scholarly journals Unbalanced Expression of Bcl-2 Family Proteins in Follicular Lymphoma: Contribution of CD40 Signaling in Promoting Survival

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 244-251 ◽  
Author(s):  
Paolo Ghia ◽  
Vassiliki A. Boussiotis ◽  
Joachim L. Schultze ◽  
Angelo A. Cardoso ◽  
David M. Dorfman ◽  
...  
Keyword(s):  
B Cells ◽  
Follicular Lymphoma ◽  
Germinal Center ◽  
Conventional Treatment ◽  
Clinical Course ◽  
Critical Role ◽  
Relative Resistance ◽  
Constitutive Overexpression

Abstract Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.

scholarly journals Unbalanced Expression of Bcl-2 Family Proteins in Follicular Lymphoma: Contribution of CD40 Signaling in Promoting Survival

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 244-251 ◽  
Author(s):  
Paolo Ghia ◽  
Vassiliki A. Boussiotis ◽  
Joachim L. Schultze ◽  
Angelo A. Cardoso ◽  
David M. Dorfman ◽  
...  
Keyword(s):  
B Cells ◽  
Follicular Lymphoma ◽  
Germinal Center ◽  
Conventional Treatment ◽  
Clinical Course ◽  
Critical Role ◽  
Relative Resistance ◽  
Constitutive Overexpression

Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.

scholarly journals Covalent Linkage of HIV-1 Trimers to Synthetic Liposomes Elicits Improved B Cell and Antibody Responses

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Shridhar Bale ◽  
Geraldine Goebrecht ◽  
Armando Stano ◽  
Richard Wilson ◽  
Takayuki Ota ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Neutralizing Antibodies ◽  
Tier 2 ◽  
Covalent Linkage ◽  
His Tag ◽  
Hiv 1

ABSTRACT We have demonstrated that a liposomal array of well-ordered trimers enhances B cell activation, germinal center formation, and the elicitation of tier-2 autologous neutralizing antibodies. Previously, we coupled well-ordered cleavage-independent NFL trimers via their C-terminal polyhistidine tails to nickel lipids integrated into the lipid bilayer. Despite favorable in vivo effects, concern remained over the potentially longer-term in vivo instability of noncovalent linkage of the trimers to the liposomes. Accordingly, we tested both cobalt coupling and covalent linkage of the trimers to the liposomes by reengineering the polyhistidine tail to include a free cysteine on each protomer of model BG505 NFL trimers to allow covalent linkage. Both cobalt and cysteine coupling resulted in a high-density array of NFL trimers that was stable in both 20% mouse serum and 100 mM EDTA, whereas the nickel-conjugated trimers were not stable under these conditions. Binding analysis and calcium flux with anti-Env-specific B cells confirmed that the trimers maintained conformational integrity following coupling. Following immunization of mice, serologic analysis demonstrated that the covalently coupled trimers elicited Env-directed antibodies in a manner statistically significantly improved compared to soluble trimers and nickel-conjugated trimers. Importantly, the covalent coupling not only enhanced gp120-directed responses compared to soluble trimers, it also completely eliminated antibodies directed to the C-terminal His tag located at the “bottom” of the spike. In contrast, soluble and noncovalent formats efficiently elicited anti-His tag antibodies. These data indicate that covalent linkage of well-ordered trimers to liposomes in high-density array displays multiple advantages in vitro and in vivo. IMPORTANCE Enveloped viruses typically encode a surface-bound glycoprotein that mediates viral entry into host cells and is a primary target for vaccine design. Liposomes with modified lipid head groups have a unique feature of capturing and displaying antigens on their surfaces, mimicking the native pathogens. Our first-generation nickel-based liposomes captured HIV-1 Env glycoprotein trimers via a noncovalent linkage with improved efficacy over soluble glycoprotein in activating germinal center B cells and eliciting tier-2 autologous neutralizing antibodies. In this study, we report the development of second-generation cobalt- and maleimide-based liposomes that have improved in vitro stability over nickel-based liposomes. In particular, the maleimide liposomes captured HIV-1 Env trimers via a more stable covalent bond, resulting in enhanced germinal center B cell responses that generated higher antibody titers than the soluble trimers and liposome-bearing trimers via noncovalent linkages. We further demonstrate that covalent coupling prevents release of the trimers prior to recognition by B cells and masks a nonneutralizing determinant located at the bottom of the trimer.

