigated the contribution of the B-cell activating factor (BAFF), a TNF family member recently implicated in the regulation of pathogenic IL-17-producing cells in autoimmune diseases. The contribution of BAFF was assessed in a murine model of lung fibrosis induced by airway administered bleomycin. We show that murine BAFF levels were strongly increased in the bronchoalveolar space and lungs after bleomycin exposure. We identified Gr1(+) neutrophils as an important source of BAFF upon BLM-induced lung inflammation and fibrosis. Genetic ablation of BAFF or BAFF neutralization by a soluble receptor significantly attenuated pulmonary fibrosis and IL-1β levels. We further demonstrate that bleomycin-induced BAFF expression and lung fibrosis were IL-1β and IL-17A dependent. BAFF was required for rIL-17A-induced lung fibrosis and augmented IL-17A production by CD3(+) T cells from murine fibrotic lungs ex vivo. Finally we report elevated levels of BAFF in bronchoalveolar lavages from IPF patients. Our data therefore support a role for BAFF in the establishment of pulmonary fibrosis and a crosstalk between IL-1β, BAFF and IL-17A.

  19. Polyclonal B-cell activation induced by extracts of Gram-negative bacteria isolated from periodontally diseased sites.

    PubMed Central

    Bick, P H; Carpenter, A B; Holdeman, L V; Miller, G A; Ranney, R R; Palcanis, K G; Tew, J G

    1981-01-01

    The objective of this research was to determine whether gram-negative bacteria frequently isolated from periodontally diseased sites contained polyclonal B-cell activators. Polyclonal B-cell activation, which results in nonspecific activation of multiple B-cell clones was analyzed by a hemolysis-in-gel assay designed to detect a broad range of antibody specificities. Extracts from numerous bacterial strains, including Bacteroides gingivalis, Bacteroides melaninogenicus subsp. melaninogenicus, B. melaninogenicus subsp. intermedius, Fusobacterium nucleatum, Selenomonas sputigena, Capnocytophaga ochracea, and Actinobacillus actinomycetemcomitans, were tested. Extracts of the above organisms were found to stimulate polyclonal antibody responses in cultures of normal human peripheral blood lymphocytes, although the magnitude of stimulation varied among the extracts. Optimal antibody-forming cell responses were found at stimulator doses between 5 and 1,000 micrograms/ml. We conclude that the resident gram-negative subgingival flora associated with periodontal lesions possesses potent polyclonal B-cell activators. These activators may contribute to disease pathogenesis by inducing B lymphocytes to produce antibody, osteolytic factors, or both and possibly other mediators of inflammation. PMID:6975240

  20. Epstein-Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease.

    PubMed

    Nagata, Keiko; Kumata, Keisuke; Nakayama, Yuji; Satoh, Yukio; Sugihara, Hirotsugu; Hara, Sayuri; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Hayashi, Kazuhiko

    2017-04-01

    Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein-Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases.

  1. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    DOE PAGES

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; ...

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, whichmore » coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.« less

  2. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    SciTech Connect

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  3. Contact dependent suppression of CD4 T cell activation and proliferation by B cells activated through IgD cross-linking.

    PubMed

    Preciado-Llanes, Lorena; Wing, James B; Foster, Rachel A; Carlring, Jennifer; Lees, Andrew; Read, Robert C; Heath, Andrew W

    2014-09-20

    Although the co-stimulatory interaction between B and T cells is well defined, recent evidence suggests that B cells also have a regulatory role. Here, we show that B cells activated using anti-IgD conjugated to dextran (α-δ-dex) directly inhibit TCR-induced CD4 T cell activation, proliferation and cytokine production. This effect was observed in CD4 T cells activated both with and without CD28 co-stimulation. T cell viability was unaffected, and the T cell suppressive effect was mediated by contact with IgD activated purified B cells and not by IL-10 or other soluble factors. This is the first evidence of IgD activated B cells mediating inhibition of activation and proliferation of CD4 T cells in humans. This article is protected by copyright. All rights reserved.

  4. MAPkinase: a second site of G-protein regulation of B-cell activation via the antigen receptors.

    PubMed Central

    Deehan, M R; Klaus, G G; Holman, M J; Harnett, W; Harnett, M M

    1998-01-01

    Ligation of the antigen receptors on B cells transduces transmembrane signals leading to the induction of DNA synthesis. We now show that a pertussis toxin-sensitive heterotrimeric G-protein(s) of the Gi class plays a key role in the regulation of surface immunoglobulin (sIg)-mediated DNA synthesis in B cells. This site of G-protein regulation is distinct from that we have previously reported to govern the coupling of the antigen receptors on B cells to the phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate. We have, moreover, identified a candidate target for this new G-protein regulation by showing that mitogen-activating protein kinase (MAPkinase) activity, which plays a key role in the transduction of sIg-mediated proliferative signals in B cells, is abrogated by pre-exposure to pertussis toxin that covalently modifies and inactivates heterotrimeric G-proteins of the Gi class. Furthermore, our data suggest that this pertussis toxin-sensitive G-protein couples the antigen receptors to MAPkinase activation, at least in part, by regulating sIg-coupling to Lyn, Syk and perhaps Blk and Fyn activity, results consistent with studies in other systems which show that classical G-protein-coupled receptors recruit such protein tyrosine kinases to tranduce MAPkinase activation. Interestingly, however, this G-protein plays no apparent role in the control of up-regulation of major histocompatibility complex class II expression on B cells, suggesting that such G-protein-regulated-tyrosine kinase and MAPkinase activation is not required for the induction of this biological response following antigen receptor ligation. Images Figure 5 PMID:9824472

  5. The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

    PubMed

    Bao, Huan; Dalal, Kush; Wang, Victor; Rouiller, Isabelle; Duong, Franck

    2013-08-01

    The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

  6. Autoreactive helper T cells alleviate the need for intrinsic TLR signaling in autoreactive B cell activation

    PubMed Central

    Giles, Josephine R.; Neves, Adriana Turqueti; Marshak-Rothstein, Ann; Shlomchik, Mark J.

    2017-01-01

    T cells play a significant role in the pathogenesis of systemic autoimmune diseases, including systemic lupus erythematosus; however, there is relatively little information on the nature and specificity of autoreactive T cells. Identifying such cells has been technically difficult because they are likely to be rare and low affinity. Here, we report a method for identifying autoreactive T cell clones that recognize proteins contained in autoantibody immune complexes, providing direct evidence that functional autoreactive helper T cells exist in the periphery of normal mice. These T cells significantly enhanced autoreactive B cell proliferation and altered B cell differentiation in vivo. Most importantly, these autoreactive T cells were able to rescue many aspects of the TLR-deficient AM14 (anti-IgG2a rheumatoid factor) B cell response, suggesting that TLR requirements can be bypassed. This result has implications for the efficacy of TLR-targeted therapy in the treatment of ongoing disease. PMID:28239656

  7. ABC relaxation theory and the factor structure of relaxation states, recalled relaxation activities, dispositions, and motivations.

    PubMed

    Smith, J C; Wedell, A B; Kolotylo, C J; Lewis, J E; Byers, K Y; Segin, C M

    2000-06-01

    ABC Relaxation Theory proposes 15 psychological relaxation-related states (R-States): Sleepiness, Disengagement, Physical Relaxation, Mental Quiet, Rested/Refreshed, At Ease/At Peace, Energized, Aware, Joy, Thankfulness and Love, Prayerfulness, Childlike Innocence, Awe and Wonder, Mystery, and Timeless/Boundless/Infinite. The present study summarizes the results of 13 separate factor analyses of immediate relaxation-related states, states associated with recalled relaxation activities, relaxation dispositions, and relaxation motivations on a combined sample of 1,904 individuals (group average ages ranged from 28-40 yr.). Four exploratory factor analyses of Smith Relaxation Inventories yielded 15 items that most consistently and exclusively load (generally at least .70) on six replicated factors. These items included happy, joyful, energized, rested, at peace, warm, limp, silent, quiet, dozing, drowsy, prayerful, mystery, distant, and indifferent. Subsequent factor analyses restricted to these items and specifying six factors were performed on 13 different data sets. Each yielded the same six-factor solution: Factor 1: Centered Positive Affect, Factor 2: Sleepiness, Factor 3: Disengagement, Factor 4: Physical Relaxation, Factor 5: Mental Quiet, and Factor 6: Spiritual. Implications for ABC Relaxation Theory are discussed.

  8. Active breathing control (ABC): Determination and reduction of breathing-induced organ motion in the chest

    SciTech Connect

    Gagel, Bernd . E-mail: BGagel@UKAachen.de; Demirel, Cengiz M.P.; Kientopf, Aline; Pinkawa, Michael; Piroth, Marc; Stanzel, Sven; Breuer, Christian; Asadpour, Branka; Jansen, Thomas; Holy, Richard; Wildberger, Joachim E.; Eble, Michael J.

    2007-03-01

    Purpose: Extensive radiotherapy volumes for tumors of the chest are partly caused by interfractional organ motion. We evaluated the feasibility of respiratory observation tools using the active breathing control (ABC) system and the effect on breathing cycle regularity and reproducibility. Methods and Materials: Thirty-six patients with unresectable tumors of the chest were selected for evaluation of the ABC system. Computed tomography scans were performed at various respiratory phases starting at the same couch position without patient movement. Threshold levels were set at minimum and maximum volume during normal breathing cycles and at a volume defined as shallow breathing, reflecting the subjective maximal tolerable reduction of breath volume. To evaluate the extent of organ movement, 13 landmarks were considering using commercial software for image coregistration. In 4 patients, second examinations were performed during therapy. Results: Investigating the differences in a normal breathing cycle versus shallow breathing, a statistically significant reduction of respiratory motion in the upper, middle, and lower regions of the chest could be detected, representing potential movement reduction achieved through reduced breath volume. Evaluating interfraction reproducibility, the mean displacement ranged between 0.24 mm (chest wall/tracheal bifurcation) to 3.5 mm (diaphragm) for expiration and shallow breathing and 0.24 mm (chest wall) to 5.25 mm (diaphragm) for normal inspiration. Conclusions: By modifying regularity of the respiratory cycle through reduction of breath volume, a significant and reproducible reduction of chest and diaphragm motion is possible, enabling reduction of treatment planning margins.

  9. Combinatorial BTK and MALT1 inhibition augments killing of CD79 mutant diffuse large B cell lymphoma.

    PubMed

    Nagel, Daniel; Bognar, Miriam; Eitelhuber, Andrea C; Kutzner, Kerstin; Vincendeau, Michelle; Krappmann, Daniel

    2015-12-08

    Survival of activated B cell-subtype (ABC) of diffuse large B cell lymphoma (DLBCL) is driven by chronic B cell receptor (BCR) signaling that activates the canonical NF-κB pathway. Inhibition of BTK by Ibrutinib has been shown to kill ABC DLBCL cells that carry activating mutations in the BCR adaptor CD79. However, mutations in BTK or in downstream components such as CARMA1/CARD11 can render lymphomas Ibrutinib resistant. Therefore, we assessed here the simultaneous inhibition of BTK and the protease MALT1 that acts downstream of CARMA1 and is essential for ABC DLBCL tumor growth. We show that in CD79 mutant cells BTK is a crucial upstream regulator of MALT1, but dispensable in CARMA1 mutant ABC DLBCL. Combined inhibition of BTK by Ibrutinib and MALT1 by S-Mepazine additively impaired MALT1 cleavage activity and expression of NF-κB pro-survival factors. Thereby, combinatorial Ibrutinib and S-Mepazine treatment enhanced killing of CD79 mutant ABC DLBCL cells. Moreover, while expression of oncogenic CARMA1 in CD79 mutant cells conferred Ibrutinib resistance, double mutant cells were still sensitive to MALT1 inhibition by S-Mepazine. Thus, based on the genetic background combinatorial BTK and MALT1 inhibition may improve effectiveness of therapeutic treatment and reduce the chances for the development of drug resistances.

  10. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation of primary human B cells.

    PubMed

    Lu, Haitian; Crawford, Robert B; Kaplan, Barbara L F; Kaminski, Norbert E

    2011-09-15

    Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells.

  11. Differential effects of interleukin-2 and interleukin-4 on immunomodulatory role of platelet-activating factor in human B cells.

    PubMed Central

    Patke, C L; Green, C G; Shearer, W T

    1994-01-01

    Platelet-activating factor (PAF), a naturally occurring phospholipid cytokine, is a potent mediator of allergic and inflammatory reactions, as well as a modulator of immune responses. In the present study we showed that PAF is involved in early B-cell activation, as demonstrated by the increased cyclic AMP (cAMP) generation by PAF in a time- and dose-dependent manner in anti-mu antibody- plus B-cell growth factor-activated normal human peripheral blood B lymphocytes. PAF also regulated differentiation by causing a biphasic response on immunoglobulin M (IgM) production with an inhibitory signal generated at 10(-6) M and a stimulatory signal generated at 10(-8) to 10(-10) M. PAF enhanced IgA secretion. The regulation exerted by PAF was shown to be specific because the addition of the PAR antagonist CV-3988 abrogated these effects and the inactive form of PAF, lyso-PAF, induced neither cAMP generation nor immunoglobulin secretion in normal human B cells. Other cytokines, interleukin-2 (IL-2) and IL-4, potent mediators of the immune response, were unable to elicit a cAMP response in B cells. However, the addition of PAF (10(-6) M) with wither IL-2 or IL-4 enhanced cAMP production above the levels enhanced by the addition of PAF alone. IL-2 or IL-4, individually, stimulated IgM production, yet costimulation with PAF resulted in a differential effect between IL-2 and IL-4. PAF down-regulated the IL-4-induced IgM secretion, whereas the IL-2-induced IgM secretion was enhanced. The presence of CV-3988 returned all valued to those obtained with IL-2 or IL-4 alone, demonstrating the specificity of PAF. These data suggest that PAF is an important B-cell immunomodulator which can interact with other leukocyte cell mediators. PMID:8556480

  12. CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics1

    PubMed Central

    Chen, Ding; Ireland, Sara J.; Remington, Gina; Alvarez, Enrique; Racke, Michael K.; Greenberg, Benjamin; Frohman, Elliot M.; Monson, Nancy L.

    2017-01-01

    CD40 interacts with CD40 ligand and plays an essential role in immune regulation and homeostasis. Recent research findings, however, support a pathogenic role of CD40 in a number of autoimmune diseases. We previously showed that memory B cells from relapsing-remitting multiple sclerosis (RRMS) patients exhibited enhanced proliferation with CD40 stimulation compared to healthy donors. In this study, we used a multi-parameter phosflow approach to analyze the phosphorylation status of NFκB and three major MAP kinases (P38, ERK and JNK), the essential components of signaling pathways downstream of CD40 engagement in B cells from MS patients. We found that memory and naïve B cells from RRMS and secondary progressive MS (SPMS) patients exhibited a significantly elevated level of phosphorylated NFκB (p-P65) following CD40 stimulation compared to healthy donor controls. Combination therapy with interferon beta-1a (Avonex) and mycophenolate mofetil (Cellcept) modulated the hyper-phosphorylation of P65 in B cells of RRMS patients at levels similar to healthy donor controls. Lower disease activity after the combination therapy correlated with the reduced phosphorylation of P65 following CD40 stimulation in treated patients. In addition, glatiramer acetate (GA) treatment also significantly reduced CD40-mediated P65 phosphorylation in RRMS patients, suggesting that reducing CD40-mediated p-P65 induction may be a general mechanism by which some current therapies modulate MS disease. PMID:27798157

  13. The Role of TLR4 on B Cell Activation and Anti-β2GPI Antibody Production in the Antiphospholipid Syndrome

    PubMed Central

    2016-01-01

    High titer of anti-β2-glycoprotein I antibodies (anti-β2GPI Ab) plays a pathogenic role in antiphospholipid syndrome (APS). Numerous studies have focused on the pathological mechanism in APS; however, little attention is paid to the immune mechanism of production of anti-β2GPI antibodies in APS. Our previous study demonstrated that Toll-like receptor 4 (TLR4) plays a vital role in the maturation of bone marrow-derived dendritic cells (BMDCs) from the mice immunized with human β2-glycoprotein I (β2GPI). TLR4 is required for the activation of B cells and the production of autoantibody in mice treated with β2GPI. However, TLR4 provides a third signal for B cell activation and then promotes B cells better receiving signals from both B cell antigen receptor (BCR) and CD40, thus promoting B cell activation, surface molecules expression, anti-β2GPI Ab production, and cytokines secretion and making B cell functioning like an antigen presenting cell (APC). At the same time, TLR4 also promotes B cells producing antibodies by upregulating the expression of B-cell activating factor (BAFF). In this paper, we aim to review the functions of TLR4 in B cell immune response and antibody production in autoimmune disease APS and try to find a new way for the prevention and treatment of APS. PMID:27868072

  14. Negative role of TAK1 in marginal zone B-cell development incidental to NF-κB noncanonical pathway activation

    PubMed Central

    Shinohara, Hisaaki; Kurosaki, Tomohiro

    2016-01-01

    The transcription factor nuclear factor-κB (NF-κB) signaling pathway is crucial in B-cell physiology. One key molecule regulating this pathway is the serine/threonine kinase TAK1 (MAP3K7). TAK1 is responsible for positive feedback mechanisms in B-cell receptor signaling that serve as an NF-κB activation threshold. This study aimed to better understand the correlation between TAK1-mediated signaling and B-cell development and humoral immune responses. Here we showed that a B-cell conditional deletion of TAK1 using mb1-cre resulted in a dramatic elimination of the humoral immune response, consistent with the absence of the B-1 B-cell subset. When monitoring the self-reactive B-cell system (the immunoglobulin hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model), we found that TAK1-deficient B cells exhibited an enhanced susceptibility to cell death that might explain the disappearance of the B1 subset. In contrast, these mice gained numerous marginal zone (MZ) B cells. We consequently examined the basal and B-cell receptor-induced activity of NF-κB2 that is reported to regulate MZ B-cell development, and demonstrated that the activity of NF-κB2 increased in TAK1-deficient B cells. Thus, our results present a novel in vivo function, the negative role of TAK1 in MZ B-cell development that is likely associated with NF-κB2 activation. PMID:27121163

  15. Social Experiences of Beginning Braille Readers in Literacy Activities: Qualitative and Quantitative Findings of the ABC Braille Study

    ERIC Educational Resources Information Center

    Sacks, Sharon Z.; Kamei-Hannan, Cheryl; Erin, Jane N.; Barclay, Lizbeth; Sitar, Debbie

    2009-01-01

    This mixed-design investigation examined the social experiences of beginning braille readers who were initially taught contracted or alphabetic braille in literacy activities as part of the ABC Braille Study. No differences in the quality or quantity of social experiences were found between the two groups over time. (Contains 4 tables.)

  16. The requirements for growth of in vivo activated autoimmune B cells are similar to those of in vitro generated lipopolysaccharide B cell blasts and dissimilar to anti-IgM plus IL-4 induced B lymphoblasts.

    PubMed

    Cabrero, E; Sánchez, M J; Gutiérrez, C

    1992-06-01

    The requirements for growth of in vivo activated B cells (natural blasts) from autoimmune NZB/W mice and of B cells from the same animals activated in vitro with either LPS or anti-IgM plus IL4 (mimicking 'in vitro' antigen induced TH cell-B cell interaction) were studied comparatively. The proliferation of natural and LPS blasts was inhibited by anti-IgM antibodies and augmented by recombinant IL-5. In contrast, anti-IgM stimulated the growth of anti-IgM plus IL-4 primed B cells but was without effect on the proliferative responses in the presence of IL-5. The growth inhibition induced by anti-IgM signalling on natural and LPS blasts seemed to be due to cross-linking of sIg rather than to binding of anti-IgM antibodies to the Fc receptors since a similar effect was observed with the F(ab)'2 fragment of this molecule. Maximum proliferation was obtained by a combination of IL-4 and IL-5 in natural and LPS blasts, whereas peak responses in anti-IgM plus IL-4 blasts were achieved by a combination of anti-IgM and IL-4. Lymphoblasts recovered after preculturing natural blasts in medium alone (more differentiated in vivo activated B cells) displayed high spontaneous proliferation which was strongly inhibited by anti-IgM. This inhibition was reversed partially by IL-4 and totally by IL-5. To define better the role of BLy+ cells in the spleen of NZB/W mice, purified Ly1+ and Ly1- cells, obtained by separation using magnetic beads, were analysed. The growth of both cell subpopulations was inhibited by anti-IgM and enhanced by IL-5. Cytotoxic elimination of Ly1+ cells from the primed B blast populations did not modify the proliferative pattern of these cells. Our results show that the growth requirements of in vivo activated autoimmune B cells resemble those of LPS blasts and differ from those following stimulation with anti-IgM plus IL-4, suggesting that B cells in systemic autoimmune diseases may have been activated by polyclonal stimulation. Nevertheless, other mechanisms

  17. Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B cell acute lymphoblastic leukemia

    PubMed Central

    Heltemes-Harris, Lynn M.; Larson, Jon D.; Starr, Timothy K.; Hubbard, Gregory K.; Sarver, Aaron L.; Largaespada, David A.; Farrar, Michael A.

    2015-01-01

    STAT5 activation occurs frequently in human progenitor B cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) to mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b–CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the JAK/STAT5 pathway (ii) progenitor B cell differentiation and (iii) the CDKN2A tumor suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, ERK and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways play in B-ALL. PMID:26500062

  18. The effect of B-cell depletion therapy on serological evidence of B-cell and plasmablast activation in patients with rheumatoid arthritis over multiple cycles of rituximab treatment.

    PubMed

    Cambridge, G; Perry, H C; Nogueira, L; Serre, G; Parsons, H M; De La Torre, I; Dickson, M C; Leandro, M J; Edwards, J C W

    2014-05-01

    B-cell depletion therapy (BCDT) based on rituximab (RTX) induces clinical remission in a majority of seropositive patients with Rheumatoid arthritis (RA). However, all patients eventually relapse. The aim of this study was to determine whether dynamic changes in combinations of serological measures of B-cell activation were associated over up to three cycles of BCDT. We included only RA patients who gave an adequate clinical response, as measured by DAS28. Twenty three patients were studied over 1 cycle, 21 over 2, and 15 over 3 cycles of BCDT. Serum analytes including isotypes of Rheumatoid factors (RhF) and anti-citrullinated protein/peptide antibodies (ACPA), B-cell activating factor (BAFF), serum free light chains (SFLC), soluble CD23 (sCD23), antibodies to tetanus toxoid (TT) and to pneumococcal capsular polysaccharide (PCP) were measured by ELISA at 4 key points in each cycle, namely: Baseline (pre-RTX in each cycle); when B-cell depleted (CD19+B-cells < 5/μl); at B-cell return (CD19+B-cells ≥ 5/μl); and at clinical relapse (ΔDAS28 > 1.2). SFLC were used as a measure of plasmablast activity. As sCD23 is cleaved from naïve B-cells coincident with attaining CD27 expression, levels were used as a novel measure of maturation of B-cells to CD27+. The most consistent changes between baseline and B-cell depletion within all 3 cycles were in SFLC, sCD23 and IgM-RhF which fell and in BAFF levels which rose. After 3 complete cycles of BCDT, both IgM autoantibodies and IgG-CCP had decreased, BAFF levels were higher (all p < 0.05); other analytes remained unchanged compared with baseline. Dynamic changes in λSFLC, sCD23, IgM-RhF and BAFF were also consistently associated with relapse in patients with longer clinical responses after B-cell return. Incremental rises in sCD23 levels in cycles 2 and 3 were correlated with time to relapse. Repopulation of the periphery after BCDT is initiated by naïve B-cells and precedes relapse. Our study showed that

  19. Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma

    PubMed Central

    Shen, Yulei; Huang, Xin; Liu, Yanyan; Wake, Laura; Liu, Cuiling; Deffenbacher, Karen; Lachel, Cynthia M.; Wang, Chao; Rohr, Joseph; Guo, Shuangping; Smith, Lynette M.; Wright, George; Bhagavathi, Sharathkumar; Dybkaer, Karen; Fu, Kai; Greiner, Timothy C.; Vose, Julie M.; Jaffe, Elaine; Rimsza, Lisa; Rosenwald, Andreas; Ott, German; Delabie, Jan; Campo, Elias; Braziel, Rita M.; Cook, James R.; Tubbs, Raymond R.; Armitage, James O.; Weisenburger, Dennis D.; Staudt, Louis M.; Gascoyne, Randy D.; McKeithan, Timothy W.; Chan, Wing C.

    2015-01-01

    We studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)–DLBCL compared with activated B-cell (ABC)–DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the “gold standard” GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL. PMID:25498913

  20. mTOR activity in AIDS-related diffuse large B-cell lymphoma

    PubMed Central

    Diaz-Perez, Julio A.; Preziosi, Michael; King, Charles C.; Jones, George A.; Jain, Sonia; Sun, Xiaoying; Reid, Erin G.; VandenBerg, Scott; Wang, Huan-You

    2017-01-01

    Background Patients infected with HIV have a significantly increased risk of developing non–Hodgkin lymphomas despite the widespread use of HAART. To investigate mTOR pathway activity in acquired immunodeficiency syndrome (AIDS) related diffuse large B-cell lymphoma AR-DLBCL, we used immunohistochemistry to examine the presence of the phosphorylated 70 ribosomal S6 protein-kinase (p70S6K), an extensively studied effector of mTOR Complex 1 (mTORC1) and the phosphorylated phosphatase and tensin homolog (pPTEN), a negative regulator of mTORC1 pathway. Materials and methods We evaluated tissue samples from 126 patients with AR-DLBCL. Among them, 98 samples were from tissue microarrays (TMAs) supplied by the Aids and Cancer Specimen Resource (ACSR), the remaining 28 samples were from cases diagnosed and treated at the University of California, San Diego (UCSD). The presence of p70S6K was evaluated with two antibodies directed against the combined epitopes Ser235/236 and Ser240/244, respectively; and additional monoclonal anti-bodies were used to identify pPTEN and phosphorylated proline-rich Akt substrate of 40kDa (pPRAS40). The degree of intensity and percentage of cells positive for p70S6K and pPTEN were assessed in all the samples. In addition, a subgroup of 28 patients from UCSD was studied to assess the presence of pPRAS40, an insulin-regulated activator of the mTORC1. The expression of each of these markers was correlated with clinical and histopathologic features. Results The majority of the patients evaluated were males (88%); only two cases (1.6%) were older than 65 years of age. We found high levels of both p70S6K-paired epitopes studied, 48% positivity against Ser235/236 (44% in ACSR and 64% in UCSD group), and 86% positivity against Ser240/244 (82% in ACSR and 100% in UCSD group). We observed more positive cells and stronger intensity with epitope Ser240/244 in comparison to Ser235/236 (p<0.0001). The degree of intensity and percentage of cells positive

  1. Serum IgD elevation is an early marker of B cell activation during infection with the human immunodeficiency viruses.

    PubMed Central

    Mizuma, H; Zolla-Pazner, S; Litwin, S; el-Sadr, W; Sharpe, S; Zehr, B; Weiss, S; Saxinger, W C; Marmor, M

    1987-01-01

    Serum IgD levels in individuals infected with the human immunodeficiency viruses (HIV) were studied as a means of monitoring the character and timing of B cell activation in individuals with this infection. Significantly increased levels of IgD were characteristic of homosexual men who were HIV seropositive but asymptomatic or mildly symptomatic. The hyper IgD globulinaemia became progressively more pronounced in patients with increasingly severe infection and reached its most marked level in patients with AIDS-related complex (ARC). In ARC patients, IgD levels were increased 8.8-fold above normal which was disproportionately greater than the 2.4-fold increase in IgG, the 1.8-fold increase in IgA and the 1.6-fold increase in IgM. IgD levels declined in AIDS patients (although remained elevated compared to controls). The data suggest that an unusual type of B cell activation is responsible for the unique pattern of hypergammaglobulinaemia seen in this disease and that the B cell activation occurs early in the pathogenesis of HIV infection, often before development of symptoms, and continues throughout the course of infection. PMID:3498566

  2. Serum IgD elevation is an early marker of B cell activation during infection with the human immunodeficiency viruses.

    PubMed

    Mizuma, H; Zolla-Pazner, S; Litwin, S; el-Sadr, W; Sharpe, S; Zehr, B; Weiss, S; Saxinger, W C; Marmor, M

    1987-04-01

    Serum IgD levels in individuals infected with the human immunodeficiency viruses (HIV) were studied as a means of monitoring the character and timing of B cell activation in individuals with this infection. Significantly increased levels of IgD were characteristic of homosexual men who were HIV seropositive but asymptomatic or mildly symptomatic. The hyper IgD globulinaemia became progressively more pronounced in patients with increasingly severe infection and reached its most marked level in patients with AIDS-related complex (ARC). In ARC patients, IgD levels were increased 8.8-fold above normal which was disproportionately greater than the 2.4-fold increase in IgG, the 1.8-fold increase in IgA and the 1.6-fold increase in IgM. IgD levels declined in AIDS patients (although remained elevated compared to controls). The data suggest that an unusual type of B cell activation is responsible for the unique pattern of hypergammaglobulinaemia seen in this disease and that the B cell activation occurs early in the pathogenesis of HIV infection, often before development of symptoms, and continues throughout the course of infection.

  3. Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency.

    PubMed

    Wille, Coral K; Li, Yangguang; Rui, Lixin; Johannsen, Eric C; Kenney, Shannon C

    2017-03-01

    Epstein-Barr virus (EBV) latently infects normal B cells and contributes to the development of certain human lymphomas. Newly infected B cells support a highly transforming form (type III) of viral latency; however, long-term EBV infection in immunocompetent hosts is limited to B cells with a more restricted form of latency (type I) in which most viral gene expression is silenced by promoter DNA methylation. How EBV converts latency type is unclear, although it is known that type I latency is associated with a germinal center (GC) B cell phenotype, and type III latency with an activated B cell (ABC) phenotype. In this study, we have examined whether expression of TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells. We found that TET2 expression is inhibited in normal GC cells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cells and ABC type lymphomas. We also demonstrate that GC type cell lines have increased 5mC levels and reduced 5hmC levels in comparison to those of ABC type lines. Finally, we show that TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected cells from type I to type III latency. These findings demonstrate that TET2 expression is repressed in GC cells independent of EBV infection and suggest that TET2 promotes type III EBV latency in B cells with an ABC or naive phenotype by enhancing EBNA2 activation of methylated EBV promoters.IMPORTANCE EBV establishes several different types of viral latency in B cells. However, cellular factors that determine whether EBV enters the highly transforming type III latency, versus the more restricted type I latency, have not been well characterized. Here we show that TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells by

  4. Soluble, but not immobilized, anti-IgM antibody inhibits post-activation events leading to T-cell-dependent B-cell differentiation.

    PubMed Central

    Zamorano, J; Rivas, D; Gayo, A; Mozo, L; Gutiérrez, C

    1995-01-01

    The potential for surface immunoglobulin-binding ligands to modify B-cell differentiation responses induced by activated T cells has been investigated. Activated T cells in human splenic mononuclear cells cultured on anti-CD3-coated plates induced B cells to produce large amounts of IgM and IgG. In this experimental system, cross-linking of B-cell antigen receptors by soluble, bivalent monoclonal or polyclonal anti-IgM antibodies completely inhibited IgM production, and greatly diminished IgG production, in a dose-dependent manner. Similar results were obtained using a F(ab')2 fragment of a goat anti-IgM antibody. Inhibition of B-cell differentiation by bivalent cross-linking reagents did not require the presence of antigen-presenting cells (APC), as comparable results were obtained in co-cultures of purified T and B cells. In contrast, enhanced immunoglobulin secretion was seen when surface IgM was cross-linked using anti-IgM antibody immobilized on the culture plate. Interestingly, activated T cells induced similar levels of expression on B cells of the activation antigens CD23, CD25 and CD71, and of class II molecules, irrespective of any treatment with soluble or immobilized anti-IgM antibody. This indicates that soluble anti-IgM specifically inhibits B-cell differentiation without altering initial events of T-cell-dependent B-cell activation. Images Figure 3 Figure 5 PMID:7642212

  5. CpG-based immunotherapy impairs antitumor activity of BRAF inhibitors in a B-cell-dependent manner.

    PubMed

    Huang, L; Wang, Z; Liu, C; Xu, C; Mbofung, R M; McKenzie, J A; Khong, H; Hwu, P; Peng, W

    2017-03-06

    Combining immunotherapy with targeted therapy has increasingly become an appealing therapeutic paradigm for cancer treatment due to its great potential for generating durable and synergistic antitumor response. In this study, however, we unexpectedly found that two types of CpG-based tumor peptide vaccine treatments consistently negated the antitumor activity of a selective BRAF inhibitor in tumors with BRAF mutation rather than showing a synergistic antitumor effect. Our further studies demonstrated that CpG alone was sufficient to dampen BRAF inhibitor-induced antitumor responses, suggesting that the impaired antitumor activity of the BRAF inhibitor observed in mice receiving CpG-based peptide vaccine is mainly dependent upon the use of CpG. Mechanistically, CpG increased the number of circulating B cells, which produced elevated amounts of tumor necrosis factor-α (TNFα) that contributed to the increased tumor resistance to BRAF inhibitors. More importantly, B-cell depletion or TNFα neutralization can restore the antitumor effect of BRAF inhibition in mice receiving CpG treatment, indicating that TNFα-secreting B cells play an indispensable role in BRAF inhibitor resistance induced by CpG. Taken together, our results strongly suggest that precautions must be implemented when designing combinatorial approaches for cancer treatment, because distinct regimens, despite their respective therapeutic benefit as monotherapy, may together provide antagonistic clinical outcomes.Oncogene advance online publication, 6 March 2017; doi:10.1038/onc.2017.35.

  6. Cytotoxic activity of the amphibian ribonucleases onconase and r-amphinase on tumor cells from B cell lymphoproliferative disorders.

    PubMed

    Smolewski, Piotr; Witkowska, Magdalena; Zwolinska, Malgorzata; Cebula-Obrzut, Barbara; Majchrzak, Agata; Jeske, Aleksandra; Darzynkiewicz, Zbigniew; Ardelt, Wojciech; Ardelt, Barbara; Robak, Tadeusz

    2014-07-01

    Although major advancements in antitumor treatment have been observed, several B cell-derived malignancies still remain incurable. A promising approach that involves targeting RNA either by the use of specific antisense oligonucleotides or cytostatic/cytotoxic ribonucleases (RNases) is being promoted. Two amphibian RNases, onconase (ONC; ranpirnase) and, more recently, r-amphinase (r-Amph), have already been tested, but thus far, mostly on solid tumors. In this study, for the first time we provide comprehensive data on ex vivo and in vivo cytotoxic activity of ONC or r-Amph against cancer cells from different B cell lymphoid malignancies, together with their detailed mode of antitumor action. Our data revealed strong pro-apoptotic activity of ONC and r-Amph in both chronic lymphocytic leukemia and aggressive B cell lymphomas, with less impact on acute lymphoblastic leukemia or multiple myeloma cells. Moreover, the antitumor action of ONC and r-Amph was markedly selective against neoplastic cells sparing normal, healthy control‑derived lymphocytes.

  7. IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL.

    PubMed

    Nogai, Hendrik; Wenzel, Sören-Sebastian; Hailfinger, Stephan; Grau, Michael; Kaergel, Eva; Seitz, Volkhard; Wollert-Wulf, Brigitte; Pfeifer, Matthias; Wolf, Annette; Frick, Mareike; Dietze, Kerstin; Madle, Hannelore; Tzankov, Alexander; Hummel, Michael; Dörken, Bernd; Scheidereit, Claus; Janz, Martin; Lenz, Peter; Thome, Margot; Lenz, Georg

    2013-09-26

    Constitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

  8. Pharmacologic inhibition of MALT1 protease by phenothiazines as a therapeutic approach for the treatment of aggressive ABC-DLBCL.

    PubMed

    Nagel, Daniel; Spranger, Stefani; Vincendeau, Michelle; Grau, Michael; Raffegerst, Silke; Kloo, Bernhard; Hlahla, Daniela; Neuenschwander, Martin; Peter von Kries, Jens; Hadian, Kamyar; Dörken, Bernd; Lenz, Peter; Lenz, Georg; Schendel, Dolores J; Krappmann, Daniel

    2012-12-11

    Proteolytic activity of the mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) paracaspase is required for survival of the activated B cell subtype of diffuse large B cell lymphoma (ABC-DLBCL). We have identified distinct derivatives of medicinal active phenothiazines, namely mepazine, thioridazine, and promazine, as small molecule inhibitors of the MALT1 protease. These phenothiazines selectively inhibit cleavage activity of recombinant and cellular MALT1 by a noncompetitive mechanism. Consequently, the compounds inhibit anti-apoptotic NF-κB signaling and elicit toxic effects selectively on MALT1-dependent ABC-DLBCL cells in vitro and in vivo. Our data provide a conceptual proof for a clinical application of distinct phenothiazines in the treatment of ABC-DLBCL.

  9. B-cell receptor-driven MALT1 activity regulates MYC signaling in mantle cell lymphoma.

    PubMed

    Dai, Beiying; Grau, Michael; Juilland, Mélanie; Klener, Pavel; Höring, Elisabeth; Molinsky, Jan; Schimmack, Gisela; Aukema, Sietse M; Hoster, Eva; Vogt, Niklas; Staiger, Annette M; Erdmann, Tabea; Xu, Wendan; Erdmann, Kristian; Dzyuba, Nicole; Madle, Hannelore; Berdel, Wolfgang E; Trneny, Marek; Dreyling, Martin; Jöhrens, Korinna; Lenz, Peter; Rosenwald, Andreas; Siebert, Reiner; Tzankov, Alexandar; Klapper, Wolfram; Anagnostopoulos, Ioannis; Krappmann, Daniel; Ott, German; Thome, Margot; Lenz, Georg

    2017-01-19

    Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor clinical outcome. Recent studies revealed the importance of B-cell receptor (BCR) signaling in maintaining MCL survival. However, it remains unclear which role MALT1, an essential component of the CARD11-BCL10-MALT1 complex that links BCR signaling to the NF-κB pathway, plays in the biology of MCL. Here we show that a subset of MCLs is addicted to MALT1, as its inhibition by either RNA or pharmacologic interference induced cytotoxicity both in vitro and in vivo. Gene expression profiling following MALT1 inhibition demonstrated that MALT1 controls an MYC-driven gene expression network predominantly through increasing MYC protein stability. Thus, our analyses identify a previously unappreciated regulatory mechanism of MYC expression. Investigating primary mouse splenocytes, we could demonstrate that MALT1-induced MYC regulation is not restricted to MCL, but represents a common mechanism. MYC itself is pivotal for MCL survival because its downregulation and pharmacologic inhibition induced cytotoxicity in all MCL models. Collectively, these results provide a strong mechanistic rationale to investigate the therapeutic efficacy of targeting the MALT1-MYC axis in MCL patients.

  10. Activation of CD4⁺ Foxp3⁺ regulatory T cells proceeds normally in the absence of B cells during EAE.

    PubMed

    Hoehlig, Kai; Shen, Ping; Lampropoulou, Vicky; Roch, Toralf; Malissen, Bernard; O'Connor, Richard; Ries, Stefanie; Hilgenberg, Ellen; Anderton, Stephen M; Fillatreau, Simon

    2012-05-01

    B cells and regulatory T (Treg) cells can both facilitate remission from experimental auto immune encephalomyelitis (EAE), a disease of the central nervous system (CNS) used as a model for multiple sclerosis (MS). Considering that B-cell-depletion therapy (BCDT) is used to treat MS patients, we asked whether Treg-cell activation depended on B cells during EAE. Treg-cell proliferation, accumulation in CNS, and augmentation of suppressive activity in the CNS were normal in B-cell-deficient mice, indicating that B cells are not essential for activation of the protective Treg-cell response and thus provide an independent layer of regulation. This function of B cells involved early suppression of the encephalitogenic CD4(+) T-cell response, which was enhanced in B-cell-deficient mice. CD4(+) T-cell depletion was sufficient to intercept the transition from acute-to-chronic EAE when applied to B-cell-deficient animals that just reached the peak of disease severity. Intriguingly, this treatment did not improve disease when applied later, implying that chronic disability was ultimately maintained independently of pathogenic CD4(+) T cells. Collectively, our data indicate that BCDT is unlikely to impair Treg-cell function, yet it might produce undesirable effects on T-cell-mediated autoimmune pathogenesis.

  11. DNA methylation signatures define molecular subtypes of diffuse large B-cell lymphoma

    PubMed Central

    Shaknovich, Rita; Geng, Huimin; Johnson, Nathalie A.; Tsikitas, Lucas; Cerchietti, Leandro; Greally, John M.

    2010-01-01

    Expression profiling has shown 2 main and clinically distinct subtypes of diffuse large B-cell lymphomas (DLBCLs): germinal-center B cell–like (GCB) and activated B cell–like (ABC) DLBCLs. Further work has shown that these subtypes are partially characterized by distinct genetic alterations and different survival. Here, we show with the use of an assay that measures DNA methylation levels of 50 000 CpG motifs distributed among more than 14 000 promoters that these 2 DLBCL subtypes are also characterized by distinct epigenetic profiles. DNA methylation and gene expression profiling were performed on a cohort of 69 patients with DLBCL. After assigning ABC or GCB labels with a Bayesian expression classifier trained on an independent dataset, a supervised analysis identified 311 differentially methylated probe sets (263 unique genes) between ABC and GCB DLBCLs. Integrated analysis of methylation and gene expression showed a core tumor necrosis factor-α signaling pathway as the principal differentially perturbed gene network. Sixteen genes overlapped between the core ABC/GCB methylation and expression signatures and encoded important proteins such as IKZF1. This reduced gene set was an accurate predictor of ABC and GCB subtypes. Collectively, the data suggest that epigenetic patterning contributes to the ABC and GCB DLBCL phenotypes and could serve as useful biomarker. PMID:20610814

  12. Similar disturbances in B cell activity and regulatory T cell function in Henoch-Schonlein purpura and systemic lupus erythematosus

    SciTech Connect

    Beale, M.G.; Nash, G.S.; Bertovich, M.J.; MacDermott, R.P.

    1982-01-01

    The immunoglobulin synthesizing activities of peripheral mononuclear cells (MNC) from five patients with Henoch-Schonlein purpura (HSP) and eight patients with active systemic lupus erythematosus (SLE) were compared. Cumulative amounts of IgM, IgG, and IgA synthesized and secreted by unstimulated and PWM-stimulated patient cells over a 12-day period were determied in a solid-phase radioimmunoassay. In unstimulated control cultures mean rates of IgM, IgG, and IgA synthesis were less than 250 ng/ml. The synthetic activities of patient MNC were markedly increased. In HSP cultures IgA was the major immunoglobulin class produced (2810 x/divide 1.33 ng/ml) followed by IgG (1754 x/divide 1.32 ng/ml) and IgM (404 x/divide 1.16 ng/ml). In SLE cultures IgA and IgG syntheses were equally elevated (4427 x/divide 1.20 and 4438 x/divide 1.49 ng/ml, respectively) whereas IgM synthesis averaged 967 x/divide 1.66 ng/ml. PWM stimulation of pateient MNC caused a sharp decline in the synthesis of all three immunoglobulin classes. After T cell depletion B cell-enriched fractions from HSP and SLE patients maintained high levels of IgA and IgG synthesis that were inhibited by PWM and by normal allogeneic but not autologous T cells. In PWM-stimulted co-cultures, patient T cells nonspecifically suppressed the synthetic activities of autologous and control B cells. in contrast patient B cells achieved normal levels of immunoglobulin synthesis when cultured with control T cells plus PWM. In longitudinal studies patient B and T cell disturbances persisted despite clinical improvement.

  13. TLR9 promotes tolerance by restricting survival of anergic anti-DNA B cells, yet is also required for their activation.

    PubMed

    Nickerson, Kevin M; Christensen, Sean R; Cullen, Jaime L; Meng, Wenzhao; Luning Prak, Eline T; Shlomchik, Mark J

    2013-02-15

    Nucleic acid-reactive B cells frequently arise in the bone marrow but are tolerized by mechanisms including receptor editing, functional anergy, and/or deletion. TLR9, a sensor of endosomal dsDNA, both promotes and regulates systemic autoimmunity in vivo, but the precise nature of its apparently contradictory roles in autoimmunity remained unclear. In this study, using the 3H9 anti-DNA BCR transgene in the autoimmune-prone MRL.Fas(lpr) mouse model of systemic lupus erythematosus, we identify the stages at which TLR9 contributes to establishing and breaking B cell tolerance. Although TLR9 is dispensable for L chain editing during B cell development in the bone marrow, TLR9 limits anti-DNA B cell life span in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer life spans and accumulate in the follicle, neither activated nor deleted. These cells retain some characteristics of anergic cells, in that they have elevated basal BCR signaling but impaired induced responses and downregulate their cell-surface BCR expression. In contrast, whereas TLR9-intact anergic B cells accumulate near the T/B border, TLR9-deficient anti-DNA B cells are somewhat more dispersed throughout the follicle. Nonetheless, in older autoimmune-prone animals, TLR9 expression specifically within the B cell compartment is required for spontaneous peripheral activation of anti-DNA B cells and their differentiation into Ab-forming cells via an extrafollicular pathway. Thus, TLR9 has paradoxical roles in regulating anti-DNA B cells: it helps purge the peripheral repertoire of autoreactive cells, yet is also required for their activation.

  14. Expression of receptor activator of nuclear factor-κB ligand by B cells in response to oral bacteria

    PubMed Central

    Han, X.; Lin, X.; Seliger, A. R.; Eastcott, J.; Kawai, T.; Taubman, M. A.

    2009-01-01

    Introduction We investigated receptor activator of nuclear factor-κB ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram-negative, anaerobic bacterium associated with aggressive periodontal disease. Methods Expression of messenger RNA transcripts (tumor necrosis factor-α, Toll-like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1-day) and late (10-day) responses in cultured rat splenocytes was examined by reverse transcription–polymerase chain reaction (RT-PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B-cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate-resistant acid phosphatase (TRAP) activity assay. Results The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL-expressing immunoglobulin G-positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans-immunized animals than from non-immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans-immunized animals. The addition of human osteoprotegerin-Fc to the culture significantly diminished such increases. Conclusion This study suggests that B-lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease. PMID

  15. Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo-activated cells.

    PubMed

    Maul, Robert W; Cao, Zheng; Venkataraman, Lakshmi; Giorgetti, Carol A; Press, Joan L; Denizot, Yves; Du, Hansen; Sen, Ranjan; Gearhart, Patricia J

    2014-10-20

    Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo-activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment.

  16. T-cell regulation of polyclonal B-cell activation induced by extracts of oral bacteria associated with periodontal diseases.

    PubMed Central

    Carpenter, A B; Sully, E C; Ranney, R R; Bick, P H

    1984-01-01

    These studies were designed to examine the role of regulatory T cells in the polyclonal antibody response of human peripheral blood lymphocytes to extracts of bacterial isolates commonly associated with periodontal disease. Polyclonal antibody responses to the organisms tested were found to be T cell dependent, as are most of the B-cell activators in the human system. Functional T helper activity was resistant to 1,500 rads of irradiation. Optimal polyclonal antibody responses to the bacterial extracts occurred at a 3:1 T-cell-to-B-cell ratio, whereas pokeweed mitogen-induced responses peaked at a 1:1 ratio, suggesting a difference in T-cell regulatory influences in response to these activators. Purified populations of T helper and suppressor cells exerted potent regulatory control of the responses to the bacterial extracts. These findings support the conclusion that regulatory T lymphocytes exert a potent modulating influence over the polyclonal response to periodontally associated bacteria and may play an important role in regulating the lymphocyte response in the diseased site. PMID:6197378

  17. Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

    SciTech Connect

    Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

    1987-05-01

    Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation.

  18. Avian Leukosis Virus Activation of an Antisense RNA Upstream of TERT in B-Cell Lymphomas

    PubMed Central

    Nehyba, Jiri; Malhotra, Sanandan; Winans, Shelby; O'Hare, Thomas H.; Justice, James

    2016-01-01

    ABSTRACT Avian leukosis virus (ALV) induces tumors by integrating its proviral DNA into the chicken genome and altering the expression of nearby genes via strong promoter and enhancer elements. Viral integration sites that contribute to oncogenesis are selected in tumor cells. Deep-sequencing analysis of B-cell lymphoma DNA confirmed that the telomerase reverse transcriptase (TERT) gene promoter is a common ALV integration target. Twenty-six unique proviral integration sites were mapped between 46 and 3,552 nucleotides (nt) upstream of the TERT transcription start site, predominantly in the opposite transcriptional orientation to TERT. Transcriptome-sequencing (RNA-seq) analysis of normal bursa revealed a transcribed region upstream of TERT in the opposite orientation, suggesting the TERT promoter is bidirectional. This transcript appears to be an uncharacterized antisense RNA. We have previously shown that TERT expression is upregulated in tumors with integrations in the TERT promoter region. We now report that the viral promoter drives the expression of a chimeric transcript containing viral sequences spliced to exons 4 through 7 of this antisense RNA. Clonal expansion of cells with ALV integrations driving overexpression of the TERT antisense RNA suggest it may have a role in tumorigenesis. IMPORTANCE The data suggest that ALV integrations in the TERT promoter region drive the overexpression of a novel antisense RNA and contribute to the development of lymphomas. PMID:27512065

  19. Distinct Patterns of B-Cell Activation and Priming by Natural Influenza Virus Infection Versus Inactivated Influenza Vaccination

    PubMed Central

    He, Xiao-Song; Holmes, Tyson H.; Sanyal, Mrinmoy; Albrecht, Randy A.; García-Sastre, Adolfo; Dekker, Cornelia L.; Davis, Mark M.; Greenberg, Harry B.

    2015-01-01

    Background. The human B-cell response to natural influenza virus infection has not been extensively investigated at the polyclonal level. Methods. The overall B-cell response of patients acutely infected with the 2009 pandemic influenza A(H1N1)pdm09 virus (A[H1N1]pdm09) was analyzed by determining the reactivity of plasmablast-derived polyclonal antibodies (PPAbs) to influenza proteins. Recipients of inactivated influenza vaccine containing the same A(H1N1)pdm09 strain were studied for comparison. Results. During acute infection, robust plasmablast responses to the infecting virus were detected, characterized by a greater PPAb reactivity to the conserved influenza virus nuclear protein and to heterovariant and heterosubtypic hemagglutinins, in comparison to responses to the inactivated A(H1N1)pdm09 vaccine. In A(H1N1)pdm09 vaccinees, the presence of baseline serum neutralizing antibodies against A(H1N1)pdm09, suggesting previous exposure to natural A(H1N1)pdm09 infection, did not affect the plasmablast response to vaccination, whereas repeated immunization with inactivated A(H1N1)pdm09 vaccine resulted in significantly reduced vaccine-specific and cross-reactive PPAb responses. Conclusions. Natural A(H1N1)pdm09 infection and inactivated A(H1N1)pdm09 vaccination result in very distinct patterns of B-cell activation and priming. These differences are likely to be associated with differences in protective immunity, especially cross-protection against heterovariant and heterosubtypic influenza virus strains. PMID:25336731

  20. The influence of the lpr gene on B cell activation: differential antibody expression in lpr congenic mouse strains.

    PubMed

    Warren, R W; Caster, S A; Roths, J B; Murphy, E D; Pisetsky, D S

    1984-04-01

    Spontaneous immunoglobulin production in four strains of lpr/lpr congenic mice was investigated to identify genetic interactions in lpr-induced polyclonal B cell activation. Sera were obtained from male and female lpr/lpr mice of the MRL, B6, C3H, and AKR strains as well as controls of +/+ genotypes. Antibody levels were compared at the time of peak response. Quantitative antibody determinations were performed by isotype-specific ELISA assays using responses to single-stranded DNA (sDNA), mouse IgG, rabbit IgG, and keyhole limpet hemocyanin as models. Among the strains studied there were significant differences in the antibody levels observed, with the strain producing highest levels dependent on the response measured. Thus, MRL-lpr/lpr produced the highest levels of IgG anti-sDNA while B6-lpr/lpr mice produced more anti-IgG than mice of other strains. Within each strain, the lpr gene appeared to affect the IgG more than the IgM response. A consistently high response by females was observed only in B6-lpr/lpr mice. These studies suggest that lpr-induced polyclonal B cell activation is influenced by the background genome with the extent of these genetic effects variable among responses.

  1. Defective disposal of immune complexes and polyclonal B cell activation persist long after exposure to bacterial lipopolysaccharide in mice

    SciTech Connect

    Granholm, N.A.; Cavallo, T. )

    1989-11-01

    Patients with systemic lupus erythematosus experience clinical exacerbation during superimposed bacterial infection. Previous studies in mice indicated that heightened immune phenomena during exposure to bacterial lipopolysaccharide (LPS) appear to be related, in part, to polyclonal B cell activation, to abnormal disposal of immune complexes (IC), and to increased localization of IC in tissues. To investigate whether such effects were reversible, we administered bacterial LPS to C57BL/6 mice for 5 weeks. Control mice received vehicle alone. We then discontinued LPS, and 6 weeks later LPS and control mice were challenged with a subsaturating dose of radiolabeled IC; the removal of IC from the circulation, their localization in the liver, spleen, and kidney were determined. In comparison to values in control mice, in mice previously exposed to LPS, serologic features of polyclonal B cell activation persisted; liver uptake of pathogenic IC (greater than Ag2Ab2) was normal, but removal of small size IC (less than or equal to Ag2Ab2) from the circulation was delayed; localization of IC in the kidneys was enhanced, and pathologic proteinuria developed. The effects of repeated exposure to bacterial LPS are partially reversible, but they last long after LPS is discontinued and may contribute to altered disposal of IC, enhanced organ localization of IC, and organ dysfunction.

  2. Effects of anti-IgM suppression on polyclonally activated murine B cells: analysis of immunoglobulin mRNA, gene specific nuclear factors and cell cycle distribution.

    PubMed Central

    Marcuzzi, A; Van Ness, B; Rouse, T; Lafrenz, D

    1989-01-01

    Polyclonal activation of murine B cells with bacterial lipopolysaccharide (LPS) and dextran sulfate (DxS) results in cell proliferation as well as increased immunoglobulin gene transcription and antibody secretion. When added to B cell cultures during mitogen activation, anti-mu antibody suppresses the rate of proliferation and immunoglobulin gene expression. Using this model system we show that co-cultures of B cells with LPS/DxS and anti-mu resulted in a decrease of both mu and kappa chain mRNA. Suppression did not prevent B cell entry into cycle nor a significant alteration in the distribution of cells in phases of cell cycle, although it did prolong the cycle transit time in a dose dependent fashion as determined by bromodeoxyuridine pulse labelling. Analysis of B cell specific nuclear binding factors, which previously have been shown to be important in regulating immunoglobulin gene transcription were examined. Results show that the kappa-specific enhancer binding activity of NF-kappa B was induced in activated as well as suppressed cultures. The lymphoid specific factor NF-A2, which recognizes the octamer sequence motif in the promoters of immunoglobulin genes, was induced by the polyclonal activation but was selectively lost in extracts from suppressed cells. Thus, specific regulation of the nuclear factor which plays a critical role in both heavy and light chain immunoglobulin gene expression may contribute to the transcriptional suppression observed in anti-mu treated B cells. Images PMID:2481271

  3. CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.

    PubMed

    von Bergwelt-Baildon, Michael; Shimabukuro-Vornhagen, Alexander; Popov, Alexey; Klein-Gonzalez, Nela; Fiore, Francesca; Debey, Svenja; Draube, Andreas; Maecker, Britta; Menezes, Isaura; Nadler, Lee M; Schultze, Joachim L

    2006-04-01

    CD40-activated B cells (CD40-B cells) have previously been introduced as an alternative source of antigen-presenting cells for immunotherapy. CD40-B cells can prime naive and expand memory T cells, and they can be generated in large numbers from very small amounts of peripheral blood derived from healthy individuals or cancer patients alike. Administration of CD40-B cells as a cellular adjuvant would require these cells to migrate toward secondary lymphoid organs and attract T cells in situ, processes guided by specific chemokines and chemokine receptors. Here, we demonstrate that primary, human CD40-B cells express a pattern of adhesion molecules and chemokine receptors necessary for homing to secondary lymphoid organs and have the capacity to migrate to cognate ligands. Furthermore, we show that CD40-B cells express important T-cell attractants and induce strong T-cell chemotaxis. These findings further support the use of CD40-B cells as cellular adjuvant for cancer immunotherapy.

  4. Clinical implications of the molecular subtypes of diffuse large B-cell lymphoma.

    PubMed

    Hill, Brian T; Sweetenham, John

    2012-05-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma and responds to standard treatment with chemoimmunotherapy in most patients. Standard prognostic scoring systems such as the International Prognostic Index (IPI) are useful for risk stratification, but are unreliable in predicting outcomes in individual patients because of the biologic heterogeneity of this disease. Gene expression profiling has revealed molecular subtypes of DLBCL: those derived from the lymph node germinal center (GCB) and others derived from an activated B-cell (ABC). A third entity, primary mediastinal B-cell lymphoma (unclassifiable DLBCL), displays pathobiologic features distinct from ABC and GCB subtypes. Patients with ABC-DLBCL have inferior progression-free survival and overall survival relative to those with the GCB subtype. In conclusion, molecular subtyping is a powerful tool for discriminating cases of DLBCL into groups that display very disparate biology and clinical outcomes. Although immunohistochemistry (IHC)-based algorithms predict both the molecular subtype as defined by gene expression profiling and clinical outcomes with reasonable concordance, not all experienced centers have been able to reproduce these findings. As techniques to subclassify DLBCL become universally adopted, large prospective trials will be needed to confirm the benefit of therapy tailored to molecular subtype.

  5. The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory, and improved activity

    PubMed Central

    Livnat-Levanon, Nurit; I. Gilson, Amy; Ben-Tal, Nir; Lewinson, Oded

    2016-01-01

    ABC transporters comprise a large and ubiquitous family of proteins. From bacteria to man they translocate solutes at the expense of ATP hydrolysis. Unlike other enzymes that use ATP as an energy source, ABC transporters are notorious for having high levels of basal ATPase activity: they hydrolyze ATP also in the absence of their substrate. It is unknown what are the effects of such prolonged and constant activity on the stability and function of ABC transporters or any other enzyme. Here we report that prolonged ATP hydrolysis is beneficial to the ABC transporter BtuC2D2. Using ATPase assays, surface plasmon resonance interaction experiments, and transport assays we observe that the constantly active transporter remains stable and functional for much longer than the idle one. Remarkably, during extended activity the transporter undergoes a slow conformational change (hysteresis) and gradually attains a hyperactive state in which it is more active than it was to begin with. This phenomenon is different from stabilization of enzymes by ligand binding: the hyperactive state is only reached through ATP hydrolysis, and not ATP binding. BtuC2D2 displays a strong conformational memory for this excited state, and takes hours to return to its basal state after catalysis terminates. PMID:26905293

  6. Polyclonal activation of rat B cells. I. A single mitogenic signal can stimulate proliferation, but three signals are required for differentiation. [Salmonella typhimurium

    SciTech Connect

    Stunz, L.L.; Feldbush, T.L.

    1986-06-01

    A water-soluble, proteinaceous preparation derived from the cell walls of Salmonella typhimurium Re mutants has recently been tested in this laboratory for its ability to act as a mitogen for rat lymphocytes. This preparation (STM) has been found to be a potent simulator of B lymphocyte proliferation, as measured both by /sup 3/H-TdR incorporation and by cell cycle analysis performed with flow cytofluorometry. STM stimulates approximately 50% of rat B cells to enter cycle. Previous investigations by others have shown that at least two sets of signals are required for B cell differentiation; (a) proliferation signals that may consist of both a stimulator of B cell conversion from G/sub 0/ to G/sub 1/ and growth factors, and (b) differentiation signals that probably include at least two B cell differentiation factors (BCDF). When STM was tested in a differentiation system it did not drive purified B cells to differentiate to PFC, either alone or when supplemented with a supernatant from concanavalin A-stimulated spleen cells (CAS). However, when both CAS and dextran sulfate (DXS) were supplied to the STM-stimulated cells, a large number of PFC resulted. DXT does not act by stimulating an additional, CAS-responsive B cell subset, since it has only a marginal effect upon /sup 3/H-TdR uptake and does not increase the number of B cells in cycle when used together with STM. The authors that the two agents may be acting sequentially: STM stimulates the B cells to proliferate, and DXS drives the proliferating cells to become responsive to CAS. This suggests that the signals for B cell differentiation must consist of at least three activities: a trigger to stimulate the cells to proliferate, a factor to drive the cells to a BCDF-responsive state, and a BCDF that can drive the cells to secrete antibody.

  7. CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4, CD8 or B cells alone are ineffective.

    PubMed

    Tishon, Antoinette; Lewicki, Hanna; Andaya, Abegail; McGavern, Dorian; Martin, Lee; Oldstone, Michael B A

    2006-03-30

    Measles virus (MV), one of the most infectious of human pathogens, still infects over 30 million humans and causes over 500,000 deaths each year [Griffin, D., 2001. Measles virus. In: Fields, B., Knipe, D., Howley, P. (Eds.), Fields Virology. Lippincott-Raven, Philadelphia, pp. 1401-1442; ]. Death is primarily due to secondary microbial infections associated with the immunosuppression caused by MV. Studies of humans with genetic or acquired deficiencies of either the humoral or cellular arm of the immune system, and rodent models have implicated T cells in the control of the ongoing MV infection but the precise role and activities of the specific T cell subset or the molecules they produce is not clear. Using a transgenic mouse model in conjunction with depletion and reconstitution of individual B and T cell subsets alone or in combination, we show that neither CD4, CD8 nor B cells per se control acute MV infection. However, combinations of either CD4 T cells and B cells, or of CD4 and CD8 T cells are essential but CD8 T with B cells are ineffective. Interferon-gamma and neutralizing antibodies, but neither perforin nor TNF-alpha alone are associated with clearance of MV infection. TNF-alpha combined with interferon-gamma is more effective in protection than interferon alone. Further, the lack of an interferon-gamma response leads to persistence of MV.

  8. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

    PubMed

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-07-09

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

  9. Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells.

    PubMed

    Pupovac, Aleta; Geraghty, Nicholas J; Watson, Debbie; Sluyter, Ronald

    2015-01-01

    Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-1(2+) uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.

  10. Anti-PEG IgM and complement system are required for the association of second doses of PEGylated liposomes with splenic marginal zone B cells.

    PubMed

    Shimizu, Taro; Mima, Yu; Hashimoto, Yosuke; Ukawa, Masami; Ando, Hidenori; Kiwada, Hiroshi; Ishida, Tatsuhiro

    2015-10-01

    The accelerated blood clearance (ABC) phenomenon makes it crucial to use PEGylated liposomes and micelles to deliver drugs. The ABC phenomenon is an immune response against an initial dose of PEGylated liposome, which causes subsequent doses to be rapidly cleared by macrophages in the liver. We recently found that in the early phase of the ABC phenomenon, subsequent doses of PEGylated liposomes were associated with splenic marginal zone (MZ)-B cells and were transported from the MZ to the follicle (FO). In this study, we investigated the underlying mechanisms behind the association of subsequent doses of PEGylated liposomes with MZ-B cells in the spleen. Serum factors, anti-PEG IgM and complement system, were crucial to the association of PEGylated liposomes with MZ-B cells, while the sensitization of MZ-B cells by the first dose of PEGylated liposomes was not significant. It was the complement receptors (CRs) on the MZ-B cells, rather than either the PEG-specific B-cell receptors or the IgM Fc receptors, that were the main contributors to the association between PEGylated liposomes and MZ-B cells. It appeared that anti-PEG IgM would bind to PEGylated liposomes and causes subsequent complement activation, resulting in the formation of immune complexes of PEGylated liposome-anti-PEG IgM-complement. The MZ-B cells then recognized these immune complexes via their CRs. Such an association via CRs might have triggered the transport of the immune complex by MZ-B cells to the FO in the spleen. The information obtained in this study might be useful in the development of an efficient antigen delivery system to usher PEGylated nanoparticles into FO dendritic cells.

  11. EBV Latent Membrane Protein 1 Activates Akt, NFκB, and Stat3 in B Cell Lymphomas

    PubMed Central

    Shair, Kathy H. Y; Bendt, Katherine M; Edwards, Rachel H; Bedford, Elisabeth C; Nielsen, Judith N; Raab-Traub, Nancy

    2007-01-01

    Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFκB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFκB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas. PMID:17997602

  12. Simultaneous activation of viral antigen-specific memory CD4+ and CD8+ T-cells using mRNA-electroporated CD40-activated autologous B-cells.

    PubMed

    Van den Bosch, Glenn A; Van Gulck, Ellen; Ponsaerts, Peter; Nijs, Griet; Lenjou, Marc; Apers, Ludwig; Kint, Ilse; Heyndrickx, Leo; Vanham, Guido; Van Bockstaele, Dirk R; Berneman, Zwi N; Van Tendeloo, Viggo F I

    2006-01-01

    Recently, it has become obvious that not only CD8 T-cells, but also CD4 T-helper cells are required for the induction of an effective, long-lasting cellular immune response. In view of the clinical importance of cytomegalovirus (CMV) and human immunodeficiency virus (HIV) infection, we developed 2 strategies to simultaneously reactivate viral antigen-specific memory CD4 and CD8 T-cells of CMV-seropositive and HIV-seropositive subjects using mRNA-electroporated autologous CD40-activated B cells. In the setting of HIV, we provide evidence that CD40-activated B cells can be cultured from HAART-naive HIV-1 seropositive patients. These cells not only express and secrete the HIV p24 antigen after electroporation with codon-optimized HIV-1 gag mRNA, but can also be used to in vitro reactivate Gag antigen-specific interferon-gamma-producing CD4 and CD8 autologous T-cells. For the CMV-specific approach, we applied mRNA coding for the pp65 protein coupled to the lysosomal-associated membrane protein-1 to transfect CD40-activated B cells to induce CMV antigen-specific CD4 and CD8 T-cells. More detailed analysis of the activated interferon-gamma-producing CMV pp65 tetramer positive CD8 T-cells revealed an effector memory phenotype with the capacity to produce interleukin-2. Our findings clearly show that the concomitant activation of both CD4 and CD8 (memory) T-cells using mRNA-electroporated CD40-B cells is feasible in CMV and HIV-1-seropositive persons, which indicates the potential value of this approach for application in cellular immunotherapy of infectious diseases.

  13. Evolution of broadly cross-reactive HIV-1-neutralizing activity: therapy-associated decline, positive association with detectable viremia, and partial restoration of B-cell subpopulations.

    PubMed

    Ferreira, Carolina B; Merino-Mansilla, Alberto; Llano, Anuska; Pérez, Ignacio; Crespo, Isabel; Llinas, Laia; Garcia, Felipe; Gatell, Jose M; Yuste, Eloisa; Sanchez-Merino, Victor

    2013-11-01

    Little is known about the stability of HIV-1 cross-neutralizing responses. Taking into account the fact that neutralization breadth has been positively associated with plasma viral load, there is no explanation for the presence of broadly neutralizing responses in a group of patients on treatment with undetectable viremia. In addition, the B-cell profile responsible for broadly cross-neutralizing responses is unknown. Here we studied the evolution of neutralizing responses and the B-cell subpopulation distribution in a group of patients with broadly cross-reactive HIV-1-neutralizing activity. We studied neutralization breadth evolution in a group of six previously identified broadly cross-neutralizing patients and six control patients during a 6-year period with a previously described minipanel of recombinant viruses from five different subtypes. B-cell subpopulation distribution during the study was also determined by multiparametric flow cytometry. Broadly cross-neutralizing activity was transient in four broad cross-neutralizers and stable, up to 4.6 years, in the other two. In four out of five broad cross-neutralizers who initiated treatment, a neutralization breadth loss occurred after viremia had been suppressed for as much as 20 months. B-cell subpopulation analyses revealed a significant increase in the frequency of naive B cells in broadly cross-reactive samples, compared with samples with less neutralization breadth (increased from 44% to 62%). We also observed a significant decrease in tissue-like and activated memory B cells (decreased from 19% to 12% and from 17% to 9%, respectively). Our data suggest that HIV-1 broadly cross-neutralizing activity is variable over time and associated with detectable viremia and partial B-cell restoration.

  14. Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics

    PubMed Central

    Robles, Eloy F.; Mena-Varas, Maria; Barrio, Laura; Merino-Cortes, Sara V.; Balogh, Péter; Du, Ming-Qing; Akasaka, Takashi; Parker, Anton; Roa, Sergio; Panizo, Carlos; Martin-Guerrero, Idoia; Siebert, Reiner; Segura, Victor; Agirre, Xabier; Macri-Pellizeri, Laura; Aldaz, Beatriz; Vilas-Zornoza, Amaia; Zhang, Shaowei; Moody, Sarah; Calasanz, Maria Jose; Tousseyn, Thomas; Broccardo, Cyril; Brousset, Pierre; Campos-Sanchez, Elena; Cobaleda, Cesar; Sanchez-Garcia, Isidro; Fernandez-Luna, Jose Luis; Garcia-Muñoz, Ricardo; Pena, Esther; Bellosillo, Beatriz; Salar, Antonio; Baptista, Maria Joao; Hernandez-Rivas, Jesús Maria; Gonzalez, Marcos; Terol, Maria Jose; Climent, Joan; Ferrandez, Antonio; Sagaert, Xavier; Melnick, Ari M.; Prosper, Felipe; Oscier, David G.; Carrasco, Yolanda R.; Dyer, Martin J. S.; Martinez-Climent, Jose A.

    2016-01-01

    NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas. PMID:27297662

  15. Stimulation of the B-cell receptor activates the JAK2/STAT3 signaling pathway in chronic lymphocytic leukemia cells

    PubMed Central

    Rozovski, Uri; Wu, Ji Yuan; Harris, David M.; Liu, Zhiming; Li, Ping; Hazan-Halevy, Inbal; Ferrajoli, Alessandra; Burger, Jan A.; O’Brien, Susan; Jain, Nitin; Verstovsek, Srdan; Wierda, William G.; Keating, Michael J.

    2014-01-01

    In chronic lymphocytic leukemia (CLL), stimulation of the B-cell receptor (BCR) triggers survival signals. Because in various cells activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway provides cells with survival advantage, we wondered whether BCR stimulation activates the JAK/STAT pathway in CLL cells. To stimulate the BCR we incubated CLL cells with anti-IgM antibodies. Anti-IgM antibodies induced transient tyrosine phosphorylation and nuclear localization of phosphorylated (p) STAT3. Immunoprecipitation studies revealed that anti-JAK2 antibodies coimmunoprecipitated pSTAT3 and pJAK2 in IgM-stimulated but not unstimulated CLL cells, suggesting that activation of the BCR induces activation of JAK2, which phosphorylates STAT3. Incubation of CLL cells with the JAK1/2 inhibitor ruxolitinib inhibited IgM-induced STAT3 phosphorylation and induced apoptosis of IgM-stimulated but not unstimulated CLL cells in a dose- and time-dependent manner. Whether ruxolitinib treatment would benefit patients with CLL remains to be determined. PMID:24778152

  16. Stimulation of the B-cell receptor activates the JAK2/STAT3 signaling pathway in chronic lymphocytic leukemia cells.

    PubMed

    Rozovski, Uri; Wu, Ji Yuan; Harris, David M; Liu, Zhiming; Li, Ping; Hazan-Halevy, Inbal; Ferrajoli, Alessandra; Burger, Jan A; O'Brien, Susan; Jain, Nitin; Verstovsek, Srdan; Wierda, William G; Keating, Michael J; Estrov, Zeev

    2014-06-12

    In chronic lymphocytic leukemia (CLL), stimulation of the B-cell receptor (BCR) triggers survival signals. Because in various cells activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway provides cells with survival advantage, we wondered whether BCR stimulation activates the JAK/STAT pathway in CLL cells. To stimulate the BCR we incubated CLL cells with anti-IgM antibodies. Anti-IgM antibodies induced transient tyrosine phosphorylation and nuclear localization of phosphorylated (p) STAT3. Immunoprecipitation studies revealed that anti-JAK2 antibodies coimmunoprecipitated pSTAT3 and pJAK2 in IgM-stimulated but not unstimulated CLL cells, suggesting that activation of the BCR induces activation of JAK2, which phosphorylates STAT3. Incubation of CLL cells with the JAK1/2 inhibitor ruxolitinib inhibited IgM-induced STAT3 phosphorylation and induced apoptosis of IgM-stimulated but not unstimulated CLL cells in a dose- and time-dependent manner. Whether ruxolitinib treatment would benefit patients with CLL remains to be determined.

  17. Optimal Medical Equipment Maintenance Service Proposal Decision Support System combining Activity Based Costing (ABC) and the Analytic Hierarchy Process (AHP).

    PubMed

    da Rocha, Leticia; Sloane, Elliot; M Bassani, Jose

    2005-01-01

    This study describes a framework to support the choice of the maintenance service (in-house or third party contract) for each category of medical equipment based on: a) the real medical equipment maintenance management system currently used by the biomedical engineering group of the public health system of the Universidade Estadual de Campinas located in Brazil to control the medical equipment maintenance service, b) the Activity Based Costing (ABC) method, and c) the Analytic Hierarchy Process (AHP) method. Results show the cost and performance related to each type of maintenance service. Decision-makers can use these results to evaluate possible strategies for the categories of equipment.

  18. Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-κB p65 subunit nuclear import

    SciTech Connect

    Niu, Mingshan; Shen, Yangling; Xu, Xiaoyu; Yao, Yao; Fu, Chunling; Yan, Zhiling; Wu, Qingyun; Cao, Jiang; Sang, Wei; Zeng, Lingyu; Li, Zhenyu; Liu, Xuejiao; and others

    2015-07-10

    Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys{sup 38} to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has been reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumine's mechanism of action and novel approach to ABC-DLBCL target therapy. - Highlights: • Current NF-κB targeting strategies lack cancer cell specificity. • Piperlongumine inhibits NF-κB p65 subunit nuclear import via directly binding to p65. • Piperlongumine selectively inhibits proliferation of ABC-DLBCL cells. • This study provides a novel approach to ABC-DLBCL target therapy.