scholarly journals Blimp-1-Dependent Repression of Pax-5 Is Required for Differentiation of B Cells to Immunoglobulin M-Secreting Plasma Cells

2002 ◽  
Vol 22 (13) ◽  
pp. 4771-4780 ◽  
Author(s):  
Kuo-I Lin ◽  
Cristina Angelin-Duclos ◽  
Tracy C. Kuo ◽  
Kathryn Calame
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Critical Role ◽  
Cell Lineage ◽  
Immunoglobulin M ◽  
Dependent Manner ◽  
Plasmacytic Differentiation

ABSTRACT B-cell lineage-specific activator protein (BSAP), encoded by the Pax-5 gene, is critical for B-cell lineage commitment and B-cell development but is not expressed in terminally differentiated B cells. We demonstrate a direct connection between BSAP and B-lymphocyte-induced maturation protein 1 (Blimp-1), a transcriptional repressor that is sufficient to drive plasmacytic differentiation. Blimp-1 binds a site on the Pax-5 promoter in vitro and in vivo and represses the Pax-5 promoter in a binding-site-dependent manner. By ectopically expressing Blimp-1 or a competitive inhibitor of Blimp-1, we show that Blimp-1 is both necessary and sufficient to repress Pax-5 during plasmacytic differentiation of primary splenic B cells. Blimp-1-dependent repression of Pax-5 is sufficient to regulate BSAP targets CD19 and J chain and is necessary but not sufficient to induce XBP-1. We further show that repression of Pax-5 is required for Blimp-1 to drive differentiation of splenocytes to immunoglobulin M-secreting cells. Thus, repression of Pax-5 plays a critical role in the Blimp-1-dependent program of plasmacytic differentiation.

Dysregulation of MMSET Is Tumorigenic In-Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 74-74 ◽  
Author(s):  
Marta Chesi ◽  
Kruti Naik ◽  
Davide F. Robbiani ◽  
Maurizio Affer ◽  
Helen D. Nickerson ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Transgenic Mice ◽  
Cell Lines ◽  
Germinal Center ◽  
Somatic Hypermutation ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Proximal Promoter

Abstract Approximately 15% of multiple myeloma (MM) is characterized by a t(4;14) translocation that causes the simultaneous dysregulation of MMSET on der(4) and fibroblast growth factor receptor 3 gene (FGFR3) on der(14). We reported several lines of evidence indicating a role for FGFR3 in myeloma tumorigenesis. First, activated FGFR3 is an oncogene capable of transforming fibroblasts. Second, FGFR3 activating mutations are acquired by MM cells during tumor progression. Third, targeted inhibition of FGFR3 leads to terminal differentiation and apoptosis in two t(4;14) MM cell lines. However, expression of FGFR3, but never of MMSET, is lost in about 25% of t(4;14) MM. Therefore, the overexpression of MMSET in all MM tumors with a t(4;14) translocation, and its homology to MLL, the oncogene on 11q23 translocated in acute leukemia suggest a critical role for MMSET in MM. To determine whether MMSET is an oncogene in vivo, we have generated transgenic mice in which MMSET expression in driven in lymphocytes by the lck proximal promoter juxtaposed to the Emu enhancer. Using the same expression vector we and others have obtained specific, high levels of transgene expression in B and T cells from spleen, bone marrow and thymus. Four transgenic lines were generated and although we detected MMSET expression in T cells in each of them, unexpectedly no expression in B cells was seen. This is consistent with our inability to ectopically express MMSET in B cell lines. Nevertheless B lymphoid tumors expressing MMSET developed at 23 month of age in each line (18/51 mice). Only 1/19 wild type matching control mice developed a splenomegaly. By Southern blot, monoclonal rearrangements of IgH, IgL and TCR β were detected within the same tumor population. In conclusion, this is the first report that MMSET is an oncogene capable of transforming lymphoid cells in an animal model. We are currently crossing these mice with FGFR3 transgenic mice to assess cooperation between these two oncogenes in tumorigenesis. Obviously a more restricted expression of MMSET in germinal center cells is required to investigate the role of MMSET in MM. Therefore, as we have done for c-myc, we are generating new transgenic mice in which MMSET expression will be activated sporadically in germinal center B cells by somatic hypermutation.

scholarly journals Expression of Recombination Activating Genes in Germinal Center B Cells: Involvement of Interleukin 7 (IL-7) and the IL-7 Receptor