  19. Processing and MHC class II presentation of exogenous soluble antigen involving a proteasome-dependent cytosolic pathway in CD40-activated B cells.

    PubMed

    Becker, Hans Jiro; Kondo, Eisei; Shimabukuro-Vornhagen, Alexander; Theurich, Sebastian; von Bergwelt-Baildon, Michael S

    2016-08-01

    Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4 + T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4 + T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway.

  20. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells

    PubMed Central

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-01-01

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes. PMID:26431330

  1. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells.

    PubMed

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-11-10

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.

  2. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  3. B-cell tolerance and autoimmunity

    PubMed Central

    Tsubata, Takeshi

    2017-01-01

    Self-reactive B cells are tolerized at various stages of B-cell development and differentiation, including the immature B-cell stage (central tolerance) and the germinal center (GC) B-cell stage, and B-cell tolerance involves various mechanisms such as deletion, anergy, and receptor editing. Self-reactive B cells generated by random immunoglobulin variable gene rearrangements are tolerized by central tolerance and anergy in the periphery, and these processes involve apoptosis regulated by Bim, a pro-apoptotic member of the Bcl-2 family, and regulation of B-cell signaling by various phosphatases, including SHIP-1 and SHP-1. Self-reactive B cells generated by somatic mutations during GC reaction are also eliminated. Fas is not directly involved in this process but prevents persistence of GC reaction that allows generation of less stringently regulated B cells, including self-reactive B cells. Defects in self-tolerance preferentially cause lupus-like disease with production of anti-nuclear antibodies, probably due to the presence of a large potential B-cell repertoire reactive to nucleic acids and the presence of nucleic acid-induced activation mechanisms in various immune cells, including B cells and dendritic cells. A feed-forward loop composed of anti-nuclear antibodies produced by B cells and type 1 interferons secreted from nucleic acid-activated dendritic cells plays a crucial role in the development of systemic lupus erythematosus.

  4. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    PubMed Central

    Manetta, Joseph; Bina, Holly; Ryan, Paul; Fox, Niles; Witcher, Derrick R; Kikly, Kristine

    2014-01-01

    B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. PMID:25258549

  5. SLP65 deficiency results in perpetual V(D)J recombinase activity in pre-B-lymphoblastic leukemia and B-cell lymphoma cells.

    PubMed

    Sprangers, M; Feldhahn, N; Liedtke, S; Jumaa, H; Siebert, R; Müschen, M

    2006-08-24

    Perpetual V(D)J recombinase activity involving multiple DNA double-strand break events in B-cell lineage leukemia and lymphoma cells may introduce secondary genetic aberrations leading towards malignant progression. Here, we investigated defective negative feedback signaling through the (pre-) B-cell receptor as a possible reason for deregulated V(D)J recombinase activity in B-cell malignancy. On studying 28 cases of pre-B-lymphoblastic leukemia and 27 B-cell lymphomas, expression of the (pre-) B-cell receptor-related linker molecule SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) was found to be defective in seven and five cases, respectively. SLP65 deficiency correlates with RAG1/2 expression and unremitting V(H) gene rearrangement activity. Reconstitution of SLP65 expression in SLP65-deficient leukemia and lymphoma cells results in downregulation of RAG1/2 expression and prevents both de novo V(H)-DJ(H) rearrangements and secondary V(H) replacement. We conclude that iterative V(H) gene rearrangement represents a frequent feature in B-lymphoid malignancy, which can be attributed to SLP65 deficiency in many cases.

  6. The frequency of naïve and early-activated hapten-specific B cell subsets dictates the efficacy of a therapeutic vaccine against prescription opioid abuse

    PubMed Central

    Laudenbach, Megan; Baruffaldi, Federico; Vervacke, Jeffrey S; Distefano, Mark D; Titcombe, Philip J; Mueller, Daniel L; Tubo, Noah J; Griffith, Thomas S; Pravetoni, Marco

    2015-01-01

    Translation of therapeutic vaccines for addiction, cancer or other chronic non-communicable diseases has been slow because only a small subset of immunized subjects achieved effective antibody levels. We hypothesize that individual variability in the number of naïve and early-activated hapten-specific B cells determines post-vaccination serum antibody levels and vaccine efficacy. Using a model vaccine against the highly abused prescription opioid oxycodone, the polyclonal B cell population specific for an oxycodone-based hapten (6OXY) was analyzed by flow cytometry paired with antigen-based magnetic enrichment. A higher frequency of 6OXY-specific B cells in either spleen biopsies or blood, before and after immunization, correlated to subsequent greater oxycodone-specific serum antibody titers and their efficacy in blocking oxycodone distribution to the brain and oxycodone-induced behavior in mice. The magnitude of 6OXY-specific B cell activation and vaccine efficacy was tightly correlated to the size of the CD4+ T cell population. The frequency of enriched 6OXY-specific B cells was consistent across various mouse tissues. These data provide novel evidence that variations in the frequency of naïve or early-activated vaccine-specific B and T cells can account for individual responses to vaccines and may predict the clinical efficacy of a therapeutic vaccine. PMID:25972483

  7. Sodium periodate-induced human suppressor cells for polyclonal B cell activation.

    PubMed

    Goust, J M

    1982-09-01

    Sodium periodate (SP) induces proliferation of mature T cells. In this study, human peripheral blood mononuclear cells (MNC) pretreated for 10 minutes at room temperature with increasing concentrations (0.1 to 5 mM) of SP before culture for 7 days in the presence of pokeweed mitogen (PWM) exhibited a dose-dependent inhibition of IgG production, contrasting with an increase in 3H-thymidine uptake. When MNC from 70 normal individuals were pretreated with 1 and 2 mM SP, IgG production in culture was suppressed by 46.8 +/- 4.5% and 60.4 +/- 4.4% (mean +/- S.E.M.), respectively, as compared to IgG synthesis in the presence of PWM alone. Longitudinal studies of MNC obtained from the same normal individuals over 6-10 months showed similar degrees of suppression, indicating that the level of SP-inducible suppressor cell activity remains relatively constant, although the degree of suppression varies among normal persons. Both T cells and monocytes were required for PWM-driven IgG production and for SP-induced suppression. A soluble factor elaborated by SP-treated monocytes was also able to suppress IgG production. This model should provide useful information about abnormal regulation of IgG synthesis in various pathological conditions.

  8. Targeting deubiquitinase activity with a novel small-molecule inhibitor as therapy for B-cell malignancies.

    PubMed

    Peterson, Luke F; Sun, Hanshi; Liu, Yihong; Potu, Harish; Kandarpa, Malathi; Ermann, Monika; Courtney, Stephen M; Young, Matthew; Showalter, Hollis D; Sun, Duxin; Jakubowiak, Andrzej; Malek, Sami N; Talpaz, Moshe; Donato, Nicholas J

    2015-06-04

    Usp9x was recently shown to be highly expressed in myeloma patients with short progression-free survival and is proposed to enhance stability of the survival protein Mcl-1. In this study, we found that the partially selective Usp9x deubiquitinase inhibitor WP1130 induced apoptosis and reduced Mcl-1 protein levels. However, short hairpin RNA-mediated knockdown (KD) of Usp9x in myeloma cells resulted in transient induction of apoptosis, followed by a sustained reduction in cell growth. A compensatory upregulation of Usp24, a deubiquitinase closely related to Usp9x, in Usp9x KD cells was noted. Direct Usp24 KD resulted in marked induction of myeloma cell death that was associated with a reduction of Mcl-1. Usp24 was found to sustain myeloma cell survival and Mcl-1 regulation in the absence of Usp9x. Both Usp9x and Usp24 were expressed and activated in primary myeloma cells whereas Usp24 protein overexpression was noted in some patients with drug-refractory myeloma and other B-cell malignancies. Furthermore, we improved the drug-like properties of WP1130 and demonstrated that the novel compound EOAI3402143 dose-dependently inhibited Usp9x and Usp24 activity, increased tumor cell apoptosis, and fully blocked or regressed myeloma tumors in mice. We conclude that small-molecule Usp9x/Usp24 inhibitors may have therapeutic activity in myeloma.

  9. Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation

    PubMed Central

    Mukhopadhyay, Chandrani; Triplett, Aleata; Bargar, Tom; Heckman, Carol; Wagner, Kay-Uwe

    2016-01-01

    Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b–null, Cbl-c–null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src. PMID:27930322

  10. Viral Double-Stranded RNA Triggers Ig Class Switching by Activating Upper Respiratory Mucosa B Cells through an Innate TLR3 Pathway Involving BAFF1

    PubMed Central

    Xu, Weifeng; Santini, Paul A.; Matthews, Allysia J.; Chiu, April; Plebani, Alessandro; He, Bing; Chen, Kang; Cerutti, Andrea

    2011-01-01

    Class switch DNA recombination (CSR) from IgM to IgG and IgA is crucial for antiviral immunity. Follicular B cells undergo CSR upon engagement of CD40 by CD40 ligand on CD4+ T cells. This T cell-dependent pathway requires 5–7 days, which is too much of a delay to block quickly replicating pathogens. To compensate for this limitation, extrafollicular B cells rapidly undergo CSR through a T cell-independent pathway that involves innate Ag receptors of the TLR family. We found that a subset of upper respiratory mucosa B cells expressed TLR3 and responded to viral dsRNA, a cognate TLR3 ligand. In the presence of dsRNA, mucosal B cells activated NF-κB, a transcription factor critical for CSR. Activation of NF-κB required TRIF (Toll/IL-1R domain-containing protein inducing IFN-β), a canonical TLR3 adapter protein, and caused germline transcription of downstream CH genes as well as expression of AID (activation-induced cytidine deaminase), a DNA-editing enzyme essential for CSR. Subsequent IgG and IgA production was enhanced by BAFF (B cell-activating factor of the TNF family), an innate mediator released by TLR3-expressing mucosal dendritic cells. Indeed, these innate immune cells triggered IgG and IgA responses upon exposure to dsRNA. By showing active TLR3 signaling and ongoing CSR in upper respiratory mucosa B cells from patients with CD40 signaling defects, our findings indicate that viral dsRNA may initiate frontline IgG and IgA responses through an innate TLR3-dependent pathway involving BAFF. PMID:18566393

  11. Deficiency of MALT1 paracaspase activity results in unbalanced regulatory and effector T and B cell responses leading to multiorgan inflammation.

    PubMed

    Bornancin, Frédéric; Renner, Florian; Touil, Ratiba; Sic, Heiko; Kolb, Yeter; Touil-Allaoui, Ismahane; Rush, James S; Smith, Paul A; Bigaud, Marc; Junker-Walker, Ursula; Burkhart, Christoph; Dawson, Janet; Niwa, Satoru; Katopodis, Andreas; Nuesslein-Hildesheim, Barbara; Weckbecker, Gisbert; Zenke, Gerhard; Kinzel, Bernd; Traggiai, Elisabetta; Brenner, Dirk; Brüstle, Anne; St Paul, Michael; Zamurovic, Natasa; McCoy, Kathy D; Rolink, Antonius; Régnier, Catherine H; Mak, Tak W; Ohashi, Pamela S; Patel, Dhavalkumar D; Calzascia, Thomas

    2015-04-15

    The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

  12. Expression of the p66Shc protein adaptor is regulated by the activator of transcription STAT4 in normal and chronic lymphocytic leukemia B cells

    PubMed Central

    Cattaneo, Francesca; Patrussi, Laura; Capitani, Nagaja; Frezzato, Federica; D'Elios, Mario Milco; Trentin, Livio; Semenzato, Gianpietro; Baldari, Cosima T.

    2016-01-01

    p66Shc attenuates mitogenic, prosurvival and chemotactic signaling and promotes apoptosis in lymphocytes. Consistently, p66Shc deficiency contributes to the survival and trafficking abnormalities of chronic lymphocytic leukemia (CLL) B cells. The mechanism of p66shc silencing in CLL B cells is methylation-independent, at variance with other cancer cell types. Here we identify STAT4 as a novel transcriptional regulator of p66Shc in B cells. Chromatin immunoprecipitation and reporter gene assays showed that STAT4 binds to and activates the p66shc promoter. Silencing or overexpression of STAT4 resulted in a co-modulation of p66Shc. IL-12-dependent STAT4 activation caused a coordinate increase in STAT4 and p66Shc expression, which correlated with enhanced B cell apoptosis. Treatment with the STAT4 inhibitor lisofylline reverted partly this effect, suggesting that STAT4 phosphorylation is not essential for but enhances p66shc transcription. Additionally, we demonstrate that CLL B lymphocytes have a STAT4 expression defect which partly accounts for their p66Shc deficiency, as supported by reconstitution experiments. Finally, we show that p66Shc participates in a positive feedback loop to promote STAT4 expression. These results provide new insights into the mechanism of p66Shc expression in B cells and its defect in CLL, identifying the STAT4/IL-12 pathway as a potential therapeutic target in this neoplasia. PMID:27494881

  13. Transient expression of Bcl6 is sufficient for oncogenic function and induction of mature B-cell lymphoma.

    PubMed

    Green, Michael R; Vicente-Dueñas, Carolina; Romero-Camarero, Isabel; Long Liu, Chih; Dai, Bo; González-Herrero, Inés; García-Ramírez, Idoia; Alonso-Escudero, Esther; Iqbal, Javeed; Chan, Wing C; Campos-Sanchez, Elena; Orfao, Alberto; Pintado, Belén; Flores, Teresa; Blanco, Oscar; Jiménez, Rafael; Martínez-Climent, Jose Angel; Criado, Francisco Javier García; Cenador, María Begoña García; Zhao, Shuchun; Natkunam, Yasodha; Lossos, Izidore S; Majeti, Ravindra; Melnick, Ari; Cobaleda, César; Alizadeh, Ash A; Sánchez-García, Isidro

    2014-06-02

    Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and can be separated into two subtypes based upon molecular features with similarities to germinal centre B-cells (GCB-like) or activated B-cells (ABC-like). Here we identify gain of 3q27.2 as being significantly associated with adverse outcome in DLBCL and linked with the ABC-like subtype. This lesion includes the BCL6 oncogene, but does not alter BCL6 transcript levels or target-gene repression. Separately, we identify expression of BCL6 in a subset of human haematopoietic stem/progenitor cells (HSPCs). We therefore hypothesize that BCL6 may act by 'hit-and-run' oncogenesis. We model this hit-and-run mechanism by transiently expressing Bcl6 within murine HSPCs, and find that it causes mature B-cell lymphomas that lack Bcl6 expression and target-gene repression, are transcriptionally similar to post-GCB cells, and show epigenetic changes that are conserved from HSPCs to mature B-cells. Together, these results suggest that BCL6 may function in a 'hit-and-run' role in lymphomagenesis.

  14. Transient expression of Bcl6 is sufficient for oncogenic function and induction of mature B-cell lymphoma

    PubMed Central

    Green, Michael R; Vicente-Dueñas, Carolina; Romero-Camarero, Isabel; Liu, Chih Long; Dai, Bo; González-Herrero, Inés; García-Ramírez, Idoia; Alonso-Escudero, Esther; Iqbal, Javeed; Chan, Wing C; Campos-Sanchez, Elena; Orfao, Alberto; Pintado, Belén; Flores, Teresa; Blanco, Oscar; Jiménez, Rafael; Martínez-Climent, Jose Angel; Criado, Francisco Javier García; Cenador, María Begoña García; Zhao, Shuchun; Natkunam, Yasodha; Lossos, Izidore S; Majeti, Ravindra; Melnick, Ari; Cobaleda, César; Alizadeh, Ash A.; Sánchez-García, Isidro

    2015-01-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and can be separated into two subtypes based upon molecular features with similarities to germinal center B-cells (GCB-like) or activated B-cells (ABC-like). Here we identify gain of 3q27.2 as being significantly associated with adverse outcome in DLBCL and linked with the ABC-like subtype. This lesion includes the BCL6 oncogene, but does not alter BCL6 transcript levels or target-gene repression. Separately, we identify expression of BCL6 in a subset of human hematopoietic stem/progenitor cells (HSPCs). We therefore hypothesize that BCL6 may act by hit-and-run oncogenesis. We model this by transiently expressing Bcl6 within murine HSPCs, and find it causes mature B-cell lymphomas that lack Bcl6 expression and target-gene repression, are transcriptionally similar to post-GCB cells, and show epigenetic changes that are conserved from HSPCs to mature B-cells. Together these results suggest that Bcl6 may function in a hit-and-run role in lymphomagenesis. PMID:24887457

  15. B lymphocyte binding to E- and P-selectins is mediated through the de novo expression of carbohydrates on in vitro and in vivo activated human B cells.

    PubMed Central

    Postigo, A A; Marazuela, M; Sánchez-Madrid, F; de Landázuri, M O

    1994-01-01

    Cell adhesion to endothelium regulates the trafficking and recruitment of leukocytes towards lymphoid organs and sites of inflammation. This phenomenon is mediated by the expression of a number of adhesion molecules on both the endothelium and circulating cells. Activation of endothelial cells (EC) with different stimuli induces the expression of several adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1), involved in their interaction with circulating cells. In this report, we have studied the binding of nonactivated and activated B cells to purified E- and P-selectins. Activated but not resting B cells were able to interact with both selectins. This binding capacity of activated B cells paralleled the induction of different carbohydrate epitopes (Lewisx, sialyl-Lewisx, CD57 and CDw65) as well as other molecules bearing these or related epitopes in myeloid cells (L-selectin, alpha L beta 2 and alpha X beta 2 integrins, and CD35) involved in the interaction of different cell types with selectins. B cells infiltrating inflamed tissues like in Hashimoto's thyroiditis, also expressed these selectin-binding carbohydrates in parallel with the expression of E-selectin by surrounding follicular dendritic cells. Moreover, the crosslinking of these selectin-binding epitopes resulted in an increased binding of B cells to different integrin ligands. Thus, in addition to the involvement of integrins, E- and P-selectins could play an important role in the interaction of B lymphocytes with the endothelium during B cell extravasation into lymphoid tissues and inflammatory foci as well as in their organization into lymphoid organs. Images PMID:7523454

  16. The ABCs of entrustable professional activities: an overview of 'entrustable professional activities' in medical education.

    PubMed

    El-Haddad, C; Damodaran, A; McNeil, H P; Hu, W

    2016-09-01

    Consultants regularly need to decide whether a trainee can be entrusted to perform a clinical activity independently. 'Entrustable professional activities' (EPA) provide a framework for justifying and better utilising supervisor entrustment decisions for trainee feedback and assessment in the workplace. Since being proposed by Olle ten Cate in 2005, EPA are emerging as an integral part of many international medical curricula, and are being considered by the Royal Australasian College of Physicians in the current review of physician training. EPA are defined as tasks or responsibilities that can be entrusted to a trainee once sufficient competence is reached to allow for unsupervised practice. An example might be to entrust a trainee to 'Initiate and co-ordinate care of the palliative patient' with only off-site or indirect supervision. Rather than attempting to measure directly each of the many separate competencies required to undertake such a complex task, EPA direct the trainee and supervisor's attention to the trainee's performance in a limited number of selected, representative, important day-to-day activities. EPA-based assessment is gaining momentum, amongst significant concerns regarding feasibility of implementation. While the optimal process for designing and implementing EPA remains to be determined, it is an assessment strategy where the over-arching goal of optimal patient care remains in clear sight. This review explores the central role of trust in medical training, the case for EPA and potential barriers to implementing EPA-based assessment.

  17. Nickel compounds induce apoptosis in human bronchial epithelial Beas-2B cells by activation of c-Myc through ERK pathway

    SciTech Connect

    Li Qin; Suen, T.-C.; Sun Hong; Arita, Adriana; Costa, Max

    2009-03-01

    Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15% of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ion-induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells.

  18. Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

    PubMed

    Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

    2015-05-01

    Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production.

  19. B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

    PubMed Central

    Mohammad, Dara K.; Ali, Raja H.; Turunen, Janne J.; Nore, Beston F.; Smith, C. I. Edvard

    2016-01-01

    Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins. PMID:27487157

  20. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies.

    PubMed

    McGuire, Andrew T; Hoot, Sam; Dreyer, Anita M; Lippy, Adriana; Stuart, Andrew; Cohen, Kristen W; Jardine, Joseph; Menis, Sergey; Scheid, Johannes F; West, Anthony P; Schief, William R; Stamatatos, Leonidas

    2013-04-08

    Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti-CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.

  1. Free Light Chains and Intrathecal B Cells Activity in Multiple Sclerosis: A Prospective Study and Meta-Analysis

    PubMed Central

    Passerini, Gabriella; Sangalli, Francesca; Moiola, Lucia; Colombo, Bruno; Locatelli, Massimo; Comi, Giancarlo

    2016-01-01

    Background. The presence of CSF oligoclonal bands (OBs) is an independent prognostic factor for multiple sclerosis (MS), but the difficulties in the standardization of the test and the interlaboratory variation in reporting have contributed to its limited use in the diagnosis of the disease. Standard nephelometric assays to measure free light chains (FLC) levels have been recently developed and the test may improve the detection of intrathecal B cells activity. Methods. The presence of OBs, kappa and lambda FLC levels, and standard indices of intrathecal inflammation were assessed in 100 consecutive patients, including patients with MS, clinically isolated syndromes (CIS), other inflammatory diseases of the CNS, and other noninflammatory diseases. Results. Both KFLC and LFLC correlated strongly with the presence of OCBs and with all common tests for intrathecal inflammation (p < 0.001 for all comparisons). KFLC and LFLC were significantly different in patients with MS and CIS compared to the other groups (p < 0.001 and p < 0.001, resp.) and had a better diagnostic accuracy than all the other tests (area under the curve 82.3 % for KFLC index and 79.3 % for LFLC index). Conclusion. Nephelometric assays for KFLC in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate MS patients. PMID:28116160

  2. Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.

    PubMed Central

    Jehuda-Cohen, T; Slade, B A; Powell, J D; Villinger, F; De, B; Folks, T M; McClure, H M; Sell, K W; Ahmed-Ansari, A

    1990-01-01

    Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals. Images PMID:2111024

  3. Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline.

    PubMed

    Pulvino, Mary; Chen, Luojing; Oleksyn, David; Li, Jing; Compitello, George; Rossi, Randy; Spence, Stephen; Balakrishnan, Vijaya; Jordan, Craig; Poligone, Brian; Casulo, Carla; Burack, Richard; Shapiro, Joel L; Bernstein, Steven; Friedberg, Jonathan W; Deshaies, Raymond J; Land, Hartmut; Zhao, Jiyong

    2015-06-20

    In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

  4. Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline

    PubMed Central

    Pulvino, Mary; Chen, Luojing; Oleksyn, David; Li, Jing; Compitello, George; Rossi, Randy; Spence, Stephen; Balakrishnan, Vijaya; Jordan, Craig; Poligone, Brian; Casulo, Carla; Burack, Richard; Shapiro, Joel L.; Bernstein, Steven; Friedberg, Jonathan W.; Deshaies, Raymond J.; Land, Hartmut; Zhao, Jiyong

    2015-01-01

    In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes. PMID:26142707

  5. STING Negatively Regulates Double-Stranded DNA-Activated JAK1-STAT1 Signaling via SHP-1/2 in B Cells.

    PubMed

    Dong, Guanjun; You, Ming; Ding, Liang; Fan, Hongye; Liu, Fei; Ren, Deshan; Hou, Yayi

    2015-05-01

    Recognition of cytosolic DNA initiates a series of innate immune responses by inducing IFN-I production and subsequent triggering JAK1-STAT1 signaling which plays critical roles in the pathogenesis of infection, inflammation and autoimmune diseases through promoting B cell activation and antibody responses. The stimulator of interferon genes protein (STING) has been demonstrated to be a critical hub of type I IFN induction in cytosolic DNA-sensing pathways. However, it still remains unknown whether cytosolic DNA can directly activate the JAK1-STAT1 signaling or not. And the role of STING is also unclear in this response. In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase. Moreover, this response is not dependent on type I IFN receptors. Interestingly, STING could inhibit dsDNA-triggered activation of JAK1-STAT1 signaling by inducing SHP-1 and SHP-2 phosphorylation. In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases. Our data provide a molecular insight into the novel role of STING in dsDNA-mediated inflammatory disorders.

  6. Efficient help for autoreactive B-cell activation requires CD4+ T-cell recognition of an agonist peptide at the effector stage.

    PubMed

    Hondowicz, Brian D; Batheja, Amrita O; Metzgar, Michele H; Pagán, Antonio J; Perng, Olivia A; Willms, Simone; Caton, Andrew J; Erikson, Jan

    2009-09-01

    T-cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4(+) T-cell priming, and subsequent T-B-cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B-cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4(+) T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4(+) T-B-cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.

  7. Are we ready to stratify treatment for diffuse large B-cell lymphoma using molecular hallmarks?

    PubMed

    Barton, Sarah; Hawkes, Eliza A; Wotherspoon, Andrew; Cunningham, David

    2012-01-01

    The division of the heterogeneous entity of diffuse large B-cell lymphoma (DLBCL) into the ontogenic phenotypes of germinal center B-cell-like (GCB) and activated B-cell-like (ABC) is optimally determined by gene expression profiling (GEP), although simpler immunohistochemistry (IHC) algorithms are alternatively being used. The cell-of-origin (COO) classification assists in prognostication and may be predictive of response to therapy. Mounting data suggests that IHC methods of classifying COO may be inaccurate. GEP categorization of COO is superior in defining prognostically and biologically distinct DLBCL subtypes, but current barriers to its widescale use include inaccessibility, cost, and lack of methodological standardization and prospective validation. The poorer prognosis of ABC-DLBCL is frequently associated with constitutive activity in the NF-κB pathway and aberrations in upstream or downstream regulators of this pathway. The molecular mechanisms underlying lymphomagenesis in GCB-DLBCL are arguably less well defined, but C-REL amplification and mutations in BCL-2 and EZH2 are common. New technologies, such as next-generation sequencing, are rapidly revealing novel pathogenic genetic aberrations, and DLBCL treatment strategies are increasingly being designed focusing on distinctive pathogenic drivers within ontogenic phenotypes. This review examines emerging molecular targets and novel therapeutic agents in DLBCL, and discusses whether stratifying therapy for DLBCL using molecular features is merited by current preclinical and clinical evidence.

  8. BRAFV600E induces ABCB1/P-glycoprotein expression and drug resistance in B-cells via AP-1 activation

    PubMed Central

    Tsai, Yo-Ting; Lozanski, Gerard; Lehman, Amy; Sass, Ellen J.; Hertlein, Erin; Salunke, Santosh B.; Chen, Ching-Shih; Grever, Michael R.; Byrd, John C.; Lucas, David M.

    2015-01-01

    A subset of patients with chronic lymphocytic leukemia (CLL) and nearly all patients with classic hairy cell leukemia (HCL) harbor somatic BRAF activating mutations. However, the pathological role of activated BRAF in B-cell leukemia development and progression remains unclear. In addition, although HCL patients respond well to the BRAFV600E inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation. To investigate the biological role of BRAFV600E in B-cell leukemia, we generated a CLL-like B-cell line, OSUCLL, with doxycycline-inducible BRAFV600E expression. Microarray and real-time PCR analysis showed that ABCB1 mRNA is upregulated in these cells, and P-glycoprotein (P-gp) expression as well as function were confirmed by immunoblot and rhodamine exclusion assays. Additionally, pharmacological inhibition of BRAFV600E and MEK alleviated the BRAFV600E-induced ABCB1/P-gp expression. ABCB1 reporter assays and gel shift assays demonstrated that AP-1 activity is crucial in this mechanism. This study therefore uncovers a pathological role for BRAFV600E in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAFV600E and MEK can be used to delay disease progression and occurrence of resistance. PMID:26350141

  9. BRAF(V600E) induces ABCB1/P-glycoprotein expression and drug resistance in B-cells via AP-1 activation.

    PubMed

    Tsai, Yo-Ting; Lozanski, Gerard; Lehman, Amy; Sass, Ellen J; Hertlein, Erin; Salunke, Santosh B; Chen, Ching-Shih; Grever, Michael R; Byrd, John C; Lucas, David M

    2015-09-05

    A subset of patients with chronic lymphocytic leukemia (CLL) and nearly all patients with classic hairy cell leukemia (HCL) harbor somatic BRAF activating mutations. However, the pathological role of activated BRAF in B-cell leukemia development and progression remains unclear. In addition, although HCL patients respond well to the BRAF(V600E) inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation. To investigate the biological role of BRAF(V600E) in B-cell leukemia, we generated a CLL-like B-cell line, OSUCLL, with doxycycline-inducible BRAF(V600E) expression. Microarray and real-time PCR analysis showed that ABCB1 mRNA is upregulated in these cells, and P-glycoprotein (P-gp) expression as well as function were confirmed by immunoblot and rhodamine exclusion assays. Additionally, pharmacological inhibition of BRAF(V600E) and MEK alleviated the BRAF(V600E)-induced ABCB1/P-gp expression. ABCB1 reporter assays and gel shift assays demonstrated that AP-1 activity is crucial in this mechanism. This study, uncovers a pathological role for BRAF(V600E) in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAF(V600E) and MEK can be used to delay disease progression and occurrence of resistance.

  10. Suppression by Δ(9)-tetrahydrocannabinol of the primary immunoglobulin M response by human peripheral blood B cells is associated with impaired STAT3 activation.

    PubMed

    Ngaotepprutaram, Thitirat; Kaplan, Barbara L F; Carney, Stephen; Crawford, Robert; Kaminski, Norbert E

    2013-08-09

    This study was undertaken to gain insights into the mechanism for Δ(9)-tetrahydrocannabinol (Δ(9)-THC)-mediated suppression of primary immunoglobulin M (IgM) responses in humans. An in vitro activation model, which employs cell surface-expressed CD40 ligand (CD40L) and recombinant cytokines (interleukin (IL)-2, -6, and -10), was used to differentiate human peripheral blood (HPB) naïve B cells into IgM secreting cells. Pretreatment with Δ(9)-THC significantly decreased the number of IgM secreting cells as determined by ELISPOT. The attenuation of IgM secretion by Δ(9)-THC involved, at least in part, the impairment of plasma cell differentiation as evidenced by suppression of immunoglobulin joining chain (IgJ) mRNA expression. The analysis at each of two different stages critically involved in plasma cell differentiation indicates that Δ(9)-THC impaired both the primary activation stage and proliferation of B cells. Interestingly, Δ(9)-THC selectively suppressed the surface expression of CD80, but not other measured B-cell activation markers (CD69, CD86, and ICAM1). Furthermore, pretreatment with Δ(9)-THC was accompanied by a robust decrease of STAT3 phosphorylation, whereas the phosphorylation of the p65 NFκB subunit was not affected. Collectively, these data provide new insights into the mechanisms for impaired B cell function by Δ(9)-THC.

  11. LRRK1 is critical in the regulation of B-cell responses and CARMA1-dependent NF-κB activation

    PubMed Central

    Morimoto, Keiko; Baba, Yoshihiro; Shinohara, Hisaaki; Kang, Sujin; Nojima, Satoshi; Kimura, Tetsuya; Ito, Daisuke; Yoshida, Yuji; Maeda, Yohei; Sarashina-Kida, Hana; Nishide, Masayuki; Hosokawa, Takashi; Kato, Yasuhiro; Hayama, Yoshitomo; Kinehara, Yuhei; Okuno, Tatsusada; Takamatsu, Hyota; Hirano, Toru; Shima, Yoshihito; Narazaki, Masashi; Kurosaki, Tomohiro; Toyofuku, Toshihiko; Kumanogoh, Atsushi

    2016-01-01

    B-cell receptor (BCR) signaling plays a critical role in B-cell activation and humoral immunity. In this study, we discovered a critical function of leucine-rich repeat kinase 1 (LRRK1) in BCR-mediated immune responses. Lrrk1−/− mice exhibited altered B1a-cell development and basal immunoglobulin production. In addition, these mice failed to produce IgG3 antibody in response to T cell–independent type 2 antigen due to defects in IgG3 class-switch recombination. Concomitantly, B cells lacking LRRK1 exhibited a profound defect in proliferation and survival upon BCR stimulation, which correlated with impaired BCR-mediated NF-κB activation and reduced expression of NF-κB target genes including Bcl-xL, cyclin D2, and NFATc1/αA. Furthermore, LRRK1 physically interacted and potently synergized with CARMA1 to enhance NF-κB activation. Our results reveal a critical role of LRRK1 in NF-κB signaling in B cells and the humoral immune response. PMID:27166870

  12. Different effect of prostaglandin E2 on B-cell activation by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2: selective inhibition of B151-TRF2-induced antibody response through increases in intracellular cyclic AMP levels

    PubMed Central

    Ishihara, K.; Ono, S.; Takahama, Y.; Hirayama, F.; Hirano, H.; Itoh, K.; Dobashi, K.; Murakami, S.; Katoh, Y.; Yamaguchi, M.; Hamaoka, T.

    1989-01-01

    Effects of prostaglandin E2 (PGE2) on murine B-cell activation induced by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2, were examined. A final differentiation of unprimed B cells into IgM-producing cells induced by B151-TRF2 was markedly inhibited by PGE2 at physiological concentrations (around 10-8 M), whereas B151-TRF1/IL-5-induced antibody responses of unprimed as well as activated B cells were not affected by PGE2, even at 10-6 M. B-cell responses induced by B151-TRF2-like factors from autoimmune-prone MRL/1pr mice were also inhibited by PGE2. Biphasic increases in intracellular cyclic AMP (cAMP) levels were induced by culturing B cells with 10-6 or 10-8 M PGE2: rapid increases within 8 min and delayed increases around 16 hr. The direct addition of dibutyryl cAMP to cultures of B cells resulted in marked inhibition of antibody responses when stimulated with B151-TRF2 but not with B151-TRF1/IL-5. The B151-TRF2-induced antibody responses were also inhibited by cAMP-elevating reagents such as forskolin, cholera toxin and theophyline. Furthermore, 2′, 5′-dideoxyadenosine, which is an inhibitor of adenylate cyclase, prevented the PGE2-mediated cAMP accumulation in unprimed B cells as well as the PGE2-mediated inhibition of B151-TRF2-induced B-cell responses when added at the initiation of culture. These results suggest that PGE2 inhibits B151-TRF2-induced antibody responses through the activation of adenylate cyclase and subsequent accumulation of intracellular cAMP, whereas B151-TRF1/IL-5-responsive B cells are resistant to the inhibitory effect of PGE2 and cAMP. PMID:2553585

  13. Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.

    PubMed

    Sarkar, Aloke; Balakrishnan, Kumudha; Chen, Jefferson; Patel, Viralkumar; Neelapu, Sattva S; McMurray, John S; Gandhi, Varsha

    2016-01-19

    The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3-7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27-43%), but not in DKO MEFs or MEFs expressing respective Casp3-7 catalytic mutants (12-13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30-73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18-36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.

  14. Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma

    PubMed Central

    Sarkar, Aloke; Balakrishnan, Kumudha; Chen, Jefferson; Patel, Viralkumar; Neelapu, Sattva S.; McMurray, John S.; Gandhi, Varsha

    2016-01-01

    The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3–7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27–43%), but not in DKO MEFs or MEFs expressing respective Casp3–7 catalytic mutants (12–13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30–73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18–36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation. PMID:26658105

  15. Asymmetrical phosphorylation and function of immunoreceptor tyrosine-based activation motif tyrosines in B cell antigen receptor signal transduction.

    PubMed

    Pao, L I; Famiglietti, S J; Cambier, J C

    1998-04-01

    CD79a and CD79b function as transducers of B cell antigen receptor signals via a cytoplasmic sequence, termed the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs contain two conserved tyrosines that may become phosphorylated upon receptor aggregation and bind distinct effectors by virtue of the distinct preference of phosphotyrosyl-containing sequences for SH2 domains. To explore the function of CD79a and CD79b ITAM tyrosines, we created membrane molecules composed of MHC class II I-Ak extracellular and transmembrane domains, and CD79a or CD79b cytoplasmic domains in which one or both of the ITAM tyrosines were mutated to phenylalanine. Functional analysis revealed that both ITAM tyrosines are required for ligand-induced Syk phosphorylation. However CD79a-ITAM and CD79b-ITAM tyrosine phosphorylations were asymmetrical, with >80% of phosphorylation occurring on the N-terminal tyrosine (Y-E-G-L). Thus, these findings suggest that following receptor ligation, only a minor proportion of phosphorylated ITAMs are doubly phosphorylated and thus can engage Syk. Only the N-terminal ITAM tyrosine of CD79a was required for ligand-mediated phosphorylation of the receptor and a subset of downstream substrates, including p62, p110, and Shc, and for Ca2+ mobilization. However, responses mediated through CD79b exhibited a greater dependence on the presence of both tyrosines. Neither tyrosine in CD79a or CD79b appeared absolutely essential for Src family kinase phosphorylation. These results indicate that phosphorylations of the tyrosines in CD79a and CD79b occur with very different stoichiometry, and the respective tyrosyl residues have distinct functions.

  16. Demonstration That Mast Cells, T Cells, and B Cells Bearing the Activating Kit Mutation D816V Occur in Clusters within the Marrow of Patients with Mastocytosis

    PubMed Central

    Taylor, Marcia L.; Sehgal, Devinder; Raffeld, Mark; Obiakor, Harold; Akin, Cem; Mage, Rose G.; Metcalfe, Dean D.

    2004-01-01

    Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering. PMID:15507672

  17. Epstein-Barr Virus–induced Molecule 1 Ligand Chemokine Is Expressed by Dendritic Cells in Lymphoid Tissues and Strongly Attracts Naive T Cells and Activated B Cells

    PubMed Central

    Ngo, Vu N.; Lucy Tang, H.; Cyster, Jason G.

    1998-01-01

    Movement of T and B lymphocytes through secondary lymphoid tissues is likely to involve multiple cues that help the cells navigate to appropriate compartments. Epstein-Barr virus– induced molecule 1 (EBI-1) ligand chemokine (ELC/MIP3β) is expressed constitutively within lymphoid tissues and may act as such a guidance cue. Here, we have isolated mouse ELC and characterized its expression pattern and chemotactic properties. ELC is expressed constitutively in dendritic cells within the T cell zone of secondary lymphoid tissues. Recombinant ELC was strongly chemotactic for naive (L-selectinhi) CD4 T cells and for CD8 T cells and weakly attractive for resting B cells and memory (L-selectinlo) CD4 T cells. After activation through the B cell receptor, the chemotactic response of B cells was enhanced. Like its human counterpart, murine ELC stimulated cells transfected with EBI-1/CC chemokine receptor 7 (CCR7). Our findings suggest a central role for ELC in promoting encounters between recirculating T cells and dendritic cells and in the migration of activated B cells into the T zone of secondary lymphoid tissues. PMID:9653094

  18. B Cell Immunity in Solid Organ Transplantation

    PubMed Central

    Karahan, Gonca E.; Claas, Frans H. J.; Heidt, Sebastiaan

    2017-01-01

    The contribution of B cells to alloimmune responses is gradually being understood in more detail. We now know that B cells can perpetuate alloimmune responses in multiple ways: (i) differentiation into antibody-producing plasma cells; (ii) sustaining long-term humoral immune memory; (iii) serving as antigen-presenting cells; (iv) organizing the formation of tertiary lymphoid organs; and (v) secreting pro- as well as anti-inflammatory cytokines. The cross-talk between B cells and T cells in the course of immune responses forms the basis of these diverse functions. In the setting of organ transplantation, focus has gradually shifted from T cells to B cells, with an increased notion that B cells are more than mere precursors of antibody-producing plasma cells. In this review, we discuss the various roles of B cells in the generation of alloimmune responses beyond antibody production, as well as possibilities to specifically interfere with B cell activation. PMID:28119695

  19. Probiotics enhance the effect of allergy immunotherapy on regulating antigen specific B cell activity in asthma patients.

    PubMed

    Liu, Jun; Chen, Feng-Hong; Qiu, Shu-Qi; Yang, Li-Tao; Zhang, Huan-Ping; Liu, Jiang-Qi; Geng, Xiao-Rui; Yang, Gui; Liu, Zhi-Qiang; Li, Jing; Liu, Zhi-Gang; Li, Hua-Bin; Yang, Ping-Chang

    2016-01-01

    Immune regulatory system dysfunction plays a role in the pathogenesis of asthma. The therapeutic effect of allergic asthma is to be improved. The immune regulatory function of probiotics has been recognized. This study tests a hypothesis that Clostridium butyricum (CB) enhances the effect of allergen specific immunotherapy (AIT) on asthma. In this study patients with allergic asthma were treated with AIT or/and CB for six months. The therapeutic effect and IgE production of the patients were observed. The results showed that administration with AIT alone alleviated the asthma symptoms; but the serum levels of interleukin (IL)-4, IL-5, IL-13 and specific IgE were not altered, which was markedly improved by the administration with CB plus AIT. Such effects were maintained only for two months in the patients treated with AIT alone; but maintained more than 12 months in those patients treated with both AIT and CB. CB facilitated AIT to induce IL-10(+) B cells (B10 cells) in asthma patients. AIT/CB therapy converted antigen specific B cells to antigen specific regulatory B cells. Butyrate modulated the gene transcription of IgE and IL-10 in the allergen specific B cells. In conclusion, administration of CB can enhance the therapeutic effect of AIT in the treatment of allergic asthma via facilitating generation of B10 cells.

  20. Probiotics enhance the effect of allergy immunotherapy on regulating antigen specific B cell activity in asthma patients

    PubMed Central

    Liu, Jun; Chen, Feng-hong; Qiu, Shu-Qi; Yang, Li-Tao; Zhang, Huan-Ping; Liu, Jiang-Qi; Geng, Xiao-Rui; Yang, Gui; Liu, Zhi-Qiang; Li, Jing; Liu, Zhi-Gang; Li, Hua-Bin; Yang, Ping-Chang

    2016-01-01

    Immune regulatory system dysfunction plays a role in the pathogenesis of asthma. The therapeutic effect of allergic asthma is to be improved. The immune regulatory function of probiotics has been recognized. This study tests a hypothesis that Clostridium butyricum (CB) enhances the effect of allergen specific immunotherapy (AIT) on asthma. In this study patients with allergic asthma were treated with AIT or/and CB for six months. The therapeutic effect and IgE production of the patients were observed. The results showed that administration with AIT alone alleviated the asthma symptoms; but the serum levels of interleukin (IL)-4, IL-5, IL-13 and specific IgE were not altered, which was markedly improved by the administration with CB plus AIT. Such effects were maintained only for two months in the patients treated with AIT alone; but maintained more than 12 months in those patients treated with both AIT and CB. CB facilitated AIT to induce IL-10+ B cells (B10 cells) in asthma patients. AIT/CB therapy converted antigen specific B cells to antigen specific regulatory B cells. Butyrate modulated the gene transcription of IgE and IL-10 in the allergen specific B cells. In conclusion, administration of CB can enhance the therapeutic effect of AIT in the treatment of allergic asthma via facilitating generation of B10 cells. PMID:28078000

  1. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing

    PubMed Central

    Quentmeier, Hilmar; Drexler, Hans G.; Hauer, Vivien; MacLeod, Roderick A. F.; Pommerenke, Claudia; Uphoff, Cord C.; Zaborski, Margarete; Berglund, Mattias

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members. PMID:27907212

  2. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing.

    PubMed

    Quentmeier, Hilmar; Drexler, Hans G; Hauer, Vivien; MacLeod, Roderick A F; Pommerenke, Claudia; Uphoff, Cord C; Zaborski, Margarete; Berglund, Mattias; Enblad, Gunilla; Amini, Rose-Marie

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members.

  3. Interaction of ABC transport proteins with toxic metals at the level of gene and transport activity in the PLHC-1 fish cell line.

    PubMed

    Della Torre, Camilla; Zaja, Roko; Loncar, Jovica; Smital, Tvrtko; Focardi, Silvano; Corsi, Ilaria

    2012-06-25

    The aim of this study was to investigate the interaction of four toxic metals with ABC transport proteins in piscine cell line PLHC-1. Cells were exposed for 24 h to 0.01-1 μM of CdCl(2), HgCl(2), As(2)O(3), or K(2)Cr(2)O(7) and the expression of a series of ABC genes (abcb1, abcc1-4) was determined using qRT-PCR. Using the fluorescent model substrates calcein-AM and monochlorbimane we measured interaction of metals with the transport activity of ABC transporters. P-glycoprotein (P-gp) activity was measured in PLHC-1/dox (P-gp overexpressing cells) while activity and interactions of metals with MRPs was measured in PLHC-1/wt cells. After 24 h exposure, abcc2-4 genes were dose-dependently up-regulated by all metals, while abcb1 and abcc1 were less affected. Up-regulation of abcc2 was more pronounced, with up to 8-fold increase in expression. Abcc3 and abcc4 were moderately inducible by HgCl(2) with 3.3-fold and 2.2-fold, respectively. All metals caused a significant inhibition of both P-gp (2.9- to 4-fold vs. controls) and MRP (1.3- to 1.8-fold) transport activities. Modulation of ABC genes and transport activities was further investigated in PLHC-1/wt cells exposed to 1 μM HgCl(2) for 72 h and in Hg resistant cells selected by long term cultivation of PLHC-1/wt cells in increasing concentrations of HgCl(2). Exposure to HgCl(2) for 72 h induced MRP genes expression and efflux activity. The long term cultivation of PLHC-1/wt cells in HgCl(2), did not cause prolonged up-regulation of the tested abc genes but resulted in higher MRP transport activities as determined by the increased sensitivity of these cells to MK571 (MRP specific inhibitor). Results of the present study indicated specific interaction of metals with selected ABC transport proteins. Modulation of ABC transporters takes place at both transcriptional and functional level. An active involvement of efflux pumps in Hg clearance in fish is suggested.

  4. A subset of annular lipids is linked to the flippase activity of an ABC transporter.

    PubMed

    Bechara, Chérine; Nöll, Anne; Morgner, Nina; Degiacomi, Matteo T; Tampé, Robert; Robinson, Carol V

    2015-03-01

    Lipids are critical components of membranes that could affect the properties of membrane proteins, yet the precise compositions of lipids surrounding membrane-embedded protein complexes is often difficult to discern. Here we report that, for the heterodimeric ABC transporter TmrAB, the extent of delipidation can be controlled by timed exposure to detergent. We subsequently characterize the cohort of endogenous lipids that are extracted in contact with the membrane protein complex, and show that with prolonged delipidation the number of neutral lipids is reduced in favour of their negatively charged counterparts. We show that lipid A is retained by the transporter and that the extent of its binding decreases during the catalytic cycle, implying that lipid A release is linked to adenosine tri-phosphate hydrolysis. Together, these results enable us to propose that a subset of annular lipids is invariant in composition, with negatively charged lipids binding tightly to TmrAB, and imply a role for this exporter in glycolipid translocation.

  5. A subset of annular lipids is linked to the flippase activity of an ABC transporter

    NASA Astrophysics Data System (ADS)

    Bechara, Chérine; Nöll, Anne; Morgner, Nina; Degiacomi, Matteo T.; Tampé, Robert; Robinson, Carol V.

    2015-03-01

    Lipids are critical components of membranes that could affect the properties of membrane proteins, yet the precise compositions of lipids surrounding membrane-embedded protein complexes is often difficult to discern. Here we report that, for the heterodimeric ABC transporter TmrAB, the extent of delipidation can be controlled by timed exposure to detergent. We subsequently characterize the cohort of endogenous lipids that are extracted in contact with the membrane protein complex, and show that with prolonged delipidation the number of neutral lipids is reduced in favour of their negatively charged counterparts. We show that lipid A is retained by the transporter and that the extent of its binding decreases during the catalytic cycle, implying that lipid A release is linked to adenosine tri-phosphate hydrolysis. Together, these results enable us to propose that a subset of annular lipids is invariant in composition, with negatively charged lipids binding tightly to TmrAB, and imply a role for this exporter in glycolipid translocation.

  6. Spatial Alterations between CD4+ TFH, B and CD8+T cells during SIV infection: T/B cell homeostasis, activation and potential mechanism for viral escape

    PubMed Central

    Hong, Jung Joo; Amancha, Praveen K; Rogers, Kenneth; Ansari, Aftab A; Villinger, Francois

    2012-01-01

    HIV/SIV infections induce chronic immune activation with remodeling of lymphoid architecture and hypergammaglobulinemia, although the mechanisms leading to such symptoms remain to be fully elucidated. Moreover, lymph nodes have been highlighted as a predilection site for SIV escape in vivo. Following 20 rhesus macaques infected with SIVmac239, as they progress from pre to acute and chronic infection, we document for the first time the local dynamics T follicular helper (TFH) cells and B cells in situ. Progression of SIV infection was accompanied with increased numbers of well delineated follicles containing germinal centers (GCs) and TFH cells with a progressive increase in the density of PD-1 expression in lymph nodes. The rise in PD-1+ TFH cells was followed by a substantial accumulation of Ki67+ B cells within GCs. However, unlike in blood, major increases in the frequency of CD27+ memory B cells were observed in lymph nodes, indicating increased turnover of these cells, correlated with increases in total and SIV specific antibody levels. Of importance, compared to T cell zones, GCs seemed to exclude CD8+ T cells while harboring increasing numbers of CD4+ T cells, many of which are positive for SIVgag, providing an environment particularly beneficial for virus replication and reservoirs. Our data highlight for the first time important spatial interactions of GC cell subsets during SIV infection, the capacity of lymphoid tissues to maintain stable relative levels of circulating B cell subsets and a potential mechanism for viral reservoirs within GCs during SIV infection. PMID:22387550

  7. NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity.

    PubMed

    Ochodnicka-Mackovicova, Katarina; Bahjat, Mahnoush; Bloedjes, Timon A; Maas, Chiel; de Bruin, Alexander M; Bende, Richard J; van Noesel, Carel J M; Guikema, Jeroen E J

    2015-09-10

    In developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.

  8. Short-term and long-term reproducibility of lung tumor position using active breathing control (ABC)

    SciTech Connect

    Koshani, Rojano . E-mail: rkashani@umich.edu; Balter, James M.; Hayman, James A.; Henning, George T.; Herk, Marcel van

    2006-08-01

    Purpose: To evaluate the short-term and long-term reproducibility of lung tumor position for scans acquired using an active breathing control (ABC) device. Methods and Materials: Ten patients with lung cancer were scanned over three sessions during the course of treatment. For each session, two scans were acquired at deep inhale, and one scan each at half of deep inhale and at exhale. Long-term reproducibility was evaluated by comparing the same breathing state scans from two sessions, with setup variation removed by skeletal alignment. Tumor alignment was based on intensity matching of a small volume around the tumor. For short-term reproducibility, the two inhale volumes from the same session were compared. Results: For the short-term reproducibility, the mean and the standard deviation (SD) of the displacement of the center of tumor were 0.0 (1.5) mm in anteroposterior (AP), 0.3 (1.4) mm in superior/inferior (SI), and 0.2 (0.7) mm in right/left (RL) directions. For long-term reproducibility, the mean (SD) were -1.3 (3.1) mm AP, -0.5 (3.8) mm SI, and 0.3 (1.6) mm RL for inhale and -0.2 (2.8) mm AP, 0.2 (2.1) mm SI, and -0.7 (1.1) mm RL for exhale. Conclusion: The ABC device demonstrates very good short-term and long-term reproducibility. Increased long-term variability in position, primarily in the SI and AP directions, indicates the role of tumor-directed localization in combination with breath-held immobilization.

  9. Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.

    PubMed

    Panapruksachat, Siribun; Iwatani, Shun; Oura, Takahiro; Vanittanakom, Nongnuch; Chindamporn, Ariya; Niimi, Kyoko; Niimi, Masakazu; Lamping, Erwin; Cannon, Richard D; Kajiwara, Susumu

    2016-07-01

    Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.

  10. CD40 ligand-mediated activation of the de novo RelB NF-kappaB synthesis pathway in transformed B cells promotes rescue from apoptosis.

    PubMed

    Mineva, Nora D; Rothstein, Thomas L; Meyers, John A; Lerner, Adam; Sonenshein, Gail E

    2007-06-15

    CD40, a tumor necrosis factor receptor family member, is expressed on B lymphocytes. Interaction between CD40 and its ligand (CD40L), expressed on activated T lymphocytes, is critical for B cell survival. Here, we demonstrate that CD40 signals B cell survival in part via transcriptional activation of the RelB NF-kappaB subunit. CD40L treatment of chronic lymphocytic leukemia cells induced levels of relB mRNA. Similarly, CD40L-mediated rescue of WEHI 231 B lymphoma cells from apoptosis induced upon B cell receptor (surface IgM) engagement led to increased relB mRNA levels. Recently, we characterized a new de novo synthesis pathway for the RelB NF-kappaB subunit, induced by the cytomegalovirus IE1 protein, in which binding of p50/p65 NF-kappaB and c-Jun/Fra-2 AP-1 complexes to the relB promoter works in synergy to potently activate transcription (Wang, X., and Sonenshein, G. E. (2005) J. Virol. 79, 95-105). CD40L treatment of WEHI 231 cells caused induction of AP-1 family members Fra-2, c-Jun, JunD, and JunB. Cotransfection of Fra-2 with the Jun AP-1 subunits and p50/c-Rel NF-kappaB led to synergistic activation of the relB promoter. Ectopic expression of relB or RelB knockdown using small interfering RNA demonstrated the important role of this subunit in control of WEHI 231 cell survival and implicated activation of the anti-apoptotic factors Survivin and manganese superoxide dismutase. Thus, CD40 engagement of transformed B cells activates relB gene transcription via a process we have termed the de novo RelB synthesis pathway, which protects these cells from apoptosis.

  11. ASK1/JNK-mediated TAp63 activation controls the cell survival signal of baicalein-treated EBV-transformed B cells.

    PubMed

    Park, Ga Bin; Kim, Yeong Seok; Lee, Hyun-Kyung; Yang, Jae Wook; Kim, Daejin; Hur, Dae Young

    2016-01-01

    Transcriptionally active p63 (TAp63) promotes cell cycle arrest, senescence, and apoptosis in several cancer cells. Migration inhibitory factor (MIF)/CD74 regulates B-cell survival through nuclear factor (NF)-κB-dependent TAp63 expression. In this study, we investigated how the level of TAp63 expression influences the induction of apoptosis in baicalein-treated EBV-transformed B cells. Baicalein induced the expression of TAp63 and apoptosis signal-regulating kinase 1 (ASK1), as well as cytotoxicity, by disrupting the mitochondrial membrane and inhibiting the activation of phosphoinositide 3-kinase (PI3K)/Akt and NF-κB. Genetic knockdown of TAp63 or ASK1 by small interfering RNA resulted in protection from apoptosis accompanied by the recovery of CD74, CD44, α4 integrin, Bcl-2, and NF-κB activation. Baicalein-induced reactive oxygen species activated the ASK1/JNK pathway with subsequent expression of TAp63. Pre-engagement with MIF/CD74 maintained the expression of CD74, CD44, and α4 integrin, as well as Syk/Src-mediated PI3K/Akt activation, in baicalein-treated EBV-transformed B cells. Meanwhile, ASK1/JNK-dependent TAp63 expression was efficiently suppressed after pre-treatment with MIF. Our results suggest that baicalein-mediated ASK1/JNK activation regulates the mitochondria-dependent apoptosis pathway through the up-regulation of TAp63 and down-regulation of NF-κB and CD74/CD44 in B-cell malignancies.

  12. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor.

    PubMed

    Foucault, Isabelle; Le Bras, Séverine; Charvet, Céline; Moon, Chéol; Altman, Amnon; Deckert, Marcel

    2005-02-01

    Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function. The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells. However, its involvement in BCR signaling is completely unknown. Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk. To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins. The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms. 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma). Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs). Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs. Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins.

  13. Nfkb1 Activation by the E26 Transformation-Specific Transcription Factors PU.1 and Spi-B Promotes Toll-Like Receptor-Mediated Splenic B Cell Proliferation

    PubMed Central

    Li, Stephen K. H.; Abbas, Ali K.; Solomon, Lauren A.; Groux, Gaëlle M. N.

    2015-01-01

    Generation of antibodies against T-independent and T-dependent antigens requires Toll-like receptor (TLR) engagement on B cells for efficient responses. However, the regulation of TLR expression and responses in B cells is not well understood. PU.1 and Spi-B (encoded by Sfpi1 and Spib, respectively) are transcription factors of the E26 transformation-specific (ETS) family and are important for B cell development and function. It was found that B cells from mice knocked out for Spi-B and heterozygous for PU.1 (Sfpi1+/− Spib−/− [PUB] mice) proliferated poorly in response to TLR ligands compared to wild-type (WT) B cells. The NF-κB family member p50 (encoded by Nfkb1) is required for lipopolysaccharide (LPS) responsiveness in mice. PUB B cells expressed reduced Nfkb1 mRNA transcripts and p50 protein. The Nfkb1 promoter was regulated directly by PU.1 and Spi-B, as shown by reporter assays and chromatin immunoprecipitation analysis. Occupancy of the Nfkb1 promoter by PU.1 was reduced in PUB B cells compared to that in WT B cells. Finally, infection of PUB B cells with a retroviral vector encoding p50 substantially restored proliferation in response to LPS. We conclude that Nfkb1 transcriptional activation by PU.1 and Spi-B promotes TLR-mediated B cell proliferation. PMID:25733685

  14. Characterization and functional studies of forkhead box protein 3− lymphocyte activation gene 3+ CD4+ regulatory T cells induced by mucosal B cells

    PubMed Central

    Chu, K-H; Chiang, B-L

    2015-01-01

    The induction of mucosal tolerance has been demonstrated to be an effective therapeutic approach for the treatment of allergic diseases. Our previous study demonstrated that Peyer's patch B cells could convert naive T cells into regulatory T cells (so-called Treg-of-B(P) cells); however, it is important to characterize this particular subset of Treg-of-B cells for future applications. This study aimed to investigate the role of lymphocyte activating gene 3 (LAG3) in mediating the regulatory function of Treg-of-B(P) cells induced by mucosal follicular B (FOB) cells. Microarray analysis and real-time polymerase chain reaction (PCR) were used to assess the gene expression pattern of Treg-of-B(P) cells. To evaluate the role of LAG3, the in-vitro suppressive function and the alleviation of airway inflammation in a murine model of asthma was assessed. Our data indicated that FOB cells isolated from Peyer's patches had the ability to generate more suppressive Treg-of-B cells with LAG3 expression, compared with CD23loCD21lo B cells. LAG3 is not only a marker for Treg-of-B(P) cells, but also participate in the suppressive ability. Moreover, CCR4 and CCR6 could be detected on the LAG3+, not LAG3−, Treg-of-B(P) cells and would help cells homing to allergic lung. In the murine model of asthma, the adoptive transfer of LAG3+ Treg-of-B(P) cells was able to sufficiently suppress T helper type 2 (Th2) cytokine production, eosinophil infiltration and alleviate asthmatic symptoms. LAG3 was expressed in Treg-of-B(P) cells and was also involved in the function of Treg-of-B(P) cells. In the future, this particular subset of Treg-of-B cells might be used to alleviate allergic symptoms. PMID:25581421

  15. Synergistic activity of Card11 mutant and Bcl6 in the development of diffuse large B-cell lymphoma in a mouse model.

    PubMed

    Takahara, Taishi; Matsuo, Keitaro; Seto, Masao; Nakamura, Shigeo; Tsuzuki, Shinobu

    2016-11-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of malignant lymphoma; it derives from germinal center B cells. Although DLBCL harbors many genetic alterations, synergistic roles between such alterations in the development of lymphoma are largely undefined. We previously established a mouse model of lymphoma by transplanting gene-transduced germinal center B cells into mice. Here, we chose one of the frequently mutated genes in DLBCL, Card11 mutant, to explore its possible synergy with other genes, using our lymphoma model. Given that BCL6 and BCL2 expression and/or function are often deregulated in human lymphoma, we examined the possible synergy between Card11, Bcl6, and Bcl2. Germinal center B cells were induced in vitro, transduced with Card11 mutant, Bcl6, and Bcl2, and transplanted. Mice rapidly developed lymphomas, with exogenously transduced Bcl2 being dispensable. Although some mice developed lymphoma in the absence of transduced Bcl6, the absence was compensated by elevated expression of endogenous Bcl6. Additionally, the synergy between Card11 mutant and Bcl6 in the development of lymphoma was confirmed by the fact that the combination of Card11 mutant and Bcl6 caused lymphoma or death significantly earlier and with higher penetrance than Card11 mutant or Bcl6 alone. Lymphoma cells expressed interferon regulatory factor 4 and PR domain 1, indicating their differentiation toward plasmablasts, which characterize activated B cell-like DLBCL that represents a clinically aggressive subtype in humans. Thus, our mouse model provides a versatile tool for studying the synergistic roles of altered genes underlying lymphoma development.

  16. CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34

    PubMed Central

    Yamane, Fumihiro; Nishikawa, Yumiko; Matsui, Kazue; Asakura, Miki; Iwasaki, Eriko; Watanabe, Koji; Tanimoto, Hikaru; Sano, Hiroki; Fujiwara, Yuki; Stanley, E. Richard; Kanayama, Naoki; Mabbott, Neil A.; Magari, Masaki; Ohmori, Hitoshi

    2014-01-01

    With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b+ monocytic cells (F4/80+, Gr-1−, Ly6C−, I-A/E−/lo, CD11c−, CD115+, CXCR4+, CCR2+, CX3CR1−) when cultured with a Lin−c-kit+ population from mouse spleen cells. The developed CD11b+ cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R. PMID:24052571

  17. Nuclear Import of JAK1 Is Mediated by a Classical NLS and Is Required for Survival of Diffuse Large B-cell Lymphoma.

    PubMed

    Zhu, Fen; Hwang, Byounghoon; Miyamoto, Shigeki; Rui, Lixin

    2017-03-01

    JAKs are non-receptor tyrosine kinases that are generally found in association with cytokine receptors. In the canonical pathway, roles of JAKs have well been established in activating STATs in response to cytokine stimulation to modulate gene transcription. In contrast, a noncanonical role of JAK2 has recently been discovered, in which JAK2 in the nucleus imparts the epigenetic regulation of gene transcription through phosphorylation of tyrosine 41 on the histone protein H3. Recent work further demonstrated that this noncanonical mechanism is conserved with JAK1, which is activated by the autocrine cytokines IL6 and IL10 in activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL), a cancer type that is particularly difficult to treat and has poor prognosis. However, how JAK1 gains access to the nucleus to enable epigenetic regulation remains undefined. Here, we investigated this question and revealed that JAK1 has a classical nuclear localization signal toward the N-terminal region, which can be recognized by multiple importin α isoforms. Moreover, the nuclear import of JAK1 is independent of its kinase activity but is required for the optimal expansion of ABC DLBCL cells in vitroImplications: This study demonstrates that the nuclear import of JAK1 is essential for the optimal fitness of ABC DLBCL cells, and targeting JAK1 nuclear localization is a potential therapeutic strategy for ABC DLBCL. Mol Cancer Res; 15(3); 348-57. ©2016 AACR.

  18. CD45R, CD44 and MHC class II are signaling molecules for the cytoskeleton-dependent induction of dendrites and motility in activated B cells.

    PubMed

    Partida-Sánchez, S; Garibay-Escobar, A; Frixione, E; Parkhouse, R M; Santos-Argumedo, L

    2000-09-01

    Anti-CD44 or anti-MHC II antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHC II, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHC II appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.

  19. Bacteroides induce higher IgA production than Lactobacillus by increasing activation-induced cytidine deaminase expression in B cells in murine Peyer's patches.

    PubMed

    Yanagibashi, Tsutomu; Hosono, Akira; Oyama, Akihito; Tsuda, Masato; Hachimura, Satoshi; Takahashi, Yoshimasa; Itoh, Kikuji; Hirayama, Kazuhiro; Takahashi, Kyoko; Kaminogawa, Shuichi

    2009-02-01

    The gut mucosal immune system is crucial in host defense against infection by pathogenic microbacteria and viruses via the production of IgA. Previous studies have shown that intestinal commensal bacteria enhance mucosal IgA production. However, it is poorly understood how these bacteria induce IgA production and which genera of intestinal commensal bacteria induce IgA production effectively. In this study, we compared the immunomodulatory effects of Bacteroides and Lactobacillus on IgA production by Peyer's patches lymphocytes. IgA production by Peyer's patches lymphocytes co-cultured with Bacteroides was higher than with Lactobacillus. In addition, the expression of activation-induced cytidine deaminase increased in co-culture with Bacteroides but not with Lactobacillus. We found that intestinal commensal bacteria elicited IgA production. In particular, Bacteroides induced the differentiation of Peyer's patches B cell into IgA(+) B cells by increasing activation-induced cytidine deaminase expression.

  20. Occurrence of autoantibodies to intermediate filament proteins in human visceral leishmaniasis and their induction by experimental polyclonal B-cell activation.

    PubMed Central

    Böhme, M W; Evans, D A; Miles, M A; Holborow, E J

    1986-01-01

    Fifteen sera of patients with visceral leishmaniasis were investigated for the occurrence of autoantibodies. They were found in high incidence and titre, and with specificity to the intermediate filament (INFIL) proteins vimentin (12 out of 15 with a titre higher than 1:10) and keratin (9 out of 15 with a titre higher than 1:10) as well as to speckled anti-nuclear antigens (ANA). Additionally, supernatants of Leishmania major and Leishmania donovani cultures containing soluble parasite-derived antigens were mitogenic to cultures of mononuclear cells (MNC) obtained from healthy donors without specific antibodies to leishmanial antigens. The activation of MNC resulted in significant immunoglobulin production, some of which demonstrated autoantibody specificity to INFIL. The co-operation of monocytes, T cells and B cells was required in order to obtain maximal stimulation. The importance of polyclonal B-cell activation for the genesis and occurrence of autoantibodies in visceral leishmaniasis is discussed. PMID:3492440

  1. Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

    PubMed

    Wild, J; Schmiedel, B J; Maurer, A; Raab, S; Prokop, L; Stevanović, S; Dörfel, D; Schneider, P; Salih, H R

    2015-08-01

    Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

  2. Elevated B-cell activating factor BAFF, but not APRIL, correlates with CSF cerebellar autoantibodies in pediatric opsoclonus-myoclonus syndrome.

    PubMed

    Fühlhuber, V; Bick, S; Kirsten, A; Hahn, A; Gerriets, T; Tschernatsch, M; Kaps, M; Preissner, K T; Blaes, F; Altenkämper, S

    2009-05-29

    Childhood opsoclonus-myoclonus syndrome (OMS) occurs idiopathic or, in association with a neuroblastoma, as a paraneoplastic syndrome. Since autoantibodies were identified in some patients, an autoimmune pathogenesis has been suspected. While the newly discovered B-cell activating factors BAFF and APRIL are involved in systemic autoimmune diseases, their association with neuroimmunological diseases is hardly understood. We here investigated the BAFF and APRIL levels in serum and cerebrospinal fluid (CSF) of OMS patients and their correlation with surface-binding autoantibodies. BAFF and APRIL were both determined by ELISA, and autoantibodies to cerebellar granular neurons (CGN) have been investigated by flow cytometry in 17 OMS patients, 16 neuroblastoma (NB) patients, 13 controls and 11 children with inflammatory neurological diseases (IND). BAFF, but no APRIL, was elevated in the CSF of OMS children and IND children. However, in contrast to IND patients, OMS patients did not have a blood-brain-barrier disturbance, indicating that BAFF was produced intrathecally in OMS patients, but not in IND patients. CSF BAFF levels showed a correlation with CSF CGN autoantibodies (r(2)=0.58, p<0.05). These data indicate that an activated B-cell system in the cerebrospinal fluid is involved in the pathogenesis of OMS, and BAFF may be a candidate parameter for the activation of B-cell immune system.

  3. Porcine B-cell activating factor promotes anti-FMDV antibodies in vitro but not in vivo after DNA vaccination of pigs.

    PubMed

    Bergamin, Fabio; Saurer, Leslie; Neuhaus, Viviane; McCullough, Kenneth C; Summerfield, Artur

    2007-12-15

    'B-cell activating factor belonging to the TNF family' (BAFF) represents a cytokine produced by antigen presenting cells promoting B-cell maturation, activation and immunoglobulin class switching. In the present study, we demonstrate expression of BAFF on cultured monocyte-derived dendritic cells, which is further enhanced by interferon-alpha or interferon-gamma treatment. From these cells, porcine BAFF was cloned and the recombinant protein was expressed in mammalian cells with and without a FLAG tag at the carboxyl terminus. Only the protein without the FLAG tag was bioactive in vitro, and promoted B-cell survival and the differentiation of foot-and-mouth disease virus (FMDV)-specific memory B cells into antibody producing cells. Based on this result it was tested whether BAFF can enhance FMDV antibody responses in the context of a DNA vaccination. To this end, pigs were immunised with the anti-FMDV DNA vaccine plasmid pcDNA3.1/P1-2A3C3D and a pCI plasmid expressing porcine BAFF. Using a needle-free transdermal application method, also referred to as 'jet injection', pigs were vaccinated three times and their humoral response quantified by ELISA and a virus neutralisation test. After the third vaccination, three out of six animals vaccinated with the pcDNA3.1/P1-2A3C3D alone but none of the animals that also received the BAFF expressing plasmid had seroconverted. These data suggest that BAFF is not appropriate as a genetic adjuvant when applied as a simple co-injection with the antigen-encoding plasmid.

  4. ROS and ERK1/2-mediated caspase-9 activation increases XAF1 expression in dexamethasone-induced apoptosis of EBV-transformed B cells.

    PubMed

    Park, Ga Bin; Choi, Yunock; Kim, Yeong Seok; Lee, Hyun-Kyung; Kim, Daejin; Hur, Dae Young

    2013-07-01

    Dexamethasone (Dex) inhibits the growth of diverse types of cancer cells and is utilized clinically for the therapy of hematological malignancies. In this study, we investigated the molecular mechanisms of Dex action in the apoptosis of Epstein-Barr virus (EBV)-transformed B cells. We showed that Dex inhibited the proliferation of EBV-transformed B cells and induced apoptosis by activating caspase-9, -3 and -8. While activation of caspase-9 was triggered as early as 2 h after Dex treatment, cleavage of caspase-8 was deferred and was found 8 h after the exposure. Dex-dependent activation of caspase-8 was blocked by the specific caspase-9 inhibitor, z-LEHD-fmk. Moreover, Dex significantly increased the expression of X-linked inhibitor of apoptosis (XIAP)‑associated factor 1 (XAF1) and induced the translocation of XAF1 into the cytosol. Cytosolic XAF1 with Puma induced the translocation of Bax into mitochondria. Dex led to up-regulation of reactive oxygen species (ROS) generation and the phosphorylation of ERK1/2 after the exposure. We speculated that ROS generation might be the first event of Dex-induced apoptosis because ROS inhibitor NAC abrogated ROS production and ERK1/2 activation, but PD98059 did not block ROS production. NAC and PD98059 also suppressed the translocation of XAF1, Puma and Bax into mitochondria. These results demonstrated that Dex-mediated activation of caspase-9 via ROS generation and ERK1/2 pathway activation resulted in the activation of caspase-8 and the increment of XAF1, thereby induced apoptosis of EBV-transformed B cells. These findings suggest that Dex constitutes a probable therapy for EBV-associated hematological malignancies.

  5. Activation of MYC in a masked t(8;17) translocation results in an aggressive B-cell leukemia.

    PubMed Central

    Gauwerky, C E; Huebner, K; Isobe, M; Nowell, P C; Croce, C M

    1989-01-01

    We have analyzed the oncogene rearrangements involving BCL2 and MYC in the leukemia cells of a patient with an aggressive prolymphocytic leukemia that had an abnormal karyotype including a t(14;18) translocation and a chromosome 17q+. Molecular analysis showed that BCL2 was rearranged in the major breakpoint cluster region and had joined into the immunoglobulin heavy chain gene as in follicular lymphoma. Cloning and sequence analysis of the rearranged MYC gene revealed that MYC was truncated at the Pvu II site at the end of the first exon of MYC and had joined into the regulatory elements of a gene that we called BCL3 (B-cell leukemia/lymphoma 3). The BCL3 locus was mapped to chromosome 17 band q22. We found BCL3 transcribed as a message of 1.7 kilobases in many hematopoietic cell lines representing all hematopoietic lineages. In the patient's leukemia cells, the truncated MYC gene was highly expressed under the influence of BCL3 regulatory elements, leading to an aggressive B-cell leukemia that presumably had been derived from an indolent lymphoma carrying a rearranged BCL2 gene. Images PMID:2682663

  6. B cell Rab7 mediates induction of activation-induced cytidine deaminase expression and class-switching in T-dependent and T-independent antibody responses.

    PubMed

    Pone, Egest J; Lam, Tonika; Lou, Zheng; Wang, Rui; Chen, Yuhui; Liu, Dongfang; Edinger, Aimee L; Xu, Zhenming; Casali, Paolo

    2015-04-01

    Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation, CSR requires activation-induced cytidine deaminase (AID), whose expression is restricted to B cells, as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice, we identified a B cell-intrinsic role for Rab7, a small GTPase involved in intracellular membrane functions, in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription, as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed, in vivo and in vitro, normal proliferation and survival, normal Blimp-1 expression and plasma cell differentiation, as well as intact activation of the noncanonical NF-κB, p38 kinase, and ERK1/2 kinase pathways. They, however, were defective in AID expression and CSR in vivo and in vitro, as induced by CD40 engagement or dual TLR1/2-, TLR4-, TLR7-, or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells, CSR was rescued by enforced AID expression. These findings, together with our demonstration that Rab7-mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.

  7. Activation of intrahepatic CD4+CXCR5+ T and CD19+ B cells is associated with viral clearance in a mouse model of acute hepatitis B virus infection.

    PubMed

    Song, Xiao-Fei; Hu, Ting-Ting; Lei, Yu; Li, Hu; Zhang, Li; Zhang, Miao; Liu, Bin; Chen, Min; Hu, Huai-Dong; Ren, Hong; Hu, Peng

    2016-08-09

    The role of immunity in the pathogenesis of acute hepatitis B virus (HBV) infection is poorly understood. The purpose of this research was to define the intrahepatic immune factors responsible for viral clearance during acute HBV infection. The model of acute HBV infection was established by hydrodynamically transfecting mice with pCDNA3.1-HBV1.3 plasmids which contained a supergenomic HBV1.3-length transgene. The frequency of CD4+ CXCR5+ T cells, CD19+ B cells and their surface molecules in livers, spleens and peripheral blood were detected using flow cytometry. The lymphomononuclear cells isolated from the livers of transfected mice were further stimulated by HBc-derived peptides and then the frequency and cytokine secretion of HBV-specific CD4+CXCR5+ T cells were detected. We found that the frequency of CXCR5+ in CD4+ T cells was specifically increased; the expression of PD-1 was decreased while the expression of ICOS was increased on intrahepatic CD4+CXCR5+ T cells. Although the frequency of CD19+ B cells was not affected, the expression of PDL-1, ICOSL and IL-21R on B cells was increased in the livers of mice. The frequency of HBV-specific CD4+CXCR5+ T cells and the production of IL-21 by intrahepatic CD4+CXCR5+ T cells of mice with acute HBV infection were increased after stimulation. Furthermore, the expression of function-related molecules of intrahepatic CD4+CXCR5+ T, including Bcl-6, CXCR5, IL-6, IL-6R, IL-21 and IL-4 in the liver was increased during acute HBV infection. In conclusion, the activation of intrahepatic CD4+CXCR5+ T cells and B cells was associated with the clearance of HBV during acute infection.

  8. Requirements for distinct steps of phospholipase Cgamma2 regulation, membrane-raft-dependent targeting and subsequent enzyme activation in B-cell signalling.

    PubMed Central

    Rodriguez, Rosie; Matsuda, Miho; Storey, Amy; Katan, Matilda

    2003-01-01

    Studies of PLCgamma (phospholipase Cgamma) have identified a number of regulatory components required for signalling; however, molecular mechanisms and the relationship between events leading to translocation and an increase of substrate hydrolysis have not been well defined. The addition of a membrane-targeting tag to many signal transducers results in constitutive activation, suggesting that these processes could be closely linked and difficult to dissect. The present study of PLCgamma2 regulation by cross-linking of the BCR (B-cell antigen receptor) or H2O2 stress in DT40 B-cells, demonstrated that the membrane targeting is a separate step from further changes that result in enzyme activation and substrate hydrolysis. Furthermore, we have defined the roles of different domains of PLCgamma2 and, using a panel of cell lines deficient in components linked to PLCgamma2 regulation, the involvement of signalling molecules with respect to each of the steps. We have found that only the lipid-raft-targeted Lyn-PLCgamma2 construct, unlike non-specific membrane targeting, overcame the requirement for the adapter protein BLNK (B-cell linker). The stable expression of Lyn-PLCgamma2 was not accompanied by an increase in substrate hydrolysis in resting cells, which followed stimulation and specifically required the presence and/or activation of Syk, Btk, phosphoinositide 3-kinase but not BLNK, as established using deficient cell lines or specific inhibitors. Based on mutational analysis of the specific tyrosine residues [Tyr753-->Phe (Y753F)/Y759F] and SH2 (Src homology 2) domains (R564A/R672A) in the context of Lyn-PLCgamma2, we found that Tyr753/Tyr759 were essential, whereas the PLCgamma2 SH2 domains did not have an important role in the transient activation of Lyn-PLCgamma2 but may serve to stabilize an activated form in sustained activation. PMID:12780340

  9. Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B-cell acute lymphoblastic leukemia.

    PubMed

    Heltemes-Harris, L M; Larson, J D; Starr, T K; Hubbard, G K; Sarver, A L; Largaespada, D A; Farrar, M A

    2016-06-30

    Signal transducer and activator of transcription 5 (STAT5) activation occurs frequently in human progenitor B-cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b-CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the Janus kinase/STAT5 pathway (ii) progenitor B-cell differentiation and (iii) the CDKN2A tumor-suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, extracellular signal-regulated kinase (Erk) and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways have in B-ALL.

  10. A proliferation-inducing ligand sustains the proliferation of human naïve (CD27⁻) B cells and mediates their differentiation into long-lived plasma cells in vitro via transmembrane activator and calcium modulator and cyclophilin ligand interactor and B-cell mature antigen.

    PubMed

    Matsuda, Yoshiko; Haneda, Masataka; Kadomatsu, Kenji; Kobayashi, Takaaki

    2015-06-01

    Long-lived plasma cells (PCs) contribute to humoral immunity through an undefined mechanism. Memory B cells, but not human naïve B cells, can be induced to differentiate into long-lived PCs in vitro. Because evidence links a proliferation-inducing ligand (APRIL), a tumor necrosis factor family member, to the ability of bone marrow to mediate long-term PC survival, we reasoned that APRIL influences the proliferation and differentiation of naïve B cells. We describe here the development of a simple cell culture system that allowed us to show that APRIL sustained the proliferation of naïve human B cells and induced them to differentiate into long-lived PCs. Blocking the transmembrane activator and calcium modulator and cyclophilin ligand interactor or B-cell mature antigen shows they were required for the differentiation of naïve B cells into long-lived PCs in vitro. Our in vitro culture system will reveal new insights into the biology of long-lived PCs.

  11. Changes in Blood B Cell-Activating Factor (BAFF) Levels in Multiple Sclerosis: A Sign of Treatment Outcome

    PubMed Central

    Kannel, Karin; Alnek, Kristi; Vahter, Liina; Gross-Paju, Katrin; Uibo, Raivo; Kisand, Kalle V.

    2015-01-01

    Multiple sclerosis (MS) is mediated primarily by autoreactive T cells. However, evidence suggesting the involvement of humoral immunity in brain diseases has increased interest in the role of B cells and their products during MS pathogenesis. The major survival factor for B cells, BAFF has been shown to play a role in several autoimmune conditions. Elevated BAFF levels have been reported in MS animal model and during MS relapse in patients. Moreover, disease-modifying treatments (DMT) reportedly influence blood BAFF levels in MS patients, but the significance of these changes remains unclear. The present study addresses how blood BAFF levels are associated with the clinical course of relapsing-remitting MS and the effectiveness of DMT and short-term steroid treatment. During a prospective longitudinal follow-up of 2.3 years, BAFF was measured in the blood of 170 MS patients in the stable phase and within 186 relapses. BAFF levels were significantly higher in MS patients compared to healthy controls. However, stable MS patients without relapses exhibited significantly higher BAFF levels than relapsing patients. Treatment with interferon-β and immunosuppressants raised BAFF blood levels. Interestingly, a similar effect was not seen in patients treated with glatiramer acetate. Short-term treatment with high doses of intravenous methylprednisolone did not significantly alter plasma BAFF levels in 65% of relapsing-remitting MS patients. BAFF were correlated weakly but significantly with monocyte and basophil counts, but not with other blood cell types (neutrophils, lymphocytes, or eosinophils) or inflammatory biomarkers. To our knowledge, this is the first report demonstrating that higher blood BAFF levels may reflect a more stable and effective MS treatment outcome. These results challenge hypotheses suggesting that elevated blood BAFF levels are associated with more severe disease presentation and could explain the recent failure of pharmaceutical trials targeting

  12. Ibrutinib Before and After Stem Cell Transplant in Treating Patients With Relapsed or Refractory Diffuse Large B-cell Lymphoma

    ClinicalTrials.gov

    2017-04-11

    Activated B-Cell-Like Diffuse Large B-Cell Lymphoma; B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma; Diffuse Large B-Cell Lymphoma, Not Otherwise Specified; Recurrent Diffuse Large B-Cell Lymphoma; Refractory Diffuse Large B-Cell Lymphoma

  13. STAT3 activation is associated with cerebrospinal fluid interleukin-10 (IL-10) in primary central nervous system diffuse large B cell lymphoma.

    PubMed

    Mizowaki, Takashi; Sasayama, Takashi; Tanaka, Kazuhiro; Mizukawa, Katsu; Takata, Kumi; Nakamizo, Satoshi; Tanaka, Hirotomo; Nagashima, Hiroaki; Nishihara, Masamitsu; Hirose, Takanori; Itoh, Tomoo; Kohmura, Eiji

    2015-09-01

    Signal transducers and activators of transcription 3 (STAT3) are activated by various cytokines and oncogenes; however, the activity and pathogenesis of STAT3 in diffuse large B cell lymphoma of the central nervous system have not been thoroughly elucidated. We investigated the phosphorylation levels of STAT3 in 40 specimens of primary central nervous system diffuse large B-cell lymphoma (PCNS DLBCL) and analyzed the association between phsopho-STAT3 (pSTAT3) expression and cerebrospinal fluid (CSF) concentration of interleukin-10 (IL-10) or IL-6. Immunohistochemistry and Western blot analysis revealed that most of the specimens in PCNS DLBCL expressed pSTST3 protein, and a strong phosphorylation levels of STAT3 was statistically associated with high CSF IL-10 levels, but not with CSF IL-6 levels. Next, we demonstrated that recombinant IL-10 and CSF containing IL-10 induced the phosphorylation of STAT3 in PCNS DLBCL cells. Furthermore, molecular subtype classified by Hans' algorithm was correlated with pSTAT3 expression levels and CSF IL-10 levels. These results suggest that the STAT3 activity is correlated with CSF IL-10 level, which is a useful marker for STAT3 activity in PCNS DLBCLs.

  14. Differential activation of the inflammasome in THP-1 cells exposed to chrysotile asbestos and Libby "six-mix" amphiboles and subsequent activation of BEAS-2B cells.

    PubMed

    Li, Muyao; Gunter, Mickey E; Fukagawa, Naomi K

    2012-12-01

    Inflammatory responses of THP-1 cells (macrophage cell line) exposed to chrysotile asbestos (Chry) and Libby six-mix (LIB) and the subsequent impact on bronchial epithelial cells were determined. Direct treatment of THP-1 cells with Chry caused cell death, activation of caspase-1 and release of IL-1β, while the addition of caspase-1 inhibitor, Z-YVAD-FMK, reduced IL-1β, suggesting that Chry activated the caspase-1 mediated Nod-like receptor protein 3 (NLRP3) inflammasome; by comparison, LIB had less effects on all of these parameters. Expression of antioxidant enzymes, protein oxidation and nitration, and lipid peroxides in THP-1 cells treated with the two particles suggest that LIB generated more reactive oxygen species (ROS) than the same dose of Chry. Differences in fiber length and surface area suggest a possible role for particulate size in the differential activation of the inflammasome. BEAS-2B cells, representing the bronchial epithelium, treated with supernatants of medium from Chry- or LIB-treated THP-1 cells (conditioned medium) activated the MAPK cascade, increased phosphorylation of ERK and Cot (MAP3K8), increased AP-1 binding activity and induced IL-6 release. To verify that IL-1β from THP-1 cells was responsible for activation of BEAS-2B, conditioned medium with added IL-1Ra, an IL-1β antagonist, was applied to BEAS-2B. Results show that IL-1Ra attenuated effects of conditioned medium, supporting a role of IL-1β, as a secondary mediator, in the transduction of inflammatory signaling from the macrophage to epithelial cells. The effects of LIB-conditioned medium appeared to be less dependent on IL-1β. In conclusion, Chry and LIB induce differential inflammatory responses in THP-1 cells that subsequently lead to differential effects in epithelial cells.

  15. Interference with immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation modulates or blocks B cell development, depending on the availability of an Igbeta cytoplasmic tail.

    PubMed

    Kraus, M; Pao, L I; Reichlin, A; Hu, Y; Canono, B; Cambier, J C; Nussenzweig, M C; Rajewsky, K

    2001-08-20

    To determine the function of immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, we generated mice in which Igalpha ITAM tyrosines were replaced by phenylalanines (Igalpha(FF/FF)). Igalpha(FF/FF) mice had a specific reduction of B1 and marginal zone B cells, whereas B2 cell development appeared to be normal, except that lambda1 light chain usage was increased. The mutants responded less efficiently to T cell-dependent antigens, whereas T cell-independent responses were unaffected. Upon B cell receptor ligation, the cells exhibited heightened calcium flux, weaker Lyn and Syk tyrosine phosphorylation, and phosphorylation of Igalpha non-ITAM tyrosines. Strikingly, when the Igalpha ITAM mutation was combined with a truncation of Igbeta, B cell development was completely blocked at the pro-B cell stage, indicating a crucial role of ITAM phosphorylation in B cell development.

  16. Abnormal kappa:lambda light chain ratio in circulating immune complexes as a marker for B cell activity in juvenile idiopathic arthritis.

    PubMed

    Low, J M; Chauhan, A K; Moore, T L

    2007-01-01

    Patients with juvenile idiopathic arthritis (JIA) have been shown to have elevated levels of circulating immune complexes (CICs) which correlated with disease activity. Our aim was to assess B cell activity by measuring the amount of and the kappa:lambda chain immunoglobulin light (L) chain ratio in CICs from JIA patients and to determine potential evidence for either an antigen-driven response or B-cell receptor editing. We used an enzyme-linked immunosorbent assay to measure kappa and lambda chains present in the CICs from the sera of patients with JIA. Statistical analysis was performed using Pearson's correlation, one-way ANOVA and Bonferroni post hoc analysis. Sera from 44 JIA patients were examined for the concentration of L chains in CICs. Healthy controls had a kappa:lambda chain ratio of 1.2:1, whereas this ratio was reversed among JIA subgroups with RF-positive polyarthritis (1:1.2), RF-negative polyarthritis (1:1.3), oligoarthritis (1:2.3) and systemic-onset arthritis (1:2.5). In addition, overall lambda chain selection was not significantly associated with a particular immunoglobulin heavy (H) chain and occurred with all immunoglobulin isotypes. We showed preferential selection of lambda chains contributing to the formation of potentially pathogenic CICs from JIA patients, of all onset types compared to healthy controls, in an H chain-independent manner. The reversal of kappa:lambda chain ratio within the JIA CICs and association with all immunoglobulin isotypes demonstrated the potential for L chain editing. Furthermore, we conclude that a reversal of the normal kappa:lambda chain ratio in JIA CICs may be used as a marker for increased B-cell activity.

  17. Ligation of CD47 induces G1 arrest in EBV-transformed B cells through ROS generation, p38 MAPK/JNK activation, and Tap73 upregulation.

    PubMed

    Park, Ga Bin; Bang, Si Ra; Lee, Hyun-Kyung; Kim, Daejin; Kim, Seonghan; Kim, Jin Kyoung; Kim, Yeong Seok; Hur, Dae Young

    2014-01-01

    CD47 is expressed in normal activated cells as well as in several tumors. It also has been implicated as having antiangiogenic and antimetastatic properties, but its roles in Epstein-Barr virus (EBV)-transformed B cells are still not fully understood. Herein, we report that EBV infection induced CD47 surface expression on B cells, and CD47 ligation with anti-CD47 mAb (B6H12) reduced cell proliferation and induced G1 arrest. CD47-induced G1 arrest was mediated through increased cyclin-dependent kinase inhibitors (CDKi) and a simultaneously decreased CDK/cyclins, and p38 MAPK/JNK activation preceded binding of CDKi-CDK. Moreover, reactive oxygen species (ROS) generation and upregulation of both TAp73 and ER stress sensor proteins were detected after CD47 ligation, and p38 inhibitor SB203580 and JNK inhibitor SP600125 blocked upregulation of TAp73 and cell cycle arrest. We investigated whether ROS generation is the initial event of CD47-mediated G1 arrest because ROS scavenger NAC effectively abrogated the majority of CD47-mediated responses but SB203580 and SP600125 did not block ROS production. Taken together, we concluded that CD47 ligation on EBV-transformed B cells led to G1 arrest by ROS generation and, subsequently, there was p38 MAPK/JNK pathway activation, ER stress triggering, and TAp73 upregulation. Our findings provide data supporting CD47 as a feasible target for EBV-associated tumor therapy.

  18. The ABC of Physical Activity for Health: a consensus statement from the British Association of Sport and Exercise Sciences.

    PubMed

    O'Donovan, Gary; Blazevich, Anthony J; Boreham, Colin; Cooper, Ashley R; Crank, Helen; Ekelund, Ulf; Fox, Kenneth R; Gately, Paul; Giles-Corti, Billie; Gill, Jason M R; Hamer, Mark; McDermott, Ian; Murphy, Marie; Mutrie, Nanette; Reilly, John J; Saxton, John M; Stamatakis, Emmanuel

    2010-04-01

    Our understanding of the relationship between physical activity and health is constantly evolving. Therefore, the British Association of Sport and Exercise Sciences convened a panel of experts to review the literature and produce guidelines that health professionals might use. In the ABC of Physical Activity for Health, A is for All healthy adults, B is for Beginners, and C is for Conditioned individuals. All healthy adults aged 18-65 years should aim to take part in at least 150 min of moderate-intensity aerobic activity each week, or at least 75 min of vigorous-intensity aerobic activity per week, or equivalent combinations of moderate- and vigorous-intensity activities. Moderate-intensity activities are those in which heart rate and breathing are raised, but it is possible to speak comfortably. Vigorous-intensity activities are those in which heart rate is higher, breathing is heavier, and conversation is harder. Aerobic activities should be undertaken in bouts of at least 10 min and, ideally, should be performed on five or more days a week. All healthy adults should also perform muscle-strengthening activities on two or more days a week. Weight training, circuit classes, yoga, and other muscle-strengthening activities offer additional health benefits and may help older adults to maintain physical independence. Beginners should work steadily towards meeting the physical activity levels recommended for all healthy adults. Even small increases in activity will bring some health benefits in the early stages and it is important to set achievable goals that provide success, build confidence, and increase motivation. For example, a beginner might be asked to walk an extra 10 min every other day for several weeks to slowly reach the recommended levels of activity for all healthy adults. It is also critical that beginners find activities they enjoy and gain support in becoming more active from family and friends. Conditioned individuals who have met the physical

  19. Production of RANKL by Memory B Cells

    PubMed Central

    Meednu, Nida; Zhang, Hengwei; Owen, Teresa; Sun, Wen; Wang, Victor; Cistrone, Christopher; Rangel-Moreno, Javier; Xing, Lianping; Anolik, Jennifer H.

    2016-01-01

    Objective Rheumatoid arthritis (RA) is a systemic autoimmune disease that often leads to joint damage. The mechanisms of bone damage in RA are complex, involving activation of bone-resorbing osteoclasts (OCs) by synoviocytes and Th17 cells. This study was undertaken to investigate whether B cells play a direct role in osteoclastogenesis through the production of RANKL, the essential cytokine for OC development. Methods RANKL production by total B cells or sorted B cell subpopulations in the peripheral blood and synovial tissue from healthy donors or anti–cyclic citrullinated peptide–positive patients with RA was examined by flow cytometry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis. To define direct effects on osteoclastogenesis, B cells were cocultured with CD14+ monocytes, and OCs were enumerated by tartrate-resistant acid phosphatase staining. Results Healthy donor peripheral blood B cells were capable of expressing RANKL upon stimulation, with switched memory B cells (CD27+IgD−) having the highest propensity for RANKL production. Notably, switched memory B cells in the peripheral blood from RA patients expressed significantly more RANKL compared to healthy controls. In RA synovial fluid and tissue, memory B cells were enriched and spontaneously expressed RANKL, with some of these cells visualized adjacent to RANK+ OC precursors. Critically, B cells supported OC differentiation in vitro in a RANKL-dependent manner, and the number of OCs was higher in cultures with RA B cells than in those derived from healthy controls. Conclusion These findings reveal the critical importance of B cells in bone homeostasis and their likely contribution to joint destruction in RA. PMID:26554541

  20. MYSM1-dependent checkpoints in B cell lineage differentiation and B cell-mediated immune response.

    PubMed

    Förster, Michael; Farrington, Kyo; Petrov, Jessica C; Belle, Jad I; Mindt, Barbara C; Witalis, Mariko; Duerr, Claudia U; Fritz, Jörg H; Nijnik, Anastasia

    2017-03-01

    MYSM1 is a chromatin-binding histone deubiquitinase. MYSM1 mutations in humans result in lymphopenia whereas loss of Mysm1 in mice causes severe hematopoietic abnormalities, including an early arrest in B cell development. However, it remains unknown whether MYSM1 is required at later checkpoints in B cell development or for B cell-mediated immune responses. We analyzed conditional mouse models Mysm1(fl/fl)Tg.mb1-cre, Mysm1(fl/fl)Tg.CD19-cre, and Mysm1(fl/fl)Tg.CD21-cre with inactivation of Mysm1 at prepro-B, pre-B, and follicular B cell stages of development. We show that loss of Mysm1 at the prepro-B cell stage in Mysm1(fl/fl)Tg.mb1-cre mice results in impaired B cell differentiation, with an ∼2-fold reduction in B cell numbers in the lymphoid organs. Mysm1(fl/fl)Tg.mb1-cre B cells also showed increased expression of activation markers and impaired survival and proliferation. In contrast, Mysm1 was largely dispensable from the pre-B cell stage onward, with Mysm1(fl/fl)Tg.CD19-cre and Mysm1(fl/fl)Tg.CD21-cre mice showing no alterations in B cell numbers and largely normal responses to stimulation. MYSM1, therefore, has an essential role in B cell lineage specification but is dispensable at later stages of development. Importantly, MYSM1 activity at the prepro-B cell stage of development is important for the normal programming of B cell responses to stimulation once they complete their maturation process.

  1. Dissociation of severe lupus-like disease from polyclonal B cell activation and IL 2 deficiency in C3H-lpr/lpr mice.

    PubMed

    Davidson, W F; Roths, J B; Holmes, K L; Rudikoff, E; Morse, H C

    1984-08-01

    The lymphoproliferation characteristic of all strains of mice homozygous for the gene lpr results from the expansion of an unusual subset of cells that express reduced levels of Ly-1 and Thy-1 antigens and high levels of Ly-5(B220), an antigen that is normally only detected on cells of the B lineage. In the present study, C3H-lpr mice were studied to determine when this population of cells first appears in lymph node (LN) and spleen and whether its appearance relates to the development of B cell activation and deficiencies in interleukin 2 (IL 2) production. The results showed that Ly-5(B220)+, sIg- cells were first detected in LN at 4 wk of age; thereafter their numbers increased exponentially until at 16 wk of age they represented more than 80% of LN cells. Two subpopulations of Ly-5(B220)+, sIg- cells were present in LN; one Ly-1+, Thy-1+ and the other Ly-1+, Thy-1-. Ly-5(B220)+, sIg- cells were not detected in C3H-lpr spleen until 6 to 10 wks after their appearance in LN, and their proportions never reached those in LN. Polyclonal B cell activation in C3H-lpr spleens was not observed until Ly-5(B220)+, sIg- cells were present, suggesting that this population may play a role in B cell stimulation. IL 2 production by C3H-lpr spleen and LN cells was normal up to 6 wk of age and was significantly impaired thereafter, with LN being more severely affected than spleen. The IL 2 defect could be significantly repaired by the addition of PMA to the cultures. Although defective IL 2 production coincided with the appearance of Ly-5(B220)+, sIg- cells in LN, it preceded the appearance of these cells in spleen. In spite of the impaired ability to produce IL 2 in vitro, CTL responses to alloantigens were normal. Although C3H-lpr mice share many of the lymphoid abnormalities observed in MRL-lpr mice, they do not develop severe, early-onset SLE-like disease characteristic of the latter strain. This suggests that factors other than defective IL 2 production and polyclonal B cell

  2. Regulatory B cells in autoimmune diseases

    PubMed Central

    Yang, Min; Rui, Ke; Wang, Shengjun; Lu, Liwei

    2013-01-01

    B cells are generally considered to be positive regulators of the immune response because of their capability to produce antibodies, including autoantibodies. The production of antibodies facilitates optimal CD4+ T-cell activation because B cells serve as antigen-presenting cells and exert other modulatory functions in immune responses. However, certain B cells can also negatively regulate the immune response by producing regulatory cytokines and directly interacting with pathogenic T cells via cell-to-cell contact. These types of B cells are defined as regulatory B (Breg) cells. The regulatory function of Breg cells has been demonstrated in mouse models of inflammation, cancer, transplantation, and particularly in autoimmunity. In this review, we focus on the recent advances that lead to the understanding of the development and function of Breg cells and the implications of B cells in human autoimmune diseases. PMID:23292280

  3. CsBAFF, a Teleost B Cell Activating Factor, Promotes Pathogen-Induced Innate Immunity and Vaccine-Induced Adaptive Immunity

    PubMed Central

    Sun, Yun; Sun, Li

    2015-01-01

    B cell activating factor (BAFF) is a member of the tumor necrosis factor family that is known to play an important role in B cell activation, proliferation, and differentiation in mammals. However, studies of BAFF in teleosts are very limited and its function, in particular that under in vivo conditions, is essentially unknown. In this study, we conducted in vivo as well as in vitro functional analyses of a BAFF homologue (CsBAFF) from the teleost fish tongue sole (Cynoglossus semilaevis). CsBAFF is composed of 261 residues and shares moderate sequence identities with known BAFFs of other teleosts. CsBAFF expression was most abundant in immune organs and was upregulated during bacterial infection. Purified recombinant CsBAFF (rCsBAFF) bound to tongue sole lymphocytes and promoted cellular proliferation and survival. The results of an in vivo study showed that CsBAFF overexpression in tongue sole significantly enhanced macrophage activation and reduced bacterial infection in fish tissues, whereas knockdown of CsBAFF expression resulted in increased bacterial dissemination and colonization in fish tissues. Furthermore, vaccination studies showed that CsBAFF enhanced the immunoprotection of a DNA vaccine and augmented the production of specific serum antibodies. Taken together, these results provide the first in vivo evidence to indicate that teleost BAFF is an immunostimulator that significantly contributes to the innate antibacterial immune response and vaccine-induced adaptive immune response. PMID:26295165

  4. The pseudo-immunoreceptor tyrosine-based activation motif of CD5 mediates its inhibitory action on B-cell receptor signaling.

    PubMed

    Gary-Gouy, H; Bruhns, P; Schmitt, C; Dalloul, A; Daëron, M; Bismuth, G

    2000-01-07

    Genetic studies revealed that CD5 could be a negative regulator of the B-cell antigen receptor (BCR). We explore here the effect of human CD5 on BCR-triggered responses. B cells were obtained expressing a chimera composed of extracellular and transmembrane domains of Fcgamma type IIB receptor fused to CD5 cytoplasmic domain (CD5cyt). Coligation of the chimera with the BCR induces CD5cyt tyrosine phosphorylation. A rapid inhibition of BCR-induced calcium response is observed, as well as a partial but delayed inhibition of phospholipase Cgamma-1 phosphorylation. Activation of extracellular regulated kinase-2 is also severely impaired. Moreover, at the functional level, interleukin-2 production is abolished. Src homology 2 domain-bearing tyrosine phosphatase SHP-1 and Src homology 2 domain-bearing inositol 5'-phosphatase SHIP usually participate in negative regulation of the BCR. We show that they do not associate with the phosphorylated CD5 chimera. We finally demonstrate that the pseudo-immunoreceptor tyrosine based activation motif present in CD5cyt is involved because its deletion eliminates the inhibitory effect of the chimera, both at biochemical and functional levels. These results demonstrate the inhibitory role of CD5 pseudo-immunoreceptor tyrosine based activation motif tyrosine phosphorylation on BCR signaling. They further support the idea that CD5 uses mechanisms different from those already described to negatively regulate the BCR pathway.

  5. Therapeutic effects of the artemisinin analog SM934 on lupus-prone MRL/lpr mice via inhibition of TLR-triggered B-cell activation and plasma cell formation

    PubMed Central

    Wu, Yanwei; He, Shijun; Bai, Bingxin; Zhang, Luyao; Xue, Lu; Lin, Zemin; Yang, Xiaoqian; Zhu, Fenghua; He, Peilan; Tang, Wei; Zuo, Jianping

    2016-01-01

    We previously reported that SM934, a water-soluble artemisinin derivative, was a viable treatment in murine lupus models. In the current study, we further investigated the therapeutic effects of a modified dosage regimen of SM934 on lupus-prone MRL/lpr mice and explored its effects on B cell responses, a central pathogenic event in systemic lupus erythematosus (SLE). When orally administered twice-daily, SM934 significantly prolonged the life-span of MRL/lpr mice, ameliorated the lymphadenopathy symptoms and decreased the levels of serum anti-nuclear antibodies (ANAs) and of the pathogenic cytokines IL-6, IL-10 and IL-21. Furthermore, SM934 treatment restored the B-cell compartment in the spleen of MRL/lpr mice by increasing quiescent B cell numbers, maintaining germinal center B-cell numbers, decreasing activated B cell numbers and reducing plasma cell (PC) numbers. Ex vivo, SM934 suppressed the Toll-like receptor (TLR)-triggered activation and proliferation of B cells, as well as antibody secretion. Moreover, the present study demonstrated that SM934 interfered with the B-cell intrinsic pathway by downregulating TLR7/9 mRNA expression, MyD88 protein expression and NF-κB phosphorylation. In human peripheral blood mononuclear cells (PBMCs), consistent with the results in MRL/lpr mice, SM934 inhibited TLR-associated B-cell activation and PC differentiation. In conclusion, a twice daily dosing regimen of SM934 had therapeutic effects on lupus-prone MRL/lpr mice by suppressing B cell activation and plasma cell formation. PMID:25942599

  6. A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*

    PubMed Central

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-01-01

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. PMID:25533467

  7. A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

    PubMed

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-02-20

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.

  8. Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function

    PubMed Central

    Simpson, Brent W.; Owens, Tristan W.; Orabella, Matthew J.; Davis, Rebecca M.; May, Janine M.; Trauger, Sunia A.

    2016-01-01

    ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. PMID:27795402

  9. Central nervous system infiltrates are characterized by features of ongoing B cell-related immune activity in MP4-induced experimental autoimmune encephalomyelitis.

    PubMed

    Batoulis, Helena; Wunsch, Marie; Birkenheier, Johannes; Rottlaender, Andrea; Gorboulev, Valentin; Kuerten, Stefanie

    2015-05-01

    In multiple sclerosis (MS) lymphoid follicle-like aggregates have been reported in the meninges of patients. Here we investigated the functional relevance of B cell infiltration into the central nervous system (CNS) in MP4-induced experimental autoimmune encephalomyelitis (EAE), a B cell-dependent mouse model of MS. In chronic EAE, B cell aggregates were characterized by the presence of CXCL13(+) and germinal center CD10(+) B cells. Germline transcripts were expressed in the CNS and particularly related to TH17-associated isotypes. We also observed B cells with restricted VH gene usage that differed from clones found in the spleen. Finally, we detected CNS-restricted spreading of the antigen-specific B cell response towards a myelin and a neuronal autoantigen. These data imply the development of autonomous B cell-mediated autoimmunity in the CNS in EAE - a concept that might also apply to MS itself.

  10. DNA-like class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Faslpr/lpr mice in vivo

    PubMed Central

    Lenert, Petar; Yasuda, Kei; Busconi, Liliana; Nelson, Patrice; Fleenor, Courtney; Ratnabalasuriar, Radhika S; Nagy, Peter L; Ashman, Robert F; Rifkin, Ian R; Marshak-Rothstein, Ann

    2009-01-01

    Introduction B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion. Methods We have developed a new generation of DNA-like compounds named class R inhibitory oligonucleotides (INH-ODNs). We tested their effectiveness in autoimmune B cells and interferon-alpha-producing dendritic cells in vitro and in lupus-prone MRL-Faslpr/lpr mice in vivo. Results Class R INH-ODNs have 10- to 30-fold higher inhibitory potency when autoreactive B cells are synergistically activated through the BCR and associated TLR7 or 9 than when stimulation occurs via non-BCR-engaged TLR7/9. Inhibition of TLR9 requires the presence of both CCT and GGG triplets in an INH-ODN, whereas the inhibition of the TLR7 pathway appears to be sequence-independent but dependent on the phosphorothioate backbone. This difference was also observed in the MRL-Faslpr/lpr mice in vivo, where the prototypic class R INH-ODN was more effective in curtailing abnormal autoantibody secretion and prolonging survival. Conclusions The increased potency of class R INH-ODNs for autoreactive B cells and dendritic cells may be beneficial for lupus patients by providing pathway-specific inhibition yet allowing them to generate protective immune response when needed. PMID:19476613

  11. Memory B cells.

    PubMed

    Kurosaki, Tomohiro; Kometani, Kohei; Ise, Wataru

    2015-03-01

    The immune system can remember a previously experienced pathogen and can evoke an enhanced response to reinfection that depends on memory lymphocyte populations. Recent advances in tracking antigen-experienced memory B cells have revealed the existence of distinct classes of cells that have considerable functional differences. Some of these differences seem to be determined by the stimulation history during memory cell formation. To induce rapid recall antibody responses, the contributions of other types of cells, such as memory T follicular helper cells, have also now begun to be appreciated. In this Review, we discuss these and other recent advances in our understanding of memory B cells, focusing on the underlying mechanisms that are required for rapid and effective recall antibody responses.

  12. Surrogate or conventional light chains are required for membrane immunoglobulin mu to activate the precursor B cell transition [published erratum appears in J Exp Med 1997 Jan 6;185(1):183

    PubMed Central

    1996-01-01

    To examine the role of light chains in early B cell development we combined RAG-1 and lambda 5 mutations to produce mice that expressed neither conventional nor surrogate light chains (RAG-1-/-, lambda 5-/- ). Unique heavy and light chain genes were then introduced into the double and single mutant backgrounds. Membrane immunoglobulin (Ig)mu (mIg mu) associated with Ig alpha-Ig beta but was unable to activate the pre-B cell transition in RAG-1-/-lambda 5-/- mice. Either lambda 5 or kappa light chains were sufficient to complement this deficiency. Therefore light chains are absolutely required for a functional Ig signaling module in early B cell development. Our data provide direct evidence for the existence of two pathways for induction of early B cell development: one which is activated through surrogate light chains and mIg mu, and an alternative pathway which uses conventional light chains and mIg mu. PMID:8920890

  13. Proteins combination on PHBV microsphere scaffold to regulate Hep3B cells activity and functionality: a model of liver tissue engineering system.

    PubMed

    Zhu, Xin Hao; Gan, Seng Keat; Wang, Chi-Hwa; Tong, Yen Wah

    2007-12-01

    The synergistic effects of extracellular matrix (ECM) protein combinations on Hep3B cell proliferation and functions are studied herein. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres were covalently conjugated with three types of proteins, collagen (type I), laminin, and fibronectin, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide cross linkers. Successful conjugations of protein molecules were verified by the presence of nitrogen peaks in X-ray photoelectron spectroscopy. The densities of grafted proteins were quantified using Micro-BCA kit. A human hepatoma cell line, Hep3B, was then cultured in vitro on the ECM proteins-modified microspheres for 2 weeks. Cell proliferation was estimated using MTT method, and two hepatic functions, albumin secretion and P-450 activity, were evaluated using ELISA and EROD assays, respectively. The results indicated that combination of the three ECM proteins on microsphere surfaces has a significant effect on the proliferation of Hep3B cells, thus better mimicking the in vivo environment for liver tissue engineering.

  14. Macrophages in T cell/histiocyte rich large B cell lymphoma strongly express metal-binding proteins and show a bi-activated phenotype.

    PubMed

    Hartmann, Sylvia; Tousseyn, Thomas; Döring, Claudia; Flüchter, Patricia; Hackstein, Holger; Herreman, An; Ponzoni, Maurilio; de Wolf-Peeters, Chris; Facchetti, Fabio; Gascoyne, Randy D; Küppers, Ralf; Steidl, Christian; Hansmann, Martin-Leo

    2013-12-01

    Abundant macrophage infiltration in tumors often correlates with a poor prognosis. T cell/histiocyte rich large B cell lymphoma (THRLBCL) is a distinct aggressive B cell lymphoma entity showing a high macrophage content. To further elucidate the role of tumor-associated macrophages in THRLBCL, we performed gene expression profiling of microdissected histiocyte subsets of THRLBCL, nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), Piringer lymphadenitis, sarcoidosis, nonspecific lymphadenitis and monocytes from peripheral blood. In a supervised principal component analysis, histiocytes from THRLBCL were most closely related to epithelioid cells from NLPHL, with both types of cells expressing genes related to proinflammatory and regulatory macrophage activity. Moreover, histiocytes from THRLBCL strongly expressed metal-binding proteins like MT2A, by which histiocytes of THRLBCL can be distinguished from the other histiocyte subsets investigated. Interestingly, the validation at the protein level showed a strong expression of TXN, CXCL9, MT2A and SOD2 not only in macrophages of THRLBCL but also in the tumor cells of NLPHL and classical Hodgkin lymphoma (cHL). Overall, the present findings indicate that macrophages in the microenvironment of THRLBCL have acquired a distinct gene expression pattern that is characterized by a mixed M1/M2 phenotype and a strong expression of several metal binding proteins. The microenvironments in NLPHL and THRLBCL appear to have a similar influence on the macrophage phenotype. The high expression of metal binding proteins in histiocytes of THRLBCL may be diagnostically useful, but a potential pathophysiological role remains to be identified.

  15. Cadmium induces carcinogenesis in BEAS-2B cells through ROS-dependent activation of PI3K/AKT/GSK-3β/β-catenin signaling

    SciTech Connect

    Son, Young-Ok; Wang, Lei; Poyil, Pratheeshkumar; Budhraja, Amit; Hitron, J. Andrew; Zhang, Zhuo; Lee, Jeong-Chae; Shi, Xianglin

    2012-10-15

    Cadmium has been widely used in industry and is known to be carcinogenic to humans. Although it is widely accepted that chronic exposure to cadmium increases the incidence of cancer, the mechanisms underlying cadmium-induced carcinogenesis are unclear. The main aim of this study was to investigate the role of reactive oxygen species (ROS) in cadmium-induced carcinogenesis and the signal transduction pathways involved. Chronic exposure of human bronchial epithelial BEAS-2B cells to cadmium induced cell transformation, as evidenced by anchorage-independent growth in soft agar and clonogenic assays. Chronic cadmium treatment also increased the potential of these cells to invade and migrate. Injection of cadmium-stimulated cells into nude mice resulted in the formation of tumors. In contrast, the cadmium-mediated increases in colony formation, cell invasion and migration were prevented by transfection with catalase, superoxide dismutase-1 (SOD1), or SOD2. In particular, chronic cadmium exposure led to activation of signaling cascades involving PI3K, AKT, GSK-3β, and β-catenin and transfection with each of the above antioxidant enzymes markedly inhibited cadmium-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the cadmium-mediated increase in total and active β-catenin proteins and colony formation. Moreover, there was a marked induction of AKT, GSK-3β, β-catenin, and carcinogenic markers in tumor tissues formed in mice after injection with cadmium-stimulated cells. Collectively, our findings suggest a direct involvement of ROS in cadmium-induced carcinogenesis and implicate a role of AKT/GSK-3β/β-catenin signaling in this process. -- Highlights: ► Chronic exposure to cadmium induces carcinogenic properties in BEAS-2B cells. ► ROS involved in cadmium-induced tumorigenicity of BEAS-2B cells. ► Cadmium activates ROS-dependent AKT/GSK-3β/β-catenin-mediated signaling. ► ROS

  16. Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

    PubMed

    Grigoryev, Yevgeniy A; Kurian, Sunil M; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M; Kantor, Aaron B; Marsh, Christopher; Salomon, Daniel R

    2010-10-14

    A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

  17. ABC transporters: bacterial exporters.

    PubMed Central

    Fath, M J; Kolter, R

    1993-01-01

    The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review. PMID:8302219

  18. The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation.

    PubMed

    Gstaiger, M; Georgiev, O; van Leeuwen, H; van der Vliet, P; Schaffner, W

    1996-06-03

    We have shown previously that both octamer binding transcription factors, namely the ubiquitous Oct-1 and the B cell-specific Oct-2A protein, can be enhanced in transcriptional activity by their association with the B cell-specific coactivator protein Bob1, also called OBF-1 or OCA-B. Here we study the structural requirements for ternary complex formation of DNA-Oct-Bob1 and coactivation function of Bob1. In analogy to DNA-bound transcription factors, Bob1 has a modular structure that includes an interaction domain (amino acids 1-65) and a C-terminal domain (amino acids 65-256), both important for transcriptional activation. A mutational analysis has resolved a region of seven amino acids (amino acids 26-32) in the N-terminus of Bob1 that are important for contacting the DNA binding POU domain of Oct-1 or Oct-2. In contrast to the viral coactivator VP16 (vmw65), which interacts with Oct-1 via the POU homeosubdomain, Bob1 association with Oct factors requires residues located in the POU-specific subdomain. Because the same residues are also involved in DNA recognition, we surmised that this association would affect the DNA binding specificity of the Oct-Bob1 complex compared with free Oct factors. While Oct-1 or Oct-2 bind to a large variety of octamer sequences, Bob1 ternary complex formation is indeed highly selective and occurs only in a subset of these sequences, leading to the differential coactivation of octamer-containing promoters. The results uncover a new level in selectivity that furthers our understanding in the regulation of cell type-specific gene expression.

  19. Trimeric G protein-CARMA1 axis links smoothened, the hedgehog receptor transducer, to NF-κB activation in diffuse large B-cell lymphoma.

    PubMed

    Qu, Changju; Liu, Yadong; Kunkalla, Kranthi; Singh, Rajesh R; Blonska, Marzenna; Lin, Xin; Agarwal, Nitin Kumar; Vega, Francisco

    2013-06-06

    Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Aberrant activation of Hedgehog (Hh) and nuclear factor (NF)-κB pathways is ubiquitously observed and known to mediate tumor growth, survival, and chemoresistance in DLBCL. Here, we find that activation of Hh signaling is positively correlated with NF-κB pathway in DLBCL tumors, and that smoothened (SMO), the signal transducer subunit of Hh pathway, contributes to NF-κB activation through recruiting G protein subunits Gαi and Gα12 to activate PKCβ/CARMA1/TRAF6/NEMO signaling axis followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO. Moreover, functional inhibition of SMO enhances the cytotoxic effects of NF-κB inhibitor. Altogether, our study reveals a noncanonical Hh signaling pathway in which SMO activates trimeric G proteins and CARMA1-associated signaling complex, leading to NF-κB activation. This signaling cascade contributes to the survival of DLBCL and may serve as a potential target for combination therapies in DLBCL.

  20. IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

    SciTech Connect

    Li, Yan; Ohms, Stephen J.; Shannon, Frances M.; Sun, Chao; Fan, Jun Y.

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer DNA methylation is dynamic and flexible and changes rapidly upon cell activation. Black-Right-Pointing-Pointer DNA methylation controls the inducible gene expression in a given cell type. Black-Right-Pointing-Pointer Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

  1. A defined fragment of bacterial protein I (OmpF) is a polyclonal B-cell activator.

    PubMed Central

    Vordermeier, M; Stäb, K; Bessler, W G

    1986-01-01

    Protein I from the outer membrane of Escherichia coli and other members of the family Enterobacteriaceae is a potent mitogen and polyclonal B-lymphocyte activator. To determine the part of the polypeptide responsible for biological activity, we cleaved the molecule into defined polypeptide fragments of approximate molecular weights 24,000, 15,000, 9,000, 7,000, and 3,000 by using the cyanogen bromide method. The fragments were purified by gel permeation chromatography and by preparative polyacrylamide gel electrophoresis. They were investigated for mitogenicity and for the induction of immunoglobulin synthesis in lymphocyte cultures from several inbred mouse strains. The fragment of molecular weight 24,000 turned out to be a potent polyclonal B-lymphocyte activator comparable to native protein I. The low-molecular-weight fragments exhibited only marginal effects. Neither purified T lymphocytes nor thymocytes were activated. Our results show that a defined fragment of protein I is responsible for its lymphocyte-stimulating activity. Images PMID:3484458

  2. Substrate recognition by the Lyn protein-tyrosine kinase. NMR structure of the immunoreceptor tyrosine-based activation motif signaling region of the B cell antigen receptor.

    PubMed

    Gaul, B S; Harrison, M L; Geahlen, R L; Burton, R A; Post, C B

    2000-05-26

    The immunoreceptor tyrosine-based activation motif (ITAM) plays a central role in transmembrane signal transduction in hematopoietic cells by mediating responses leading to proliferation and differentiation. An initial signaling event following activation of the B cell antigen receptor is phosphorylation of the CD79a (Ig-alpha) ITAM by Lyn, a Src family protein-tyrosine kinase. To elucidate the structural basis for recognition between the ITAM substrate and activated Lyn kinase, the structure of an ITAM-derived peptide bound to Lyn was determined using exchange-transferred nuclear Overhauser NMR spectroscopy. The bound substrate structure has an irregular helix-like character. Docking based on the NMR data into the active site of the closely related Lck kinase strongly favors ITAM binding in an orientation similar to binding of cyclic AMP-dependent protein kinase rather than that of insulin receptor tyrosine kinase. The model of the complex provides a rationale for conserved ITAM residues, substrate specificity, and suggests that substrate binds only the active conformation of the Src family tyrosine kinase, unlike the ATP cofactor, which can bind the inactive form.

  3. Transitional B cells are the target of negative selection in the B cell compartment

    PubMed Central

    1995-01-01

    B lymphocytes recognize antigen through membrane-bound antigen- receptors, membrane IgM and IgD (mIgM and mIgD). Binding to foreign antigens initiates a cascade of biochemical events that lead to activation and differentiation. In contrast, binding to self-antigens leads to death or to inactivation. It is commonly believed that the B cells acquire the ability to discriminate between self and nonself in the early phases of development. We report here that immature B cells, which have just emerged from the mIgMneg, B220pos pool, are not deleted upon binding of self-antigen. In vivo, developing B cells become sensitive to tolerance induction in a relatively late window of differentiation, when they are in transition from the immature (HSAbright, B220dull) to the mature (HSAdull, B220bright) stage. In the transitional B cells, early markers of differentiation such as Pgp1 (CD44) and ThB reach the highest level of expression, while the expression of CD23 and mIgD, late markers of differentiation, and expression of class II MHC, progressively increases. Most of the transitional B cells, but only few of the mature and of the immature B cells, express the fas antigen, while mature B cells, but not immature and transitional B cells, express bcl-2 protein. mIgM is present in low amounts in immature B cells, reaches the highest level of expression in transitional B cells and is down-regulated in mature resting B cells, where it is coexpressed with mIgD. The high expression of mIgM, the presence of the fas antigen and the absence of bcl-2 protein is compatible with the high sensitivity of transitional B cells to negative selection. In vitro, immature B cells die rapidly by apoptosis after cross-linking of mIgM. This result, combined with the resistance of immature B cells to elimination in vivo, suggests that early in development the stroma cell microenvironment modulates signals transduced through mIgM. The functional and phenotypic division of IgMpos bone marrow B cells in

  4. A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys

    PubMed Central

    Smith, Eric J.; Olson, Kara; Haber, Lauric J.; Varghese, Bindu; Duramad, Paurene; Tustian, Andrew D.; Oyejide, Adelekan; Kirshner, Jessica R.; Canova, Lauren; Menon, Jayanthi; Principio, Jennifer; MacDonald, Douglas; Kantrowitz, Joel; Papadopoulos, Nicholas; Stahl, Neil; Yancopoulos, George D.; Thurston, Gavin; Davis, Samuel

    2015-01-01

    Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies. PMID:26659273

  5. Differential role of reactive oxygen species in the activation of mitogen-activated protein kinases and Akt by key receptors on B-lymphocytes: CD40, the B cell antigen receptor, and CXCR4

    PubMed Central

    Lee, Rosaline L.; Westendorf, Jens

    2007-01-01

    Background Antibodies produced by B-lymphocytes play a key role in the host defense against infection. The development, survival, and activation of B cell is regulated by multiple receptors including the B cell antigen receptor (BCR), which detects the presence of pathogens, CD40, which binds co-stimulatory molecules on activated T cells, and chemokines such as SDF-1 (CXCL12) that play key roles in B cell development and trafficking. Signaling by many receptors results in the generation of reactive oxygen species (ROS) that function as second messengers by regulating the activity of redox-sensitive kinases and phosphatases. We investigated the role of ROS in signaling by the BCR, CD40, and CXCR4, the receptor for SDF-1. We focused on activation of ERK, JNK, p38, and Akt, kinases that regulate multiple processes including cell survival, proliferation, and migration. Results Using the anti-oxidants N-acetyl L-cysteine (NAC) and ebselen to deplete intracellular ROS, we identified a differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by these receptors. We found that CD40 activated JNK, p38, and Akt via redox-dependent pathways that were sensitive to ROS depletion by NAC and ebselen. In contrast, BCR-induced activation of ERK, JNK, p38, and Akt was not affected by ROS depletion. We also found that CXCR4-induced Akt activation was ROS-dependent even though activation of the ERK, JNK, and p38 MAP kinases by CXCR4 occurred via ROS-independent pathways. Conclusion The differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by the BCR, CD40, and CXCR4 likely reflects the multiplicity of upstream activators for each of these kinases, only some of which may be regulated in a redox-dependent manner. These findings support the idea that ROS are important second messengers in B cells and suggest that oxidants or anti-oxidants could be used to modulate B cell activation. PMID:18481208

  6. Oncogenic CARMA1 couples NF-κB and β-catenin signaling in diffuse large B-cell lymphomas

    PubMed Central

    Bognar, M K; Vincendeau, M; Erdmann, T; Seeholzer, T; Grau, M; Linnemann, J R; Ruland, J; Scheel, C H; Lenz, P; Ott, G; Lenz, G; Hauck, S M; Krappmann, D

    2016-01-01

    Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of β-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3β to oncogenic CARMA1. Recruitment of the β-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of β-catenin. The β-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated β-catenin expression. In line, β-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased β-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, β-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that β-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and β-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis. PMID:26776161

  7. Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A.

    PubMed

    Pulvino, Mary; Liang, Yue; Oleksyn, David; DeRan, Michael; Van Pelt, Elise; Shapiro, Joel; Sanz, Ignacio; Chen, Luojing; Zhao, Jiyong

    2012-08-23

    Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, remains a partially curable disease. Genetic alterations affecting components of NF-κB signaling pathways occur frequently in DLBCL. Almost all activated B cell-like (ABC) DLBCL, which is the least curable group among the 3 major subtypes of this malignancy, and a substantial fraction of germinal center B cell-like (GCB) DLBCL exhibit constitutive NF-κB pathway activity. It has been demonstrated that ABC-DLBCL cells require such activity for proliferation and survival. Therefore, inhibition of NF-κB activation in DLBCL may provide an efficient and targeted therapy. In screening for small-molecule compounds that may inhibit NF-κB activation in DLBCL cells, we identified a compound, NSC697923, which inhibits the activity of the ubiquitin-conjugating (E2) enzyme Ubc13-Uev1A. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells. Importantly, NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells and GCB-DLBCL cells, suggesting the Ubc13-Uev1A may be crucial for DLBCL growth. Consistently, knockdown of Ubc13 expression also inhibited DLBCL cell survival. The results of the present study indicate that Ubc13-Uev1A may represent a potential therapeutic target in DLBCL. In addition, compound NSC697923 may be exploited for the development of DLBCL therapeutic agents.

  8. Brentuximab vedotin activity in diffuse large B-cell lymphoma with CD30 undetectable by visual assessment of conventional immunohistochemistry.

    PubMed

    Bartlett, Nancy L; Smith, Mitchell R; Siddiqi, Tanya; Advani, Ranjana H; O'Connor, Owen A; Sharman, Jeff P; Feldman, Tatyana; Savage, Kerry J; Shustov, Andrei R; Diefenbach, Catherine S; Oki, Yasuhiro; Palanca-Wessels, Maria Corinna; Uttarwar, Mayur; Li, Martha; Yang, Jing; Jacobsen, Eric D

    2017-07-01

    This phase 2 study evaluated brentuximab vedotin monotherapy in CD30-expressing DLBCL; after several patients with little to no CD30 achieved a complete remission (CR), the study evaluated treatment of DLBCL with undetectable CD30 (CD30u) by local visual immunohistochemistry (vIHC). Sixteen of 52 CD30u DLBCL patients (31%) had an objective response (6 CRs [12%]). Median progression-free survival (PFS) was 1.4 months (range, 0.4-15.6) and median overall survival (OS) was 7.5 months (range, 0.7-18.6+). Subsequent CD30 expression quantitated by computer-assisted digital image analysis (cIHC) showed that 11 of 16 CD30u DLBCL responders had ≥1% CD30. Correlative analyses of CD30u and CD30-expressing DLBCL combined demonstrated that ≥1% CD30 expression by cIHC resulted in a trend toward a higher response rate and significantly longer median PFS and OS. A minimum CD30 expression threshold appears to be required for antitumor activity in DLBCL; however, other factors also likely contribute to activity. (NCT01421667).

  9. Antitumor activity of SAHA, a novel histone deacetylase inhibitor, against murine B cell lymphoma A20 cells in vitro and in vivo.

    PubMed

    Yang, Bohan; Yu, Dandan; Liu, Jingwen; Yang, Kunyu; Wu, Gang; Liu, Hongli

    2015-07-01

    Suberoylanilide hydroxamic acid (SAHA; vorinostat), the second generation of histone deacetylase (HDAC) inhibitor, has been approved for the treatment of cutaneous manifestations of cutaneous T cell lymphoma (CTCL). It has also shown its anticancer activity over a large range of other hematological and solid malignancies, but few studies have been reported in B cell lymphoma. In this study, we aimed to investigate the antitumor activity of SAHA on murine B cell lymphoma cell line A20 cells. We treated A20 cells with different concentrations of SAHA. The effect of SAHA on the proliferation of A20 cells was studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay in vitro; the anti-proliferation activity in vivo was evaluated by proliferating cell nuclear antigen (PCNA) of xenograft tumor tissues through immunocytochemical staining. Apoptosis were detected by Hoechst 33258 staining and Annexin V/propidium iodide (PI) double-labeled cytometry in vitro. The effect of SAHA on cell cycle of A20 cells was studied by a propidium iodide method. Autophagic cell death induced by SAHA was confirmed by transmission electron microscopy (TEM). Angiogenesis marker (CD31) was measured by immunocytochemical staining to investigate the anti-angiogenic effect of SAHA. Western blot was used to detect the expression of signaling pathway factors (phospho-AKT, phospho-ERK, AKT, ERK, Nur77, HIF-1α, and VEGF). Our results showed that SAHA inhibited the proliferation of A20 cells in a time- and dose-dependent manner, induced cell apoptosis and G0/G1 phase arrest of cell cycle, promoted autophagic cell death, and suppressed tumor progress in NCI-A20 cells nude mice xenograft model in vivo. SAHA decreased the activation of AKT (phospho-AKT: p-AKT) and ERK1/2 (phospho-ERK: p-ERK) proteins and inhibited the expression of pro-angiogenic factors (VEGF and HIF-1α), downregulated its downstream signaling factor (Nur77), which might be contributed to the antitumor mechanisms

  10. Roles of MAPK pathway activation during cytokine induction in BEAS-2B cells exposed to fine World Trade Center (WTC) dust.

    PubMed

    Wang, Shang; Prophete, Colette; Soukup, Joleen M; Chen, Lung-Chi; Costa, Max; Ghio, Andrew; Qu, Qingshan; Cohen, Mitchell D; Chen, Haobin

    2010-01-01

    The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter (PM) into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM₂.₅ fraction or WTC₂.₅). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the mitogen-activated protein kinase (MAPK) signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC₂.₅. Our results showed that exposure to WTC₂.₅ for 5 hr increased interleukin-6 (IL-6) mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, cytokine multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal protein kinase, signaling pathways were found to be activated in cells exposed to WTC₂.₅. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC₂.₅; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC₂.₅-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other

  11. Germinal Center Marker GL7 Probes Activation-Dependent Repression of N-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation▿ †

    PubMed Central

    Naito, Yuko; Takematsu, Hiromu; Koyama, Susumu; Miyake, Shizu; Yamamoto, Harumi; Fujinawa, Reiko; Sugai, Manabu; Okuno, Yasushi; Tsujimoto, Gozoh; Yamaji, Toshiyuki; Hashimoto, Yasuhiro; Itohara, Shigeyoshi; Kawasaki, Toshisuke; Suzuki, Akemi; Kozutsumi, Yasunori

    2007-01-01

    Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the α2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing α2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells. PMID:17296732

  12. Comparative In Vitro Immune Stimulation Analysis of Primary Human B Cells and B Cell Lines

    PubMed Central

    Van Belle, Kristien; Herman, Jean; Boon, Louis; Waer, Mark

    2016-01-01

    B cell specific immunomodulatory drugs still remain an unmet medical need. Utilisation of validated simplified in vitro models would allow readily obtaining new insights in the complexity of B cell regulation. For this purpose we investigated which human B lymphocyte stimulation assays may be ideally suited to investigate new B lymphocyte immunosuppressants. Primary polyclonal human B cells underwent in vitro stimulation and their proliferation, production of immunoglobulins (Igs) and of cytokines, and expression of cell surface molecules were analysed using various stimuli. ODN2006, a toll-like receptor 9 (TLR9) agonist, was the most potent general B cell stimulus. Subsequently, we investigated on which human B cell lines ODN2006 evoked the broadest immunostimulatory effects. The Namalwa cell line proved to be the most responsive upon TLR9 stimulation and hence may serve as a relevant, homogeneous, and stable B cell model in an in vitro phenotypic assay for the discovery of new targets and inhibitors of the B cell activation processes. As for the read-out for such screening assay, it is proposed that the expression of activation and costimulatory surface markers reliably reflects B lymphocyte activation. PMID:28116319

  13. Roles of MAPK Pathway Activation During Cytokine Induction in BEAS-2B Cells Exposed to Fine World Trade Center (WTC) Dust

    PubMed Central

    Wang, Shang; Prophete, Colette; Soukup, Joleen M.; Chen, Lung-chi; Costa, Max; Ghio, Andrew; Qu, Qingshan; Chen, Haobin

    2014-01-01

    The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM2.5 fraction or WTC2.5). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the MAPK signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC2.5. Our results showed that exposure to WTC2.5 for 5 hr increased IL-6 mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, Cytokine Multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both ERK and p38, but not JNK, signaling pathways were found to be activated in cells exposed to WTC2.5. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC2.5; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC2.5-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other inflammation-associated respiratory dysfunctions in the individuals exposed to the dusts released from the WTC collapse. PMID:20731619

  14. Carbohydrate kinase (RhaK)-dependent ABC transport of rhamnose in Rhizobium leguminosarum demonstrates genetic separation of kinase and transport activities.

    PubMed

    Rivers, Damien; Oresnik, Ivan J

    2013-08-01

    In Rhizobium leguminosarum the ABC transporter responsible for rhamnose transport is dependent on RhaK, a sugar kinase that is necessary for the catabolism of rhamnose. This has led to a working hypothesis that RhaK has two biochemical functions: phosphorylation of its substrate and affecting the activity of the rhamnose ABC transporter. To address this hypothesis, a linker-scanning random mutagenesis of rhaK was carried out. Thirty-nine linker-scanning mutations were generated and mapped. Alleles were then systematically tested for their ability to physiologically complement kinase and transport activity in a strain carrying an rhaK mutation. The rhaK alleles generated could be divided into three classes: mutations that did not affect either kinase or transport activity, mutations that eliminated both transport and kinase activity, and mutations that affected transport activity but not kinase activity. Two genes of the last class (rhaK72 and rhaK73) were found to have similar biochemical phenotypes but manifested different physiological phenotypes. Whereas rhaK72 conferred a slow-growth phenotype when used to complement rhaK mutants, the rhaK73 allele did not complement the inability to use rhamnose as a sole carbon source. To provide insight to how these insertional variants might be affecting rhamnose transport and catabolism, structural models of RhaK were generated based on the crystal structure of related sugar kinases. Structural modeling suggests that both rhaK72 and rhaK73 affect surface-exposed residues in two distinct regions that are found on one face of the protein, suggesting that this protein's face may play a role in protein-protein interaction that affects rhamnose transport.

  15. Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation

    PubMed Central

    Liu, Ta-Ming; Woyach, Jennifer A.; Zhong, Yiming; Lozanski, Arletta; Lozanski, Gerard; Dong, Shuai; Strattan, Ethan; Lehman, Amy; Zhang, Xiaoli; Jones, Jeffrey A.; Flynn, Joseph; Andritsos, Leslie A.; Maddocks, Kami; Jaglowski, Samantha M.; Blum, Kristie A.; Byrd, John C.; Dubovsky, Jason A.

    2015-01-01

    Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib’s capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2R665W confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance. PMID:25972157

  16. Mitochondrial ABC transporters.

    PubMed

    Lill, R; Kispal, G

    2001-01-01

    In contrast to bacteria, mitochondria contain only a few ATP binding cassette (ABC) transporters in their inner membrane. The known mitochondrial ABC proteins fall into two major classes that, in the yeast Saccharomyces cerevisiae, are represented by the half-transporter Atm1p and the two closely homologous proteins Mdl1p and Mdl2p. In humans two Atm1p orthologues (ABC7 and MTABC3) and two proteins homologous to Mdll/2p have been localized to mitochondria. The Atm1p-like proteins perform an important function in mitochondrial iron homeostasis and in the maturation of Fe/S proteins in the cytosol. Mutations in ABC7 are causative of hereditary X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). MTABC3 may be a candidate gene for the lethal neonatal syndrome. The function of the mitochondrial Mdl1/2p-like proteins is not clear at present with the notable exception of murine ABC-me that may transport intermediates of heme biosynthesis from the matrix to the cytosol in erythroid tissues.

  17. Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells.

    PubMed

    Bermejo, Daniela A; Jackson, Shaun W; Gorosito-Serran, Melisa; Acosta-Rodriguez, Eva V; Amezcua-Vesely, Maria C; Sather, Blythe D; Singh, Akhilesh K; Khim, Socheath; Mucci, Juan; Liggitt, Denny; Campetella, Oscar; Oukka, Mohamed; Gruppi, Adriana; Rawlings, David J

    2013-05-01

    Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17(+) B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17(+) B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

  18. The PI3K pathway in B cell metabolism.

    PubMed

    Jellusova, Julia; Rickert, Robert C

    2016-09-01

    B cell growth and proliferation is tightly regulated by signaling through the B cell receptor and by other membrane bound receptors responding to different cytokines. The PI3K signaling pathway has been shown to play a crucial role in B cell activation, differentiation and survival. Activated B cells undergo metabolic reprograming in response to changing energetic and biosynthetic demands. B cells also need to be able to coordinate metabolic activity and proliferation with nutrient availability. The PI3K signaling network has been implicated in regulating nutrient acquisition, utilization and biosynthesis, thus integrating receptor-mediated signaling with cell metabolism. In this review, we discuss the current knowledge about metabolic changes induced in activated B cells, strategies to adapt to metabolic stress and the role of PI3K signaling in these processes.

  19. CNS accumulation of regulatory B cells is VLA-4-dependent

    PubMed Central

    Lehmann-Horn, Klaus; Sagan, Sharon A.; Winger, Ryan C.; Spencer, Collin M.; Bernard, Claude C.A.; Sobel, Raymond A.

    2016-01-01

    Objective: To investigate the role of very late antigen-4 (VLA-4) on regulatory B cells (Breg) in CNS autoimmune disease. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in mice selectively deficient for VLA-4 on B cells (CD19cre/α4f/f) by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p)35–55 or recombinant human (rh) MOG protein. B-cell and T-cell populations were examined by flow cytometry and immunohistochemistry. Breg were evaluated by intracellular IL-10 staining of B cells and, secondly, by coexpression of CD1d and CD5. Results: As previously reported, EAE was less severe in B-cell VLA-4-deficient vs control CD19cre mice when induced by rhMOG, a model that is B-cell-dependent and leads to efficient B-cell activation and antibody production. Paradoxically, B-cell VLA-4-deficient mice developed more severe clinical disease than control mice when EAE was induced with MOG p35-55, a B-cell-independent encephalitogen that does not efficiently activate B cells. Peripheral T-cell and humoral immune responses were not altered in B-cell VLA-4-deficient mice. In MOG p35-55-induced EAE, B-cell VLA-4 deficiency reduced CNS accumulation of B but not T cells. Breg were detected in the CNS of control mice with MOG p35-55-induced EAE. However, more severe EAE in B-cell VLA-4-deficient mice was associated with virtual absence of CNS Breg. Conclusions: Our results demonstrate that CNS accumulation of Breg is VLA-4-dependent and suggest that Breg may contribute to regulation of CNS autoimmunity in situ. These observations underscore the need to choose the appropriate encephalitogen when studying how B cells contribute to pathogenesis or regulation of CNS autoimmunity. PMID:27027096

  20. Switched-memory B cells remodel B cell receptors within secondary germinal centers

    PubMed Central

    Okitsu, Shinji L.; McHeyzer-Williams, Michael G.

    2015-01-01

    Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive B cell receptor (BCR) re-diversification, but underlying mechanisms remain unresolved. Here integrated specificity and function of individual memory B cell progeny reveal ongoing evolution of polyclonal antibody specificities through germinal center (GC) specific transcriptional activity. At the clonal and sub-clonal levels, single cell expression of Cd83 and Pol□ segregates the secondary GC transcriptional program into 4 stages that regulate divergent mechanisms of memory BCR evolution. These studies demonstrate that vaccine boosts re-activate a cyclic program of GC function in switched-memory B cells to remodel existing antibody specificities and enhance durable immune protection. PMID:25642821

  1. B Cell-Activating Transcription Factor Plays a Critical Role in the Pathogenesis of Anti-Major Histocompatibility Complex-Induced Obliterative Airway Disease.

    PubMed

    Xu, Z; Ramachandran, S; Gunasekaran, M; Nayak, D; Benshoff, N; Hachem, R; Gelman, A; Mohanakumar, T

    2016-04-01

    Antibodies (Abs) against major histocompatibility complex (MHC) results in T helper-17 (Th17)-mediated immunity against lung self-antigens (SAgs), K-α1 tubulin and collagen V and obliterative airway disease (OAD). Because B cell-activating transcription factor (BATF) controls Th17 and autoimmunity, we proposed that BATF may play a critical role in OAD. Anti-H2K(b) was administered intrabronchially into Batf (-/-) and C57BL/6 mice. Histopathology of the lungs on days 30 and 45 after Ab administration to Batf (-/-) mice resulted in decreased cellular infiltration, epithelial metaplasia, fibrosis, and obstruction. There was lack of Abs to SAgs, reduction of Sag-specific interleukin (IL)-17 T cells, IL-6, IL-23, IL-17, IL-1β, fibroblast growth factor-6, and CXCL12 and decreased Janus kinase 2, signal transducer and activator of transcription 3 (STAT3), and retinoid-related orphan receptor γT. Further, micro-RNA (miR)-301a, a regulator of Th17, was reduced in Batf (-/-) mice in contrast to upregulation of miR-301a and downregulation of protein inhibitor of activated STAT3 (PIAS3) in anti-MHC-induced OAD animals. We also demonstrate an increase in miR-301a in the bronchoalveolar lavage cells from lung transplant recipients with Abs to human leukocyte antigen. This was accompanied by reduction in PIAS3 mRNA. Therefore, we conclude that BATF plays a critical role in the immune responses to SAgs and pathogenesis of anti-MHC-induced rejection. Targeting BATF should be considered for preventing chronic rejection after human lung transplantation.

  2. Memory B cells in Transplantation

    PubMed Central

    Chong, Anita S.; Sciammas, Roger

    2014-01-01

    Much of the research on the humoral response to allografts has focused on circulating serum antibodies and the long-lived plasma cells that produce these antibodies. In contrast, the interrogation of the quiescent memory B cell compartment is technically more challenging and thus has not been incorporated into the clinical diagnostic or prognostic toolkit. In this review, we discuss new technologies that have allowed this heretofore enigmatic subset of B cells to be identified at quiescence and during a recall response. These technologies in experimental models are providing new insights into memory B cell heterogeneity with respect to their phenotype, cellular function and the antibodies they produce. Similar technologies are also allowing for the identification of comparable memory alloreactive B cells in transplant recipients. While much of the focus in transplant immunology has been on controlling the alloreactive B cell population, long-term transplant patient survival is critically dependent on protection by pathogen-specific memory B cells. Techniques are also available that allow the interrogation of memory B cell response to pathogen re-encounter. Thus we are poised in our ability toinvestigate how immunosuppression affects allo- as well as pathogen-specific memory B cells, and reason that these investigation can yield new insights that will be beneficial for graft as well as patient survival. PMID:25525921

  3. Inflammatory monocytes hinder antiviral B cell responses

    PubMed Central

    Sammicheli, Stefano; Kuka, Mirela; Di Lucia, Pietro; de Oya, Nereida Jimenez; De Giovanni, Marco; Fioravanti, Jessica; Cristofani, Claudia; Maganuco, Carmela G.; Fallet, Benedict; Ganzer, Lucia; Sironi, Laura; Mainetti, Marta; Ostuni, Renato; Larimore, Kevin; Greenberg, Philip D.; de la Torre, Juan Carlos; Guidotti, Luca G.; Iannacone, Matteo

    2016-01-01

    Antibodies are critical for protection against viral infections. However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms. Here we analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of the draining lymph node where they extensively interact with CD11b+Ly6Chi inflammatory monocytes. These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon-, CCR2-dependent fashion and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide. Depletion of inflammatory monocytes, inhibition of their lymph node recruitment or impairment of their nitric oxide-producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production. In conclusion, our results identify inflammatory monocytes as critical gatekeepers that prevent antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host. PMID:27868108

  4. SU-E-T-326: The Oxygen Saturation (SO2) and Breath-Holding Time Variation Applied Active Breathing Control (ABC)

    SciTech Connect

    Gong, G; Yin, Y

    2014-06-01

    Purpose: To study the oxygen saturation (SO2) and breath-holding time variation applied active breathing control (ABC) in radiotherapy of tumor. Methods: 24 volunteers were involved in our trials, and they all did breath-holding motion assisted by ELEKTA Active Breathing Coordinator 2.0 for 10 times respectively. And the patient monitor was used to observe the oxygen saturation (SO2) variation. The variation of SO2, and length of breath-holding time and the time for recovering to the initial value of SO2 were recorded and analyzed. Results: (1) The volunteers were divided into two groups according to the SO2 variation in breath-holding: A group, 14 cases whose SO2 reduction were more than 2% (initial value was 97% to 99%, while termination value was 91% to 96%); B group, 10 cases were less than 2% in breath-holding without inhaling oxygen. (2) The interfraction breath holding time varied from 8 to 20s for A group compared to the first breath-holding time, and for B group varied from 4 to 14s. (3) The breathing holding time of B group prolonged mean 8s, compared to A group. (4) The time for restoring to the initial value of SO2 was from 10s to 30s. And the breath-holding time shortened obviously for patients whose SO2 did not recover to normal. Conclusion: It is very obvious that the SO2 reduction in breath-holding associated with ABC for partial people. It is necessary to check the SO2 variation in breath training, and enough time should be given to recover SO2.

  5. ABC's of Being Smart

    ERIC Educational Resources Information Center

    Foster, Joanne

    2011-01-01

    Determining what giftedness is all about means focusing on many aspects of the individual. In this paper, the author focuses on letter D of the ABC's of being smart. She starts with specifics about giftedness (details), and then moves on to some ways of thinking (dispositions).

  6. 1968 ABC Summer Program.

    ERIC Educational Resources Information Center

    Kerr, Frances M.; Russell, Valerie E.

    A talent development project at Mount Holyoke College, part of A Better Chance (ABC)-Independent Schools Talent Search program, was offered during the summer of 1968 to 71 disadvantaged high school students from 13 states. Major aims of the program were to help these students with college potential to strengthen their academic skills and…

  7. Plant ABC Transporters

    PubMed Central

    Kang, Joohyun; Park, Jiyoung; Choi, Hyunju; Burla, Bo; Kretzschmar, Tobias; Lee, Youngsook; Martinoia, Enrico

    2011-01-01

    ABC transporters constitute one of the largest protein families found in all living organisms. ABC transporters are driven by ATP hydrolysis and can act as exporters as well as importers. The plant genome encodes for more than 100 ABC transporters, largely exceeding that of other organisms. In Arabidopsis, only 22 out of 130 have been functionally analyzed. They are localized in most membranes of a plant cell such as the plasma membrane, the tonoplast, chloroplasts, mitochondria and peroxisomes and fulfill a multitude of functions. Originally identified as transporters involved in detoxification processes, they have later been shown to be required for organ growth, plant nutrition, plant development, response to abiotic stresses, pathogen resistance and the interaction of the plant with its environment. To fulfill these roles they exhibit different substrate specifies by e.g. depositing surface lipids, accumulating phytate in seeds, and transporting the phytohormones auxin and abscisic acid. The aim of this review is to give an insight into the functions of plant ABC transporters and to show their importance for plant development and survival. PMID:22303277

  8. The ABC transporter ABC40 encodes a phenylacetic acid export system in Penicillium chrysogenum.

    PubMed

    Weber, Stefan S; Kovalchuk, Andriy; Bovenberg, Roel A L; Driessen, Arnold J M

    2012-11-01

    The filamentous fungus Penicillium chrysogenum is used for the industrial production of β-lactam antibiotics. The pathway for β-lactam biosynthesis has been resolved and involves the enzyme phenylacetic acid CoA ligase that is responsible for the CoA activation of the side chain precursor phenylacetic acid (PAA) that is used for the biosynthesis of penicillin G. To identify ABC transporters related to β-lactam biosynthesis, we analyzed the expression of all 48 ABC transporters present in the genome of P. chryso-genum when grown in the presence and absence of PAA. ABC40 is significantly upregulated when cells are grown or exposed to high levels of PAA. Although deletion of this transporter did not affect β-lactam biosynthesis, it resulted in a significant increase in sensitivity to PAA and other weak acids. It is concluded that ABC40 is involved in weak acid detoxification in P. chrysogenum including resistance to phenylacetic acid.

  9. Antigen-processing organelles from DRB1*1101 and DRB1*1104 B cell lines display a differential degradation activity.

    PubMed

    Barbey, C; Watts, C; Corradin, G

    1995-01-01

    We have developed an in vitro assay for tetanus toxin (tt) C fragment (C-fr) degradation. Purified endosomes (abbreviated endosomes 1101 or 1104) and lysosomes (abbreviated lysosomes 1101 or 1104) from the DRB1*1101 (Gly 86) and DRB1*1104 (Val 86) B cell lines were used to degrade 125I-labeled C-fr in vitro. Using three distinct methods of analysis, we show that the capacity of endosomes and lysosomes to degrade the tt C-fr or tt synthetic Y-P30 peptide differed. Using sodium dodecylsulfate-polyacrylamide gel electrophoresis, 125I-labeled C-fr degradation patterns observed either with endosomes 1101/1104 or lysosomes 1101/1104 are distinct both in terms of the number of fragments and the kinetics of generation of the fragments. These results were confirmed by high-performance liquid chromatography analysis, where we observed that the elution profiles of the 125I-labeled Y-P30 peptide digested by endosomes 1101/1104 were different compared to those obtained with lysosomes 1101/1104. Furthermore, the kinetics of degradation of 125I-labeled Y-P30 were faster with lysosomes 1104 than with lysosomes 1101. This difference in activity of the 1101 and 1104 organelles was also found in a functional assay where we showed that the activation capacity of the P30 peptide was diminished when digested by lysosome 1104, regardless of the antigen-presenting cell (APC) used, whereas endosomes 1101 or lysosomes 1101 modified P30 peptide in a form that discriminated between presentation by 1101 or 1104 APC. Taken together, these results suggest that the differential processing and presentation displayed by the DRB1*1101 and DRB1*1104 APC is due partly to a different enzymatic content and partly to the dimorphism at position DR beta 86.

  10. MicroRNA-21 plays an oncogenic role by targeting FOXO1 and activating the PI3K/AKT pathway in diffuse large B-cell lymphoma

    PubMed Central

    Kim, Pil-Jong; Kim, Young-Goo; Nam, Soo Jeong; Paik, Jin Ho; Kim, Tae Min; Heo, Dae Seog; Kim, Chul-Woo; Jeon, Yoon Kyung

    2015-01-01

    The prognostic implications of miR-21, miR-17-92 and miR-155 were evaluated in diffuse large B-cell lymphoma (DLBCL) patients, and novel mechanism by which miR-21 contributes to the oncogenesis of DLBCL by regulating FOXO1 and PI3K/AKT/mTOR pathway was investigated. The expressions of miR-21, miR-17-92 and miR-155 measured by quantitative reverse-transcription-PCR were significantly up-regulated in DLBCL tissues (n=200) compared to control tonsils (P=0.012, P=0.001 and P<0.0001). Overexpression of miR-21 and miR-17-92 was significantly associated with shorter progression-free survival (P=0.003 and P=0.014) and overall survival (P=0.004 and P=0.012). High miR-21 was an independent prognostic factor in DLBCL patients treated with rituximab-combined chemotherapy. MiR-21 level was inversely correlated with the levels of FOXO1 and PTEN in DLBCL cell lines. Reporter-gene assay showed that miR-21 directly targeted and suppressed the FOXO1 expression, and subsequently inhibited Bim transcription in DLBCL cells. MiR-21 also down-regulated PTEN expression and consequently activated the PI3K/AKT/mTOR pathway, which further decreased FOXO1 expression. Moreover, miR-21 inhibitor suppressed the expression and activity of MDR1, thereby sensitizing DLBCL cells to doxorubicin. These data demonstrated that miR-21 plays an important oncogenic role in DLBCL by modulating the PI3K/AKT/mTOR/FOXO1 pathway at multiple levels resulting in strong prognostic implication. Therefore, targeting miR-21 may have therapeutic relevance in DLBCL. PMID:25909227

  11. Progenitors for Ly-1 B cells are distinct from progenitors for other B cells

    PubMed Central

    1985-01-01

    Data from previous multiparameter fluorescence-activated cell sorter (FACS) analysis and sorting studies define a subset of murine B cells that expresses the Ly-1 surface determinant in conjunction with IgM, IgD, Ia, and other typical B cell markers. These Ly-1 B cells are physically and functionally distinct. They express more IgM and less IgD than most other B cells; they are not normally found in lymph node or bone marrow; they are always present at low frequencies (1-5%) in normal spleens, and, as we show here, they comprise about half of the B cells (10-20% of total cells) recovered from the peritoneal cavity in normal mice. Furthermore, most of the commonly studied IgM autoantibodies in normal and autoimmune mice are produced by these Ly-1 B cells, even though they seldom produce antibodies to exogenous antigens such as trinitrophenyl-Ficoll or trinitrophenyl-keyhole limpet hemocyanin. Cell transfer studies presented here demonstrate that the progenitors of Ly-1 B cells are different from the progenitors of the predominant B cell populations in spleen and lymph node. In these studies, we used FACS analysis and functional assays to characterize donor-derived (allotype-marked) B cells present in lethally irradiated recipients 1-2 mo after transfer. Surprisingly, adult bone marrow cells typically used to reconstitute B cells in irradiated recipients selectively failed to reconstitute the Ly-1 B subset. Liver, spleen, and bone marrow cells from young mice, in contrast, reconstituted all B cells (including Ly-1 B), and peritoneal "washout" cells (PerC) from adult mice uniquely reconstituted Ly-1 B. Bone marrow did not block Ly- 1 B development, since PerC and newborn liver still gave rise to Ly-1 B when jointly transferred with marrow. These findings tentatively assign Ly-1 B to a distinct developmental lineage originating from progenitors that inhabit the same locations as other B cell progenitors in young animals, but move to unique location(s) in adults. PMID

  12. The IgD-binding domain of the Moraxella IgD-binding protein MID (MID962-1200) activates human B cells in the presence of T cell cytokines.

    PubMed

    Nordström, Therése; Jendholm, Johan; Samuelsson, Martin; Forsgren, Arne; Riesbeck, Kristian

    2006-02-01

    Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is an outer membrane protein with specific affinity for soluble and cell-bound human IgD. Here, we demonstrate that mutated M. catarrhalis strains devoid of MID show a 75% decreased activation of human B cells as compared with wild-type bacteria. In contrast to MID-expressing Moraxella, the MID-deficient Moraxella mutants did not bind to human CD19+ IgD+ B cells. The smallest MID fragment with preserved IgD-binding capacity comprises 238 amino acids (MID(962-1200)). To prove the specificity of MID(962-1200) for IgD, a Chinese hamster ovary (CHO) cell line expressing membrane-anchored human IgD was manufactured. MID(962-1200) bound strongly to the recombinant IgD on CHO cells. Moreover, MID(962-1200) stimulated peripheral blood lymphocyte (PBL) proliferation 5- and 15-fold at 0.1 and 1.0 microg/ml, respectively. This activation could be blocked completely by antibodies directed against the CD40 ligand (CD154). MID(962-1200) also activated purified B cells in the presence of interleukin (IL)-2 or IL-4. An increased IL-6 production was seen after stimulation with MID(962-1200), as revealed by a human cytokine protein array. MID(962-1200) fused to green fluorescent protein (GFP) bound to human B cells and activated PBL to the same degree as MID(962-1200). Taken together, MID is the only IgD-binding protein in Moraxella. Furthermore, the novel T cell-independent antigen MID(962-1200) may, together with MID(962-1200)-GFP, be considered as promising reagents in the study of IgD-dependent B cell activation.

  13. The 3BP2 Adapter Protein Is Required for Optimal B-Cell Activation and Thymus-Independent Type 2 Humoral Response▿

    PubMed Central

    Chen, Grace; Dimitriou, Ioannis D.; La Rose, Jose; Ilangumaran, Subburaj; Yeh, Wen-Chen; Doody, Gina; Turner, Martin; Gommerman, Jennifer; Rottapel, Robert

    2007-01-01

    3BP2 is a pleckstrin homology domain- and Src homology 2 (SH2) domain-containing adapter protein that is mutated in the rare human bone disorder cherubism and which has also been implicated in immunoreceptor signaling. However, a function for this protein has yet to be established. Here we show that mice lacking 3BP2 exhibited a perturbation in the peritoneal B1 and splenic marginal-zone B-cell compartments and diminished thymus-independent type 2 antigen response. 3BP2−/− B cells demonstrated a proliferation defect in response to antigen receptor cross-linking and a heightened sensitivity to B-cell receptor-induced death via a caspase-3-dependent apoptotic pathway. We show that 3BP2 binds via its SH2 domain to the CD19 signaling complex and is required for optimum Syk phosphorylation and calcium flux. PMID:17283041

  14. RP105-Negative B Cells in Systemic Lupus Erythematosus

    PubMed Central

    Koarada, Syuichi; Tada, Yoshifumi

    2012-01-01

    Systemic lupus erythematosus (SLE) is a multisystem disease characterized by B cells producing autoantibodies against nuclear proteins and DNA, especially anti-double-strand DNA (dsDNA) antibodies. RP105 (CD180), the toll-like receptor- (TLR-) associated molecule, is expressed on normal B cells. However, RP105-negative B cells increase in peripheral blood from patients with active SLE. RP105 may regulate B-cell activation, and RP105-negative B cells produce autoantibodies and take part in pathophysiology of SLE. It is possible that targeting RP105-negative B cells is one of the treatments of SLE. In this paper, we discuss the RP105 biology and clinical significance in SLE. PMID:21941580

  15. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

    PubMed

    Lin, Hong Ting; Bavro, Vassiliy N; Barrera, Nelson P; Frankish, Helen M; Velamakanni, Saroj; van Veen, Hendrik W; Robinson, Carol V; Borges-Walmsley, M Inês; Walmsley, Adrian R

    2009-01-09

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

  16. The Role of the Atypical Kinases ABC1K7 and ABC1K8 in Abscisic Acid Responses

    PubMed Central

    Manara, Anna; DalCorso, Giovanni; Furini, Antonella

    2016-01-01

    The ABC1K family of atypical kinases (activity of bc1 complex kinase) is represented in bacteria, archaea, and eukaryotes. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. ABC1K7 and ABC1K8 are probably involved in oxidative stress responses, isoprenyl lipid synthesis and distribution of iron within chloroplasts. Because reactive oxygen species take part in abscisic acid (ABA)-mediated processes, we investigated the functions of ABC1K7 and ABC1K8 during germination, stomatal movement, and leaf senescence. Both genes were upregulated by ABA treatment and some ABA-responsive physiological processes were affected in abc1k7 and abc1k8 mutants. Germination was more severely affected by ABA, osmotic stress and salt stress in the single and double mutants; the stomatal aperture was smaller in the mutants under standard growth conditions and was not further reduced by exogenous ABA application; ABA-induced senescence symptoms were more severe in the leaves of the single and double mutants compared to wild type leaves. Taken together, our results suggest that ABC1K7 and ABC1K8 might be involved in the cross-talk between ABA and ROS signaling. PMID:27047531

  17. Impairment of B-cell functions during HIV-1 infection.

    PubMed

    Amu, Sylvie; Ruffin, Nicolas; Rethi, Bence; Chiodi, Francesca

    2013-09-24

    A variety of B-cell dysfunctions are manifested during HIV-1 infection, as reported early during the HIV-1 epidemic. It is not unusual that the pathogenic mechanisms presented to elucidate impairment of B-cell responses during HIV-1 infection focus on the impact of reduced T-cell numbers and functions, and lack of germinal center formation in lymphoid tissues. To our understanding, however, perturbation of B-cell phenotype and function during HIV-1 infection may begin at several different B-cell developmental stages. These impairments can be mediated by intrinsic B-cell defects as well as by the lack of proper T-cell help. In this review, we will highlight some of the pathways and molecular interactions leading to B-cell impairment prior to germinal center formation and B-cell activation mediated through the B-cell receptor in response to HIV-1 antigens. Recent studies indicate a regulatory role for B cells on T-cell biology and immune responses. We will discuss some of these novel findings and how these regulatory mechanisms could potentially be affected by the intrinsic defects of B cells taking place during HIV-1 infection.

  18. B Cell Subsets in Atherosclerosis

    PubMed Central

    Perry, Heather M.; Bender, Timothy P.; McNamara, Coleen A.

    2012-01-01

    Atherosclerosis, the underlying cause of heart attacks and strokes, is a chronic inflammatory disease of the artery wall. Immune cells, including lymphocytes modulate atherosclerotic lesion development through interconnected mechanisms. Elegant studies over the past decades have begun to unravel a role for B cells in atherosclerosis. Recent findings provide evidence that B cell effects on atherosclerosis may be subset-dependent. B-1a B cells have been reported to protect from atherosclerosis by secretion of natural IgM antibodies. Conventional B-2 B cells can promote atherosclerosis through less clearly defined mechanism that may involve CD4 T cells. Yet, there may be other populations of B cells within these subsets with different phenotypes altering their impact on atherosclerosis. Additionally, the role of B cell subsets in atherosclerosis may depend on their environmental niche and/or the stage of atherogenesis. This review will highlight key findings in the evolving field of B cells and atherosclerosis and touch on the potential and importance of translating these findings to human disease. PMID:23248624

  19. Gender-Specific Mechanisms Underlying the Amelioration of High-Fat Diet-Induced Glucose Intolerance in B-Cell-Activating Factor Deficient Mice

    PubMed Central

    Kim, Bobae; Hyun, Chang-Kee

    2016-01-01

    It has recently been found that B cell activating factor (BAFF) plays an important role in the regulation of energy homeostasis. We also have previously reported that BAFF deficiency reverses high-fat (HF) diet-induced glucose intolerance by potentiating adipose tissue function. In the present study, we found that BAFF deficient (BAFF-/-) mice exhibit gender-specific differences in protection against diet-induced glucose intolerance, and aimed to characterize the gender-dependent molecular alterations in energy metabolism. Under HF feeding conditions, serum BAFF level of female wild-type (WT) mice was considerably higher than that of male mice. Despite increased body weight gain, both male and female BAFF-/- mice showed significantly improved glucose tolerance compared to their WT counterparts. Expressions of genes involved in glucose transport, thermogenesis and lipid oxidation were up-regulated in brown adipose tissues of both male and female BAFF-/- mice. Interestingly, the expression of thermogenic genes in subcutaneous adipose tissue was significantly enhanced in female BAFF-/- compared to WT mice, but the difference was not observed between male BAFF-/- and WT mice. The enhanced thermogenic program was confirmed by higher protein levels of UCP1 and irisin in female BAFF-/- than in WT mice. Additionally, adiponectin production in white adipose tissues and AMPK phosphorylation in subcutaneous adipose tissue were also significantly elevated in female BAFF-/- compared to WT mice, but not in male BAFF-/- mice. Our findings define a comprehensive scenario for the enhancing effect of BAFF depletion on glucose tolerance wherein the underlying mechanism is, at least in part, gender-specific, and suggest that gender difference should be considered as an important factor in the use of BAFF blockade as a therapeutic approach for the prevention and treatment of type 2 diabetes. PMID:27814392

  20. B Cell Activating Transcription Factor (BATF) plays a critical role in the pathogenesis of anti-MHC induced Obliterative Airway Disease

    PubMed Central

    Xu, Z.; Ramachandran, S.; Gunasekaran, M.; Nayak, D.; Benshoff, N.; Hachem, R.; Gelman, A.; Mohanakumar, T.

    2016-01-01

    Antibodies (Abs) against MHC results in T helper-17 (Th17) mediated immunity against lung self-antigens (SAgs), K-α1 tubulin and collagen V and Obliterative Airway Disease (OAD). Since B cell activating transcription factor (BATF) controls Th17 and autoimmunity, we proposed that BATF may play a critical role in OAD. Anti-H2Kb was administered intrabronchially into Batf−/− and C57BL/6 mice. Histopathology of the lungs on days 30 and 45 following Abs administration to Batf−/− mice resulted in decreased cellular infiltration, epithelial metaplasia, fibrosis and obstruction. There was lack of Abs to SAgs, reduction of SAgs specific IL17 T cells, IL-6, IL-23, IL-17, IL-1β, FGF-6 and CXCL12 and decreased Janus kinase 2, STAT3, and Retinoid-related orphan receptor gamma-T. Further, miR-301a, a regulator of Th17, was reduced in Batf−/− mice in contrast to up regulation of miR-301a and down regulation of PIAS3 in anti-MHC induced OAD animals. We also demonstrate increase in miR-301a in the bronchoalveolar lavage cells from lung transplant (LTx) recipients with Abs to HLA. This was accompanied by reduction in PIAS3 mRNA. Therefore, we conclude that BATF plays a critical role in the immune responses to SAgs and pathogenesis of anti-MHC induced rejection. Targeting BATF should be considered for preventing chronic rejection following human LTx. PMID:26844425

  1. In aged mice, low surrogate light chain promotes pro-B-cell apoptotic resistance, compromises the PreBCR checkpoint, and favors generation of autoreactive, phosphorylcholine-specific B cells.

    PubMed

    Ratliff, Michelle; Alter, Sarah; McAvoy, Kelly; Frasca, Daniela; Wright, Jacqueline A; Zinkel, Sandra S; Khan, Wasif N; Blomberg, Bonnie B; Riley, Richard L

    2015-06-01

    In aged mice, new B-cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B-cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5(low) B-cell precursors generate new B cells which show increased reactivity to the self-antigen/bacterial antigen phosphorylcholine (PC). Pro-B cells in old bone marrow as well as pro-B cells from young adult λ5-deficient mice are resistant to cytokine-induced apoptosis (TNFα; TGFβ), indicating that low λ5 expression in pro-B cells is sufficient to cause increased survival. Transfer of TNFα-producing 'age-associated B cells' (ABC; CD21/35(-) CD23(-)) or follicular (FO) B cells from aged mice into RAG-2 KO recipients led to preferential loss of λ5(high) pro-B cells, but retention of λ5(low), apoptosis-resistant pro-B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα-producing B cells, results in preferential loss of SLC(high) pro-B cells within the bone marrow. Further B-cell development then occurs via an 'SLC(low)' pathway that not only impairs B-cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery.

  2. Regulatory B cells inhibit cytotoxic T lymphocyte (CTL) activity and elimination of infected CD4 T cells after in vitro reactivation of HIV latent reservoirs.

    PubMed

    Siewe, Basile; Wallace, Jennillee; Rygielski, Sonya; Stapleton, Jack T; Martin, Jeffrey; Deeks, Steven G; Landay, Alan

    2014-01-01

    During HIV infection, IL-10/IL-10 receptor and programmed death-1 (PD-1)/programmed death-1-ligand (PD-L1) interactions have been implicated in the impairment of cytotoxic T lymphocyte (CTL) activity. Despite antiretroviral therapy (ART), attenuated anti-HIV CTL functions present a major hurdle towards curative measures requiring viral eradication. Therefore, deeper understanding of the mechanisms underlying impaired CTL is crucial before HIV viral eradication is viable. The generation of robust CTL activity necessitates interactions between antigen-presenting cells (APC), CD4+ and CD8+ T cells. We have shown that in vitro, IL-10hiPD-L1hi regulatory B cells (Bregs) directly attenuate HIV-specific CD8+-mediated CTL activity. Bregs also modulate APC and CD4+ T cell function; herein we characterize the Breg compartment in uninfected (HIVNEG), HIV-infected "elite controllers" (HIVEC), ART-treated (HIVART), and viremic (HIVvir), subjects, and in vitro, assess the impact of Bregs on anti-HIV CTL generation and activity after reactivation of HIV latent reservoirs using suberoylanilide hydroxamic acid (SAHA). We find that Bregs from HIVEC and HIVART subjects exhibit comparable IL-10 expression levels significantly higher than HIVNEG subjects, but significantly lower than HIVVIR subjects. Bregs from HIVEC and HIVART subjects exhibit comparable PD-L1 expression, significantly higher than in HIVVIR and HIVNEG subjects. SAHA-treated Breg-depleted PBMC from HIVEC and HIVART subjects, displayed enhanced CD4+ T-cell proliferation, significant upregulation of antigen-presentation molecules, increased frequency of CD107a+ and HIV-specific CD8+ T cells, associated with efficient elimination of infected CD4+ T cells, and reduction in integrated viral DNA. Finally, IL-10-R and PD-1 antibody blockade partially reversed Breg-mediated inhibition of CD4+ T-cell proliferation. Our data suggest that, possibly, via an IL-10 and PD-L1 synergistic mechanism; Bregs likely inhibit APC function

  3. Machine learning-based classification of diffuse large B-cell lymphoma patients by eight gene expression profiles.

    PubMed

    Zhao, Shuangtao; Dong, Xiaoli; Shen, Wenzhi; Ye, Zhen; Xiang, Rong

    2016-05-01

    Gene expression profiling (GEP) had divided the diffuse large B-cell lymphoma (DLBCL) into molecular subgroups: germinal center B-cell like (GCB), activated B-cell like (ABC), and unclassified (UC) subtype. However, this classification with prognostic significance was not applied into clinical practice since there were more than 1000 genes to detect and interpreting was difficult. To classify cancer samples validly, eight significant genes (MYBL1, LMO2, BCL6, MME, IRF4, NFKBIZ, PDE4B, and SLA) were selected in 414 patients treated with CHOP/R-CHOP chemotherapy from Gene Expression Omnibus (GEO) data sets. Cutoffs for each gene were obtained using receiver-operating characteristic curves (ROC) new model based on the support vector machine (SVM) estimated the probability of membership into one of two subgroups: GCB and Non-GCB (ABC and UC). Furtherly, multivariate analysis validated the model in another two cohorts including 855 cases in all. As a result, patients in the training and validated cohorts were stratified into two subgroups with 94.0%, 91.0%, and 94.4% concordance with GEP, respectively. Patients with Non-GCB subtype had significantly poorer outcomes than that with GCB subtype, which agreed with the prognostic power of GEP classification. Moreover, the similar prognosis received in the low (0-2) and high (3-5) IPI scores group demonstrated that the new model was independent of IPI as well as GEP method. In conclusion, our new model could stratify DLBCL patients with CHOP/R-CHOP regimen matching GEP subtypes effectively.

  4. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells

    NASA Astrophysics Data System (ADS)

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B.

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

  5. Age effects on B cells and humoral immunity in humans

    PubMed Central

    Frasca, Daniela; Diaz, Alain; Romero, Maria; Landin, Ana Marie; Blomberg, Bonnie B

    2010-01-01

    Both humoral and cellular immune responses are impaired in aged individuals, leading to decreased vaccine responses. Although T cell defects occur, defects in B cells play a significant role in age-related humoral immune changes. The ability to undergo class switch recombination (CSR), the enzyme for CSR, AID (activation-induced cytidine deaminase) and the transcription factor E47 are all decreased in aged stimulated B cells. We here present an overview of age-related changes in human B cell markers and functions, and also discuss some controversies in the field of B cell aging. PMID:20728581

  6. Targeting B cells with biologics in SLE

    PubMed Central

    La Cava, Antonio

    2010-01-01

    Importance of the field The use of biologics as immune modulators in several autoimmune diseases has provided new tools to the physician's therapeutic armamentiarium and has led to improved patients' outcomes and quality of life. By producing autoantibodies, B cells in SLE are key players in the pathogenesis of the disease and in its clinical manifestations. Therefore, biologics that target B cells in SLE aims at reducing the activity of these cells for the induction of remissions and/or amelioration of disease activity, reduction of organ involvement, and limitation of the complications and side effects caused by immunosuppressive therapies. Areas covered in this review This review describes the past and current clinical trials with B cell-targeted biologics in SLE, to provide a historical perspective and the state-of-the-art on the topic. What the reader will gain We will review how the disappointment in the field from promising agents has been instrumental in providing valuable lessons leading to an improved design of new trials that are now giving encouraging results Take home message In systemic lupus erythematosus (SLE), the use of B cell-based biologics in clinical trials has shown both disappointment and promise PMID:20919800

  7. Beyond RCHOP: A Blueprint for Diffuse Large B Cell Lymphoma Research.

    PubMed

    Nowakowski, Grzegorz S; Blum, Kristie A; Kahl, Brad S; Friedberg, Jonathan W; Baizer, Lawrence; Little, Richard F; Maloney, David G; Sehn, Laurie H; Williams, Michael E; Wilson, Wyndham H; Leonard, John P; Smith, Sonali M

    2016-12-01

    Diffuse large B cell lymphoma (DLBCL) comprises multiple molecular and biological subtypes, resulting in a broad range of clinical outcomes. With standard chemoimmunotherapy, there remains an unacceptably high treatment failure rate in certain DLBCL subsets: activated B cell (ABC) DLBCL, double-hit lymphoma defined by the dual translocation of MYC and BCL2, dual protein-expressing lymphomas defined by the overexpression of MYC and BCL2, and older patients and those with central nervous system involvement. The main research challenges for DLBCL are to accurately identify molecular subsets and to determine if specific chemotherapy platforms and targeted agents offer differential benefit. The ultimate goal should be to maximize initial cure rates to improve long-term survival while minimizing toxicity. In particular, a frontline trial should focus on biologically defined risk groups not likely to be cured with cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab (R-CHOP). An additional challenge is to develop effective and personalized strategies in the relapsed setting, for which there is no current standard other than autologous stem cell transplantation, which benefits a progressively smaller proportion of patients. Relapsed/refractory DLBCL is the ideal setting for testing novel agents and new biomarker tools and will require a national call for biopsies to optimize discovery in this setting. Accordingly, the development of tools with both prognostic and predictive utility and the individualized application of new therapies should be the main priorities. This report identifies clinical research priorities for critical areas of unmet need in this disease.

  8. The A Allele of the Single-Nucleotide Polymorphism rs630923 Creates a Binding Site for MEF2C Resulting in Reduced CXCR5 Promoter Activity in B-Cell Lymphoblastic Cell Lines.

    PubMed

    Mitkin, Nikita A; Muratova, Alisa M; Schwartz, Anton M; Kuprash, Dmitry V

    2016-01-01

    Chemokine receptor CXCR5 is highly expressed in B-cells and under normal conditions is involved in their migration to specific areas of secondary lymphoid organs. B-cells are known to play an important role in various autoimmune diseases including multiple sclerosis (MS) where areas of demyelinating lesions attract B-cells by overexpressing CXCL13, the CXCR5 ligand. In this study, we aimed to determine the functional significance of single-nucleotide polymorphism rs630923 (A/C), which is located in cxcr5 gene promoter, and its common allele is associated with increased risk of MS. Using bioinformatics and pull-down assay in B-lymphoblastic cell lines, we showed that protective minor rs630923 "A" allele created functional binding site for MEF2C transcription factor. Elevated MEF2C expression in B-cells correlated with reduced activity of cxcr5 promoter containing rs630923 "A" allele. This effect that was fully neutralized by MEF2C-directed siRNA may mechanistically explain the protective role of the rs630923 minor allele in MS. Using site-directed mutagenesis of the cxcr5 gene promoter, we were unable to find any experimental evidence for the previously proposed role of NFκB transcription factors in rs630923-mediated CXCR5 promoter regulation. Thus, our results identify MEF2C as a possible mediator of protective function of the rs630923 "A" allele in MS.

  9. CD23 can negatively regulate B-cell receptor signaling

    PubMed Central

    Liu, Chaohong; Richard, Katharina; Wiggins, Melvin; Zhu, Xiaoping; Conrad, Daniel H.; Song, Wenxia

    2016-01-01

    CD23 has been implicated as a negative regulator of IgE and IgG antibody responses. However, whether CD23 has any role in B-cell activation remains unclear. We examined the expression of CD23 in different subsets of peripheral B cells and the impact of CD23 expression on the early events of B-cell receptor (BCR) activation using CD23 knockout (KO) mice. We found that in addition to marginal zone B cells, mature follicular B cells significantly down regulate the surface expression level of CD23 after undergoing isotype switch and memory B-cell differentiation. Upon stimulation with membrane-associated antigen, CD23 KO causes significant increases in the area of B cells contacting the antigen-presenting membrane and the magnitude of BCR clustering. This enhanced cell spreading and BCR clustering is concurrent with increases in the levels of phosphorylation of tyrosine and Btk, as well as the levels of F-actin and phosphorylated Wiskott Aldrich syndrome protein, an actin nucleation promoting factor, in the contract zone of CD23 KO B cells. These results reveal a role of CD23 in the negative regulation of BCR signaling in the absence of IgE immune complex and suggest that CD23 down-regulates BCR signaling by influencing actin-mediated BCR clustering and B-cell morphological changes. PMID:27181049

  10. The rap GTPases regulate B cell morphology, immune-synapse formation, and signaling by particulate B cell receptor ligands.

    PubMed

    Lin, Kevin B L; Freeman, Spencer A; Zabetian, Saba; Brugger, Hayley; Weber, Michele; Lei, Victor; Dang-Lawson, May; Tse, Kathy W K; Santamaria, Rene; Batista, Facundo D; Gold, Michael R

    2008-01-01

    B lymphocytes spread and extend membrane processes when searching for antigens and form immune synapses upon contacting cells that display antigens on their surface. Although these dynamic morphological changes facilitate B cell activation, the signaling pathways underlying these processes are not fully understood. We found that activation of the Rap GTPases was essential for these changes in B cell morphology. Rap activation was important for B cell receptor (BCR)- and lymphocyte-function-associated antigen-1 (LFA-1)-induced spreading, for BCR-induced immune-synapse formation, and for particulate BCR ligands to induce localized F-actin assembly and membrane-process extension. Rap activation and F-actin assembly were also required for optimal BCR signaling in response to particulate antigens but not soluble antigens. Thus by controlling B cell morphology and cytoskeletal organization, Rap might play a key role in the activation of B cells by particulate and cell-associated antigens.

  11. Nivolumab With or Without Varlilumab in Treating Patients With Relapsed or Refractory Aggressive B-cell Lymphomas

    ClinicalTrials.gov

    2017-03-13

    Activated B-Cell-Like Diffuse Large B-Cell Lymphoma; ALK-Positive Large B-Cell Lymphoma; Atypical Burkitt/Burkitt-Like Lymphoma; Diffuse Large B-Cell Lymphoma Associated With Chronic Inflammation; Diffuse Large B-Cell Lymphoma, Not Otherwise Specified; Epstein-Barr Virus Positive Diffuse Large B-Cell Lymphoma of the Elderly; Epstein-Barr Virus-Positive Mucocutaneous Ulcer; Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma; High-Grade B-Cell Lymphoma With MYC and BCL2 and/or BCL6 Rearrangements; Human Herpesvirus-8-Positive Neoplastic Cells Present; Intravascular Large B-Cell Lymphoma; MYC-Negative B-Cell Lymphoma With 11q Aberration Resembling Burkitt Lymphoma; Plasmablastic Lymphoma; Primary Cutaneous Diffuse Large B-Cell Lymphoma; Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type; Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System; Primary Effusion Lymphoma; Recurrent Adult Burkitt Lymphoma; Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Lymphomatoid Granulomatosis; Recurrent Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Refractory Burkitt Lymphoma; Refractory Diffuse Large B-Cell Lymphoma; Refractory Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Skin Ulcer; Small Intestinal B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma; T-Cell/Histiocyte-Rich Large B-Cell Lymphoma

  12. The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

    SciTech Connect

    Muscarella, Donna E. Bloom, Stephen E.

    2008-04-01

    The c-Jun N-terminal kinase (JNK) pathway can play paradoxical roles as either a pro-survival or a pro-cell death pathway depending on type of stress and cell type. The goal of the present study was to determine the role of JNK pathway signaling for regulating B-cell apoptosis in two important but contrasting situations-global proteotoxic damage, induced by arsenite and hyperthermia, versus specific microtubule inhibition, induced by the anti-cancer drug vincristine, using the EW36 B-cell line. This cell line over-expresses the Bcl-2 protein and is a useful model to identify treatments that can overcome multi-drug resistance in lymphoid cells. Exposure of EW36 B-cells to arsenite or lethal hyperthermia resulted in activation of the JNK pathway and induction of apoptosis. However, pharmacological inhibition of the JNK pathway did not inhibit apoptosis, indicating that JNK pathway activation is not required for apoptosis induction by these treatments. In contrast, vincristine treatment of EW36 B-cells resulted in JNK activation and apoptosis that was suppressed by JNK inhibition. A critical difference between the two types of stress treatments was that only vincristine-induced JNK activation resulted in phosphorylation of Bcl-2 at threonine-56, a modification that can block its anti-apoptotic function. Importantly, Bcl-2 phosphorylation was attenuated by JNK inhibition implicating JNK as the upstream kinase. Furthermore, arsenite and hyperthermia treatments activated a p53/p21 pathway associated with apoptosis induction, whereas vincristine did not activate this pathway. These results reveal two stress-activated pathways, one JNK-dependent and another JNK-independent, either of which can bypass Bcl-2 mediated resistance, resulting in cell death.

  13. Design of Coltuximab Ravtansine, a CD19-Targeting Antibody-Drug Conjugate (ADC) for the Treatment of B-Cell Malignancies: Structure-Activity Relationships and Preclinical Evaluation.

    PubMed

    Hong, E Erica; Erickson, Hans; Lutz, Robert J; Whiteman, Kathleen R; Jones, Gregory; Kovtun, Yelena; Blanc, Veronique; Lambert, John M

    2015-06-01

    , suggesting that hepatic catabolism of the payload to less cytotoxic maytansinoid species contributes to the overall therapeutic window of SAR3419. This compound is currently in phase II clinical evaluation for the treatment of diffuse large B cell lymphoma.

  14. Predicting falls within the elderly community: comparison of postural sway, reaction time, the Berg balance scale and the Activities-specific Balance Confidence (ABC) scale for comparing fallers and non-fallers.

    PubMed

    Lajoie, Y; Gallagher, S P

    2004-01-01

    Simple reaction time, the Berg balance scale, the Activities-specific Balance Confidence (ABC) scale and postural sway were studied in order to determine cut-off scores as well as develop a model used in the prevention of fallers within the elderly community. One hundred and twenty-five subjects, 45 fallers and 80 non-fallers were evaluated throughout the study and results indicated that non-fallers have significantly faster reaction times, have higher scores on the Berg balance scale and the ABC scale as well as sway at slower frequencies when compared to fallers. Furthermore, all risk factors were subsequently entered into a logistic regression analysis and results showed that reaction time, the total Berg score and the total ABC score contributed significantly to the prediction of falls with 89% sensitivity and 96% specificity. A second logistic regression was carried out with the same previous variables as well as all questions of the Berg and ABC scales. Results from the logistic analysis revealed that three variables were associated with fall status with 91% sensitivity and 97% specificity. Results from the following study would seem rather valuable as an assessment tool for health care professionals in the identification and monitoring of potential fallers within nursing homes and throughout the community.

  15. Stimulation of Src family protein-tyrosine kinases as a proximal and mandatory step for SYK kinase-dependent phospholipase Cgamma2 activation in lymphoma B cells exposed to low energy electromagnetic fields.

    PubMed

    Dibirdik, I; Kristupaitis, D; Kurosaki, T; Tuel-Ahlgren, L; Chu, A; Pond, D; Tuong, D; Luben, R; Uckun, F M

    1998-02-13

    Here, we present evidence that exposure of DT40 lymphoma B cells to low energy electromagnetic field (EMF) results in a tyrosine kinase-dependent activation of phospholipase Cgamma2 (PLC-gamma2) leading to increased inositol phospholipid turnover. B cells rendered PLC-gamma2-deficient by targeted disruption of the PLC-gamma2 gene as well as PLC-gamma2-deficient cells reconstituted with Src homology domain 2 (SH2) domain mutant PLC-gamma2 did not show any increase in inositol-1,4,5-trisphosphate levels after EMF exposure, providing direct evidence that PLC-gamma2 is responsible for EMF-induced stimulation of inositol phospholipid turnover, and its SH2 domains are essential for this function. B cells rendered SYK-deficient by targeted disruption of the syk gene did not show PLC-gamma2 activation in response to EMF exposure. The C-terminal SH2 domain of SYK kinase is essential for its ability to activate PLC-gamma2. SYK-deficient cells reconstituted with a C-terminal SH2 domain mutant syk gene failed to elicit increased inositol phospholipid turnover after EMF exposure, whereas SYK-deficient cells reconstituted with an N-terminal SH2 domain mutant syk gene showed a normal EMF response. LYN kinase is essential for the initiation of this biochemical signaling cascade. Lymphoma B cells rendered LYN-deficient through targeted disruption of the lyn gene did not elicit enhanced inositol phospholipid turnover after EMF exposure. Introduction of the wild-type (but not a kinase domain mutant) mouse fyn gene into LYN-deficient B cells restored their EMF responsiveness. B cells reconstituted with a SH2 domain mutant fyn gene showed a normal EMF response, whereas no increase in inositol phospholipid turnover in response to EMF was noticed in LYN-deficient cells reconstituted with a SH3 domain mutant fyn gene. Taken together, these results indicate that EMF-induced PLC-gamma2 activation is mediated by LYN-regulated stimulation of SYK, which acts downstream of LYN kinase and

  16. Involvement of B cells in non-infectious uveitis

    PubMed Central

    Smith, Justine R; Stempel, Andrew J; Bharadwaj, Arpita; Appukuttan, Binoy

    2016-01-01

    Non-infectious uveitis—or intraocular inflammatory disease—causes substantial visual morbidity and reduced quality of life amongst affected individuals. To date, research of pathogenic mechanisms has largely been focused on processes involving T lymphocyte and/or myeloid leukocyte populations. Involvement of B lymphocytes has received relatively little attention. In contrast, B-cell pathobiology is a major field within general immunological research, and large clinical trials have showed that treatments targeting B cells are highly effective for multiple systemic inflammatory diseases. B cells, including the terminally differentiated plasma cell that produces antibody, are found in the human eye in different forms of non-infectious uveitis; in some cases, these cells outnumber other leukocyte subsets. Recent case reports and small case series suggest that B-cell blockade may be therapeutic for patients with non-infectious uveitis. As well as secretion of antibody, B cells may promote intraocular inflammation by presentation of antigen to T cells, production of multiple inflammatory cytokines and support of T-cell survival. B cells may also perform various immunomodulatory activities within the eye. This translational review summarizes the evidence for B-cell involvement in non-infectious uveitis, and considers the potential contributions of B cells to the development and control of the disease. Manipulations of B cells and/or their products are promising new approaches to the treatment of non-infectious uveitis. PMID:26962453

  17. Germinal center B cells recognize antigen through a specialized immune synapse architecture

    PubMed Central

    Nowosad, Carla R.; Spillane, Katelyn M.; Tolar, Pavel

    2016-01-01

    B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we show that in contrast to naive and memory B cells, which gathered antigen towards the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β (PKC-β)–NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T-cell dependent selection of high-affinity B cells in GCs. PMID:27183103

  18. Retinoic acid-induced IgG production in TLR-activated human primary B cells involves ULK1-mediated autophagy.

    PubMed

    Eriksen, Agnete Bratsberg; Torgersen, Maria Lyngaas; Holm, Kristine Lillebø; Abrahamsen, Greger; Spurkland, Anne; Moskaug, Jan Øivind; Simonsen, Anne; Blomhoff, Heidi Kiil

    2015-01-01

    In the present study we have established a vital role of autophagy in retinoic acid (RA)-induced differentiation of toll-like receptor (TLR)-stimulated human B cells into Ig-secreting cells. Thus, RA enhanced autophagy in TLR9- and CD180-stimulated peripheral blood B cells, as revealed by increased levels of the autophagosomal marker LC3B-II, enhanced colocalization between LC3B and the lysosomal marker Lyso-ID, by a larger percentage of cells with more than 5 characteristic LC3B puncta, and by the concomitant reduction in the level of SQSTM1/p62. Furthermore, RA induced expression of the autophagy-inducing protein ULK1 at the transcriptional level, in a process that required the retinoic acid receptor RAR. By inhibiting autophagy with specific inhibitors or by knocking down ULK1 by siRNA, the RA-stimulated IgG production in TLR9- and CD180-mediated cells was markedly reduced. We propose that the identified prominent role of autophagy in RA-mediated IgG-production in normal human B cells provides a novel mechanism whereby vitamin A exerts its important functions in the immune system.

  19. A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells

    PubMed Central

    Lorenzo, Daniela; Duarte, Alejandra; Mundiñano, Juliana; Berguer, Paula; Nepomnaschy, Irene; Piazzon, Isabel

    2016-01-01

    B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed. PMID:27603942

  20. B cells and Autoantibodies: Complex Roles in CNS Injury

    PubMed Central

    Ankeny, Daniel P.; Popovich, Phillip G.

    2010-01-01

    Emerging data indicate that traumatic injury to the brain or spinal cord activates B lymphocytes, culminating in the production of antibodies specific for antigens found within and outside the central nervous system (CNS). In this article, we summarize what is known about the effects of CNS injury on B cells. We outline the potential mechanisms for CNS trauma-induced B cell activation and discuss the potential consequences of these injury-induced B cell responses. Based on recent data, we hypothesize that a subset of autoimmune B cell responses initiated by CNS injury are pathogenic and that targeted inhibition of B cells could improve recovery in brain and spinal cord injured patients. PMID:20691635

  1. Evolution of B Cell Immunity

    PubMed Central

    Sunyer, J. Oriol

    2013-01-01

    Two types of adaptive immune strategies are known to have evolved in vertebrates: the VLR-based system, which is present in jawless organisms and is mediated by VLRA and VLRB lymphocytes, and the BCR/TCR-based system, which is present in jawed species and is provided by B and T cell receptors expressed on B and T cells, respectively. Here we summarize features of B cells and their predecessors in the different animal phyla, focusing the review on B cells from jawed vertebrates. We point out the critical role of nonclassical species and comparative immunology studies in the understanding of B cell immunity. Because nonclassical models include species relevant to veterinary medicine, basic science research performed in these animals contributes to the knowledge required for the development of more efficacious vaccines against emerging pathogens. PMID:25340015

  2. B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes

    PubMed Central

    Packard, Thomas A.; Smith, Mia J.; Conrad, Francis J.; Johnson, Sara A.; Getahun, Andrew; Lindsay, Robin S.; Hinman, Rochelle M.; Friedman, Rachel S.; Thomas, James W.; Cambier, John C.

    2016-01-01

    B cells have been strongly implicated in the development of human type 1 diabetes and are required for disease in the NOD mouse model. These functions are dependent on B cell antigen receptor (BCR) specificity and expression of MHC, implicating linked autoantigen recognition and presentation to effector T cells. BCR-antigen affinity requirements for participation in disease are unclear. We hypothesized that BCR affinity for the autoantigen insulin differentially affects lymphocyte functionality, including tolerance modality and the ability to acquire and become activated in the diabetogenic environment. Using combined transgenic and retrogenic heavy and light chain to create multiple insulin-binding BCRs, we demonstrate that affinity for insulin is a critical determinant of the function of these autoreactive cells. We show that both BCR affinity for insulin and genetic background affect tolerance induction in immature B cells. We also find new evidence that may explain the enigmatic ability of B cells expressing 125 anti-insulin BCR to support development of TID in NOD mice despite a reported affinity beneath requirements for binding insulin at in vivo concentrations. We report that when expressed as an antigen receptor the affinity of 125 is much higher than determined by measurements of the soluble form. Finally, we show that in vivo acquisition of insulin requires both sufficient BCR affinity and permissive host/tissue environment. We propose that a confluence of BCR affinity, pancreas environment, and B cell tolerance-regulating genes in the NOD animal allows acquisition of insulin and autoimmunity. PMID:27834793

  3. Subtle Structural Differences Trigger Inhibitory Activity of Propafenone Analogues at the Two Polyspecific ABC Transporters: P‐Glycoprotein (P‐gp) and Breast Cancer Resistance Protein (BCRP)

    PubMed Central

    Schwarz, Theresa; Montanari, Floriane; Cseke, Anna; Wlcek, Katrin; Visvader, Lene; Palme, Sarah; Chiba, Peter; Kuchler, Karl; Urban, Ernst

    2016-01-01

    Abstract The transmembrane ABC transporters P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are widely recognized for their role in cancer multidrug resistance and absorption and distribution of compounds. Furthermore, they are linked to drug–drug interactions and toxicity. Nevertheless, due to their polyspecificity, a molecular understanding of the ligand‐transporter interaction, which allows designing of both selective and dual inhibitors, is still in its infancy. This study comprises a combined approach of synthesis, in silico prediction, and in vitro testing to identify molecular features triggering transporter selectivity. Synthesis and testing of a series of 15 propafenone analogues with varied rigidity and basicity of substituents provide first trends for selective and dual inhibitors. Results indicate that both the flexibility of the substituent at the nitrogen atom, as well as the basicity of the nitrogen atom, trigger transporter selectivity. Furthermore, inhibitory activity of compounds at P‐gp seems to be much more influenced by logP than those at BCRP. Exploiting these differences further should thus allow designing specific inhibitors for these two polyspecific ABC‐transporters. PMID:26970257

  4. Transformation of Lettuce with rol ABC Genes: Extracts Show Enhanced Antioxidant, Analgesic, Anti-Inflammatory, Antidepressant, and Anticoagulant Activities in Rats.

    PubMed

    Ismail, Hammad; Dilshad, Erum; Waheed, Mohammad Tahir; Mirza, Bushra

    2017-03-01

    Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91-102 % increase in total phenolic contents and 53-65 % increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133 % in transformed plants, respectively. Results of DPPH assay showed maximum 51 % increase, and lipid peroxidation assay exhibited 20 % increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80 % inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45 % inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.

  5. Characterisation and expression analysis of B-cell activating factor (BAFF) in spiny dogfish (Squalus acanthias): cartilaginous fish BAFF has a unique extra exon that may impact receptor binding.

    PubMed

    Li, Ronggai; Dooley, Helen; Wang, Tiehui; Secombes, Christopher J; Bird, Steve

    2012-04-01

    B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system.

  6. B-Cell-Mediated Strategies to Fight Chronic Allograft Rejection

    PubMed Central

    Dalloul, Ali

    2013-01-01

    Solid organs have been transplanted for decades. Since the improvement in graft selection and in medical and surgical procedures, the likelihood of graft function after 1 year is now close to 90%. Nonetheless even well-matched recipients continue to need medications for the rest of their lives hence adverse side effects and enhanced morbidity. Understanding Immune rejection mechanisms, is of increasing importance since the greater use of living-unrelated donors and genetically unmatched individuals. Chronic rejection is devoted to T-cells, however the role of B-cells in rejection has been appreciated recently by the observation that B-cell depletion improve graft survival. By contrast however, B-cells can be beneficial to the grafted tissue. This protective effect is secondary to either the secretion of protective antibodies or the induction of B-cells that restrain excessive inflammatory responses, chiefly by local provision of IL-10, or inhibit effector T-cells by direct cellular interactions. As a proof of concept B-cell-mediated infectious transplantation tolerance could be achieved in animal models, and evidence emerged that the presence of such B-cells in transplanted patients correlate with a favorable outcome. Among these populations, regulatory B-cells constitute a recently described population. These cells may develop as a feedback mechanism to prevent uncontrolled reactivity to antigens and inflammatory stimuli. The difficult task for the clinician, is to quantify the respective ratios and functions of “tolerant” vs. effector B-cells within a transplanted organ, at a given time point in order to modulate B-cell-directed therapy. Several receptors at the B-cell membrane as well as signaling molecules, can now be targeted for this purpose. Understanding the temporal expansion of regulatory B-cells in grafted patients and the stimuli that activate them will help in the future to implement specific strategies aimed at fighting chronic allograft

  7. CD23 surface density on B cells is associated with IgE levels and determines IgE-facilitated allergen uptake, as well as activation of allergen-specific T cells

    PubMed Central

    Selb, Regina; Eckl-Dorna, Julia; Neunkirchner, Alina; Schmetterer, Klaus; Marth, Katharina; Gamper, Jutta; Jahn-Schmid, Beatrice; Pickl, Winfried F.; Valenta, Rudolf; Niederberger, Verena

    2017-01-01

    Background Increasing evidence suggests that the low-affinity receptor for IgE, CD23, plays an important role in controlling the activity of allergen-specific T cells through IgE-facilitated allergen presentation. Objective We sought to determine the number of CD23 molecules on immune cells in allergic patients and to investigate whether the number of CD23 molecules on antigen-presenting cells is associated with IgE levels and influences allergen uptake and allergen-specific T-cell activation. Methods Numbers of CD23 molecules on immune cells of allergic patients were quantified by using flow cytometry with QuantiBRITE beads and compared with total and allergen-specific IgE levels, as well as with allergen-induced immediate skin reactivity. Allergen uptake and allergen-specific T-cell activation in relation to CD23 surface density were determined by using flow cytometry in combination with confocal microscopy and T cells transfected with the T-cell receptor specific for the birch pollen allergen Bet v 1, respectively. Defined IgE-allergen immune complexes were formed with human monoclonal allergen-specific IgE and Bet v 1. Results In allergic patients the vast majority of CD23 molecules were expressed on naive IgD+ B cells. The density of CD23 molecules on B cells but not the number of CD23+ cells correlated with total IgE levels (RS = 0.53, P = .03) and allergen-induced skin reactions (RS = 0.63, P = .008). Uptake of allergen-IgE complexes into B cells and activation of allergen-specific T cells depended on IgE binding to CD23 and were associated with CD23 surface density. Addition of monoclonal IgE to cultured PBMCs significantly (P = .04) increased CD23 expression on B cells. Conclusion CD23 surface density on B cells of allergic patients is correlated with allergen-specific IgE levels and determines allergen uptake and subsequent activation of T cells. PMID:27372566

  8. Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23.

    PubMed Central

    Cordier, M; Calender, A; Billaud, M; Zimber, U; Rousselet, G; Pavlish, O; Banchereau, J; Tursz, T; Bornkamm, G; Lenoir, G M

    1990-01-01

    A set of B-cell activation molecules, including the Epstein-Barr virus (EBV) receptor CR2 (CD21) and the B-cell activation antigen CD23 (Blast2/Fc epsilon RII), is turned on by infecting EBV-negative B-lymphoma cell lines with immortalizing strains of the viruslike B95-8 (BL/B95 cells). This up regulation may represent one of the mechanisms involved in EBV-mediated B-cell immortalization. The P3HR1 nonimmortalizing strain of the virus, which is deleted for the entire Epstein-Barr nuclear antigen 2 (EBNA2) protein open reading frame, is incapable of inducing the expression of CR2 and CD23, suggesting a crucial role for EBNA2 in the activation of these molecules. In addition, lymphoma cells containing the P3HR1 genome (BL/P3HR1 cells) do not express the viral latent membrane protein (LMP), which is regularly expressed in cells infected with immortalizing viral strains. Using electroporation, we have transfected the EBNA2 gene cloned in an episomal vector into BL/P3HR1 cells and have obtained cell clones that stably express the EBNA2 protein. In these clones, EBNA2 expression was associated with an increased amount of CR2 and CD23 steady-state RNAs. Of the three species of CD23 mRNAs described, the Fc epsilon RIIa species was preferentially expressed in these EBNA2-expressing clones. An increased cell surface expression of CR2 but not of CD23 was observed, and the soluble form of CD23 molecule (SCD23) was released. We were, however, not able to detect any expression of LMP in these cell clones. These data demonstrate that EBNA2 gene is able to complement P3HR1 virus latent functions to induce the activation of CR2 and CD23 expression, and they emphasize the role of EBNA2 protein in the modulation of cellular gene implicated in B-cell proliferation and hence in EBV-mediated B-cell immortalization. Nevertheless, EBNA2 expression in BL/P3HR1 cells is not able to restore the level of CR2 and CD23 expression observed in BL/B95 cells, suggesting that other cellular or viral

  9. Gallium arsenide exposure impairs splenic B cell accessory function.

    PubMed

    Gondre-Lewis, Timothy A; Hartmann, Constance B; Caffrey, Rebecca E; McCoy, Kathleen L

    2003-03-01

    Gallium arsenide (GaAs) is utilized in industries for its semiconductor and optical properties. Chemical exposure of animals systemically suppresses several immune functions. The ability of splenic B cells to activate antigen-specific helper CD4(+) T cell hybridomas was assessed, and various aspects of antigen-presenting cell function were examined. GaAs-exposed murine B cells were impaired in processing intact soluble protein antigens, and the defect was antigen dependent. In contrast, B cells after exposure competently presented peptides to the T cells, which do not require processing. Cell surface expression of major histocompatibility complex (MHC) class II molecules and several costimulatory molecules on splenic B cells, which are critical for helper T cell activation, was not affected by chemical exposure. GaAs exposure also did not influence the stability of MHC class II heterodimers, suggesting that the defect may precede peptide exchange. GaAs-exposed B cells contained a normal level of aspartyl cathepsin activity; however, proteolytic activities of thiol cathepsins B and L were approximately half the control levels. Furthermore, two cleavage fragments of invariant chain, a molecular chaperone of MHC class II molecules, were increased in GaAs-exposed B cells, indicative of defective degradation. Thus, diminished thiol proteolytic activity in B cells may be responsible for their impaired antigen processing and invariant chain degradation, which may contribute to systemic immunosuppression caused by GaAs exposure.

  10. Do You Know Your ABC?

    ERIC Educational Resources Information Center

    Neale, Claire

    2013-01-01

    Within primary schools, the core subjects of literacy and numeracy are highly regarded, and rightly so, as children need to learn to read, write and be numerically literate. This means that all children learn their ABCs at an early age, But, what about the "other ABC"--"Airway, Breathing and Circulation?" Accidents and medical…

  11. Essential role for B cells in transplantation tolerance

    PubMed Central

    Redfield, Robert R; Rodriguez, Eduardo; Parsons, Ronald; Vivek, Kumar; Mustafa, Moiz M; Noorchashm, Hooman; Naji, Ali

    2017-01-01

    T lymphocytes are the primary targets of immunotherapy in clinical transplantation. However, B lymphocytes are detrimental to graft survival by virtue of their capacity to present antigen to T cells via the indirect pathway of allorecognition and the generation of donor specific alloantibody. Furthermore, the long-term survival of organ allografts remains challenged by chronic rejection, a process in which activated B cells have been found to play a significant role. Therefore, the achievement of transplantation tolerance will likely require induction of both T and B cell tolerance to alloantigens. Moreover, human and animal investigations have shown that subsets of B cells, Transitional and Regulatory, are inherently tolerogenic. Developing therapeutic strategies that exploit these populations may be key to achieving transplantation tolerance. In this review we describe the current evidence for the essential role of B cells in transplant tolerance and discuss emerging B cell directed strategies to achieve allograft tolerance. PMID:21982511

  12. Manassantin A and B from Saururus chinensis inhibit interleukin-6-induced signal transducer and activator of transcription 3 activation in Hep3B cells.

    PubMed

    Chang, Jong Sun; Lee, Seung Woong; Kim, Myo Sun; Yun, Bo Ra; Park, Mi Hye; Lee, Seok-Geun; Park, Su-Jin; Lee, Woo Song; Rho, Mun-Chual

    2011-01-01

    Inhibition of interleukin-6 (IL-6) has been postulated to be an effective therapy in the pathogenesis of several inflammatory diseases. The current study was performed to examine potential effects of manassantin A and B isolated from Saururus chinensis on the IL-6-induced response to human hepatoma cells. We found that manassantin A and B inhibit signal transducer and activator of transcription 3 (Stat3) activity stimulated by IL-6. We also found that both compounds decreased IL-6-induced Stat3 phosphorylation and nuclear translocation. Both compounds blocked suppressor of cytokine signaling 3 (SOCS-3)-mRNA expression induced by IL-6. In addition, we found that Stat3 inhibitory effects of these compounds could be related to protein tyrosine phosphatase. These findings suggest that manassantin A and B could be useful remedies for treatment of inflammatory diseases by inhibiting IL-6 action.

  13. Spatial alterations between CD4(+) T follicular helper, B, and CD8(+) T cells during simian immunodeficiency virus infection: T/B cell homeostasis, activation, and potential mechanism for viral escape.

    PubMed

    Hong, Jung Joo; Amancha, Praveen K; Rogers, Kenneth; Ansari, Aftab A; Villinger, Francois

    2012-04-01

    HIV/SIV infections induce chronic immune activation with remodeling of lymphoid architecture and hypergammaglobulinemia, although the mechanisms leading to such symptoms remain to be fully elucidated. Moreover, lymph nodes have been highlighted as a predilection site for SIV escape in vivo. Following 20 rhesus macaques infected with SIVmac239 as they progress from pre-infection to acute and chronic infection, we document for the first time, to our knowledge, the local dynamics of T follicular helper (T(FH)) cells and B cells in situ. Progression of SIV infection was accompanied by increased numbers of well-delineated follicles containing germinal centers (GCs) and T(FH) cells with a progressive increase in the density of programmed death-1 (PD-1) expression in lymph nodes. The rise in PD-1(+) T(FH) cells was followed by a substantial accumulation of Ki67(+) B cells within GCs. However, unlike in blood, major increases in the frequency of CD27(+) memory B cells were observed in lymph nodes, indicating increased turnover of these cells, correlated with increases in total and SIV specific Ab levels. Of importance, compared with T cell zones, GCs seemed to exclude CD8(+) T cells while harboring increasing numbers of CD4(+) T cells, many of which are positive for SIVgag, providing an environment particularly beneficial for virus replication and reservoirs. Our data highlight for the first time, to our knowledge, important spatial interactions of GC cell subsets during SIV infection, the capacity of lymphoid tissues to maintain stable relative levels of circulating B cell subsets, and a potential mechanism for viral reservoirs within GCs during SIV infection.

  14. PDK1 regulates B cell differentiation and homeostasis.

    PubMed

    Baracho, Gisele V; Cato, Matthew H; Zhu, Zilu; Jaren, Olav R; Hobeika, Elias; Reth, Michael; Rickert, Robert C

    2014-07-01

    Successful B cell differentiation and prevention of cell transformation depends on balanced and fine-tuned activation of cellular signaling pathways. The phosphatidyl inositol-3 kinase (PI3K) signaling pathway has emerged as a major regulator of B lymphocyte homeostasis and function. Phosphoinositide-dependent protein kinase-1 (PDK1) is the pivotal node in the PI3K pathway, regulating the stability and activity of downstream AGC kinases (including Akt, RSK, S6K, SGK, and PKC). Although the importance of PI3K activity in B cell differentiation is well documented, the role of PDK1 and other downstream effectors is underexplored. Here we used inducible and stage-specific gene targeting approaches to elucidate the role of PDK1 in early and peripheral B cell differentiation. PDK1 ablation enhanced cell cycle entry and apoptosis of IL-7-dependent pro-B cells, blocking Ig synthesis and B cell maturation. PDK1 also was essential for the survival and activation of peripheral B cells via regulation of PKC and Akt-dependent downstream effectors, such as GSK3α/β and Foxo1. We found that PDK1 deletion strongly impaired B cell receptor (BCR) signaling, but IL-4 costimulation was sufficient to restore BCR-induced proliferation. IL-4 also normalized PKCβ activation and hexokinase II expression in BCR-stimulated cells, suggesting that this signaling pathway can act independent of PDK1 to support B cell growth. In summary, our results demonstrate that PDK1 is indispensable for B cell survival, proliferation, and growth regulation.

  15. ABC transporters in fish species: a review

    PubMed Central

    Ferreira, Marta; Costa, Joana; Reis-Henriques, Maria A.

    2014-01-01

    ATP-binding cassette (ABC) proteins were first recognized for their role in multidrug resistance (MDR) in chemotherapeutic treatments, which is a major impediment for the successful treatment of many forms of malignant tumors in humans. These proteins, highly conserved throughout vertebrate species, were later related to cellular detoxification and accounted as responsible for protecting aquatic organisms from xenobiotic insults in the so-called multixenobiotic resistance mechanism (MXR). In recent years, research on these proteins in aquatic species has highlighted their importance in the detoxification mechanisms in fish thus it is necessary to continue these studies. Several transporters have been pointed out as relevant in the ecotoxicological context associated to the transport of xenobiotics, such as P-glycoproteins (Pgps), multidrug-resistance-associated proteins (MRPs 1-5) and breast cancer resistance associated protein (BCRP). In mammals, several nuclear receptors have been identified as mediators of phase I and II metabolizing enzymes and ABC transporters. In aquatic species, knowledge on co-regulation of the detoxification mechanism is scarce and needs to be addressed. The interaction of emergent contaminants that can act as chemosensitizers, with ABC transporters in aquatic organisms can compromise detoxification processes and have population effects and should be studied in more detail. This review intends to summarize the recent advances in research on MXR mechanisms in fish species, focusing in (1) regulation and functioning of ABC proteins; (2) cooperation with phase I and II biotransformation enzymes; and (3) ecotoxicological relevance and information on emergent pollutants with ability to modulate ABC transporters expression and activity. Several lines of evidence are clearly suggesting the important role of these transporters in detoxification mechanisms and must be further investigated in fish to underlay the mechanism to consider their use as

  16. Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking

    PubMed Central

    Flores-Fernández, Rocio; Blanco-Favela, Francisco; Fuentes-Pananá, Ezequiel M.; Chávez-Sánchez, Luis; Gorocica-Rosete, Patricia; Pizaña-Venegas, Alberto; Chávez-Rueda, Adriana Karina

    2016-01-01

    Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE). In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR) as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease. PMID:27314053

  17. [The role of IRA B cells in selected inflammatory processes].

    PubMed

    Zasada, Magdalena; Rutkowska-Zapała, Magdalena; Lenart, Marzena; Kwinta, Przemko

    2016-03-16

    The first report about the discovery of new, previously unknown immune cells named IRA B cells (innate response activator B cells) appeared in 2012. So far, their presence has been verified in both mice and humans. However, IRA B cells belong to the family of B lymphocytes and have a number of characteristics unique to this group of cells. IRA B cells are formed from activated B1a lymphocytes after their contact with a pathogen. B1a lymphocytes mainly reside within body cavities. Activated by the pathogen, they move on into secondary lymphoid organs (spleen, lymph nodes) where they differentiate into IRA B cells. IRA B cells are a rich source of granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF can stimulate IRA B cells in an autocrine manner for the secretion of intracellular stocks of immunoglobulin M (IgM), which can facilitate pathogens' phagocytosis by neutrophils. GM-CSF also stimulates neutrophils into active phagocytosis. Rapid eradication of the pathogen can prevent the development of an excessive inflammatory response, which can be dangerous for the organism. Until now the involvement of IRA B lymphocytes in the pathogenesis of sepsis and pneumonia has been proven, as well as their role in the progression of atherosclerotic lesions in mice. There is research in progress on the possibility of increasing the number of IRA B cells, for example by intravenous supply of modified immunoglobulins. It is necessary to characterize human IRA B cells and to determine their role in the functioning of the immune system.

  18. Analysis of the linker for activation of T cells and the linker for activation of B cells in natural killer cells reveals a novel signaling cassette, dual usage in ITAM signaling, and influence on development of the Ly49 repertoire.

    PubMed

    Whittaker, Gillian C; Burshtyn, Deborah N; Orr, Selinda J; Quigley, Laura; Hodge, Deborah L; Pascal, Véronique; Zhang, Weiguo; McVicar, Daniel W

    2008-10-01

    The linker for activation of T cells (LAT) and the linker for activation of B cells (LAB/NTAL/LAT2) are integral proteins in receptor coupling to downstream events. Both proteins are expressed in natural killer (NK) cells and LAT is phosphorylated during target cell interactions or ligation of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled CD16. Regardless, Lat(-/-) mice exhibit normal natural and antibody-mediated killing. Here we place both LAT and LAB in the DAP12 pathway of NK cells. Moreover, we unveil a LAT-independent pathway that requires expression of Syk. Mice lacking either LAT or LAB have a skewed Ly49 repertoire, and activated NK cells from Lat(-/-) mice have reduced responses to the ITAM-coupled receptor NK1.1. In contrast, resting Lat(-/-) NK cells show intact NK1.1 responses, whereas NK cells without LAB are hyperactive. Elimination of both adaptors severely reduces NK1.1 signaling under both conditions. Together these data show that NK ITAMs preferentially use a signaling cassette regulated by interplay between LAT and LAB. Activation by interleukin-2 causes a shift to greater dependency on LAT due to suppression of Syk signaling. The overlapping use of multiple adaptors permits fine-tuning of NK-cell ITAM responses over the course of an immune response.

  19. Memory B cells: total recall.

    PubMed

    Phan, Tri Giang; Tangye, Stuart G

    2017-03-28

    Immunological memory is a cornerstone of adaptive immune responses in higher vertebrates. The remarkable ability to generate memory cells following Ag exposure, in the context of natural infection or immunization, provides long-lived protection against infectious diseases, often for the hosts' lifetime. Indeed, the generation of memory B cells and long-lived plasma cells underpins the success of most vaccines. The concept of immunological memory is not new-it was first proposed nearly 2500 years ago. While our understanding of the complexities of humoral and cell-mediated memory continues to evolve, important aspects of this process remain unresolved. Here, we will provide an overview of recent advances in B-cell memory in mice and humans, and in health and disease.

  20. Salmonella induces PD-L1 expression in B cells.

    PubMed

    Lopez-Medina, Marcela; Perez-Lopez, Araceli; Alpuche-Aranda, Celia; Ortiz-Navarrete, Vianney

    2015-10-01

    Salmonella persists for a long time in B cells; however, the mechanism(s) through which infected B cells avoid effector CD8 T cell responses has not been characterized. In this study, we show that Salmonella infects and survives within all B1 and B2 cell subpopulations. B cells are infected with a Salmonella typhimurium strain expressing an ovalbumin (OVA) peptide (SIINFEKL) to evaluate whether B cells process and present Salmonella antigens in the context of MHC-I molecules. Our data showed that OVA peptides are presented by MHC class I K(b)-restricted molecules and the presented antigen is generated through proteasomal degradation and vacuolar processing. In addition, Salmonella-infected B cells express co-stimulatory molecules such as CD40, CD80, and CD86 as well as inhibitory molecules such as PD-L1. Thus, the cross-presentation of Salmonella antigens and the expression of activation molecules suggest that infected B cells are able to prime and activate specific CD8(+) T cells. However, the Salmonella infection-stimulated expression of PD-L1 suggests that the PD-1/PD-L1 pathway may be involved in turning off the cytotoxic effector response during Salmonella persistent infection, thereby allowing B cells to become a reservoir for the bacteria.

  1. Human Memory B Cells in Healthy Gingiva, Gingivitis, and Periodontitis.

    PubMed

    Mahanonda, Rangsini; Champaiboon, Chantrakorn; Subbalekha, Keskanya; Sa-Ard-Iam, Noppadol; Rattanathammatada, Warattaya; Thawanaphong, Saranya; Rerkyen, Pimprapa; Yoshimura, Fuminobu; Nagano, Keiji; Lang, Niklaus P; Pichyangkul, Sathit

    2016-08-01

    The presence of inflammatory infiltrates with B cells, specifically plasma cells, is the hallmark of periodontitis lesions. The composition of these infiltrates in various stages of homeostasis and disease development is not well documented. Human tissue biopsies from sites with gingival health (n = 29), gingivitis (n = 8), and periodontitis (n = 21) as well as gingival tissue after treated periodontitis (n = 6) were obtained and analyzed for their composition of B cell subsets. Ag specificity, Ig secretion, and expression of receptor activator of NF-κB ligand and granzyme B were performed. Although most of the B cell subsets in healthy gingiva and gingivitis tissues were CD19(+)CD27(+)CD38(-) memory B cells, the major B cell component in periodontitis was CD19(+)CD27(+)CD38(+)CD138(+)HLA-DR(low) plasma cells, not plasmablasts. Plasma cell aggregates were observed at the base of the periodontal pocket and scattered throughout the gingiva, especially apically toward the advancing front of the lesion. High expression of CXCL12, a proliferation-inducing ligand, B cell-activating factor, IL-10, IL-6, and IL-21 molecules involved in local B cell responses was detected in both gingivitis and periodontitis tissues. Periodontitis tissue plasma cells mainly secreted IgG specific to periodontal pathogens and also expressed receptor activator of NF-κB ligand, a bone resorption cytokine. Memory B cells resided in the connective tissue subjacent to the junctional epithelium in healthy gingiva. This suggested a role of memory B cells in maintaining periodontal homeostasis.

  2. Tissue and developmental expression of a gene from Hessian fly encoding an ABC-active-transporter protein during interactions with wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report on the transcriptional patterns of a putative white (w) gene encoding an ABC-transporter protein during development in Hessian fly, Mayetiola destructor. The deduced amino acid sequence for the Hessian fly white showed 77 to 74% similarities to white/ATP-binding-cassette proteins and 57 t...

  3. The protease activity of the paracaspase MALT1 is controlled by monoubiquitination.

    PubMed

    Pelzer, Christiane; Cabalzar, Katrin; Wolf, Annette; Gonzalez, Montserrat; Lenz, Georg; Thome, Margot

    2013-04-01

    The protease activity of the paracaspase MALT1 is central to lymphocyte activation and lymphomagenesis, but how this activity is controlled remains unknown. Here we identify a monoubiquitination of MALT1 on Lys644 that activated the protease function of MALT1. Monoubiquitinated MALT1 had enhanced protease activity, whereas a ubiquitination-deficient MALT1 mutant with replacement of that lysine with arginine (MALT1(K644R)) had less protease activity, which correlated with impaired induction of interleukin 2 (IL-2) via the T cell antigen receptor in activated T cells. Expression of MALT1(K644R) diminished the survival of cells derived from diffuse large B cell lymphoma of the activated B cell-like subtype (ABC DLBCL), which require constitutive protease activity of MALT1 for survival. Thus, monoubiquitination of MALT1 is essential for its catalytic activation and is therefore a potential target for the treatment of ABC-DLBCL and for immunomodulation.

  4. Regulatory B cells in human inflammatory and autoimmune diseases: from mouse models to clinical research.

    PubMed

    Miyagaki, Tomomitsu; Fujimoto, Manabu; Sato, Shinichi

    2015-10-01

    B cells have been generally considered to be positive regulators of immune responses because of their ability to produce antigen-specific antibodies and to activate T cells through antigen presentation. Impairment of B cell development and function may cause inflammatory and autoimmune diseases. Recently, specific B cell subsets that can negatively regulate immune responses have been described in mouse models of a wide variety of inflammatory and autoimmune diseases. The concept of those B cells, termed regulatory B cells, is now recognized as important in the murine immune system. Among several regulatory B cell subsets, IL-10-producing regulatory B cells are the most widely investigated. On the basis of discoveries from studies of such mice, human regulatory B cells that produce IL-10 in most cases are becoming an active area of research. There have been emerging data suggesting the importance of human regulatory B cells in various diseases. Revealing the immune regulation mechanisms of human regulatory B cells in human inflammatory and autoimmune diseases could lead to the development of novel B cell targeted therapies. This review highlights the current knowledge on regulatory B cells, mainly IL-10-producing regulatory B cells, in animal models of inflammatory and autoimmune diseases and in clinical research using human samples.

  5. Distinct functions for the transcription factor Foxo1 at various stages of B cell differentiation.

    PubMed

    Dengler, Hart S; Baracho, Gisele V; Omori, Sidne A; Bruckner, Shane; Arden, Karen C; Castrillon, Diego H; DePinho, Ronald A; Rickert, Robert C

    2008-12-01

    The transcription factors Foxo1, Foxo3 and Foxo4 modulate cell fate 'decisions' in diverse systems. Here we show that Foxo1-dependent gene expression was critical at many stages of B cell differentiation. Early deletion of Foxo1 caused a substantial block at the pro-B cell stage due to a failure to express interleukin 7 receptor-alpha. Foxo1 inactivation in late pro-B cells resulted in an arrest at the pre-B cell stage due to lower expression of the recombination-activating genes Rag1 and Rag2. Deletion of Foxo1 in peripheral B cells led to fewer lymph node B cells due to lower expression of L-selectin and failed class-switch recombination due to impaired upregulation of the gene encoding activation-induced cytidine deaminase. Thus, Foxo1 regulates a transcriptional program that is essential for early B cell development and peripheral B cell function.

  6. Gene-expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy.

    PubMed

    Gutiérrez-García, Gonzalo; Cardesa-Salzmann, Teresa; Climent, Fina; González-Barca, Eva; Mercadal, Santiago; Mate, José L; Sancho, Juan M; Arenillas, Leonor; Serrano, Sergi; Escoda, Lourdes; Martínez, Salomé; Valera, Alexandra; Martínez, Antonio; Jares, Pedro; Pinyol, Magdalena; García-Herrera, Adriana; Martínez-Trillos, Alejandra; Giné, Eva; Villamor, Neus; Campo, Elías; Colomo, Luis; López-Guillermo, Armando

    2011-05-05

    Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell-like (GCB) and activated-B cell-like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

  7. Citalopram protects against cold-restraint stress-induced activation of brain-derived neurotrophic factor and expression of nuclear factor kappa-light-chain-enhancer of activated B cells in rats.

    PubMed

    Garabadu, Debapriya; Reddy, B C M Harshavardhan; Krishnamurthy, Sairam

    2015-02-01

    The present study evaluates the protective effect of citalopram against cold-restraint stress (CRS) paradigm. Rats were pretreated with citalopram (0.1, 1.0, and 10.0 mg/kg) acutely and repeatedly for 21 days before exposure to the CRS procedure. None of the doses of citalopram attenuated CRS-induced gastric ulcers in the acute study. In contrast, repeated pretreatment of citalopram at a dose level of 0.1 mg/kg attenuated the CRS-induced gastric ulcers. Citalopram (0.1 mg/kg) diminished CRS-induced increase in plasma corticosterone, but not plasma norepinephrine level in the chronic study indicating its effect on hypothalamic-pituitary-adrenal axis function. Repeated citalopram (0.1 mg/kg) pretreatment attenuated CRS-induced changes in serotonin turnover in the hippocampus and amygdala. Moreover, repeated pretreatment with citalopram (0.1 mg/kg) mitigated the CRS-induced increase in the expression of brain-derived neurotrophic factor (BDNF) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the hippocampus and amygdala. These results suggest that there is a region- and a dose-specific effect of citalopram on CRS-induced BDNF-NFκB activation. Therefore, citalopram showed antistress activity in the CRS model through changes in the stress-responsive pathways such as hypothalamic-pituitary-adrenal-axis and brain serotonergic system apart from decreasing the expression of BDNF and NFκB.

  8. YY1 Is Required for Germinal Center B Cell Development

    PubMed Central

    Vuyyuru, Raja; Jha, Vibha; Hodewadekar, Suchita; Manser, Tim; Atchison, Michael L.

    2016-01-01

    YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during