1998 ◽  
Vol 188 (2) ◽  
pp. 365-372 ◽  
Author(s):  
Masaki Hikida ◽  
Yasunori Nakayama ◽  
Yumi Yamashita ◽  
Yoshio Kumazawa ◽  
Shin-Ichi Nishikawa ◽  
...  
Keyword(s):  
B Cells ◽  
Germinal Center ◽  
Receptor Editing ◽  
Recombination Activating Genes ◽  
Interleukin 7 ◽  
Recombination Activating Gene ◽  
Draining Lymph Node ◽  
Rag 1

Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD+ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti– IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4–deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.

scholarly journals Nuclear Factor-κB Binds to the Epstein-Barr Virus LMP1 Promoter and Upregulates Its Expression

2008 ◽  
Vol 83 (3) ◽  
pp. 1393-1401 ◽  
Author(s):  
Pegah Johansson ◽  
Ann Jansson ◽  
Ulla Rüetschi ◽  
Lars Rymo
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Critical Role ◽  
Mobility Shift ◽  
Barr Virus ◽  
Exogenous Expression ◽  
Epstein Barr

ABSTRACT The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-κB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-κB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-κB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-κB factors in both B cells and epithelial cells. Exogenous expression of NF-κB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-κB activation and LMP1 expression.

scholarly journals Role of B Cells in Host Defense against Primary Coxiella burnetii Infection

2015 ◽  
Vol 83 (12) ◽  
pp. 4826-4836 ◽  
Author(s):  
Laura Schoenlaub ◽  
Alexandra Elliott ◽  
Danielle Freches ◽  
William J. Mitchell ◽  
Guoquan Zhang
Keyword(s):  
B Cells ◽  
Host Defense ◽  
Coxiella Burnetii ◽  
Critical Role ◽  
Interleukin 10 ◽  
Content Type ◽  
B1a Cells

DespiteCoxiella burnetiibeing an obligate intracellular bacterial pathogen, our recent study demonstrated that B cells play a critical role in vaccine-induced immunity toC. burnetiiinfection by producing protective antibodies. However, the role of B cells in host defense against primaryC. burnetiiinfection remains unclear. In this study, we investigated whether B cells play an important role in host defense against primaryC. burnetiiinfection. The results showed that peritoneal B cells were able to phagocytose virulentC. burnetiibacteria and formCoxiella-containing vacuoles (CCVs) and thatC. burnetiican infect and replicate in peritoneal B1a subset B cellsin vitro, demonstrating a potential role for peritoneal B cells in host defense againstC. burnetiiinfectionin vivo. In addition, the results showing that B1a cells secreted a high level of interleukin-10 (IL-10) in response toC. burnetiiinfectionin vitrosuggest that B1a cells may play an important role in inhibiting theC. burnetiiinfection-induced inflammatory response. The observation that adoptive transfer of peritoneal B cells did not significantly affect the severity ofC. burnetiiinfection-induced diseases in both severe combined immunity-deficient (SCID) and μMT mice indicates that peritoneal B cells alone may not be able to controlC. burnetiiinfection. In contrast, our finding thatC. burnetiiinfection induced more-severe splenomegaly and a higher bacterial burden in the spleens of B1a cell-deficient Bruton's tyrosine kinase x-linked immunity-deficient (BTKxid) mice than in their wild-type counterparts further suggests that B1a cells play an important role in host defense against primaryC. burnetiiinfection.

scholarly journals Protein Kinase R Is a Novel Mediator of CD40 Signaling and Plays a Critical Role in Modulating Immunoglobulin Expression during Respiratory Syncytial Virus Infection

2011 ◽  
Vol 18 (12) ◽  
pp. 2060-2066 ◽  
Author(s):  
Sheetal A. Thakur ◽  
Zachary B. Zalinger ◽  
Teresa R. Johnson ◽  
Farhad Imani
Keyword(s):  
Respiratory Syncytial Virus ◽  
B Cells ◽  
Protein Kinase ◽  
Critical Role ◽  
Vital Role ◽  
Protein Kinase R ◽  
Cd40 Ligation ◽  
Syncytial Virus

ABSTRACTEffective immunoglobulin responses play a vital role in protection against most pathogens. However, the molecular mediators and mechanisms responsible for signaling and selective expression of immunoglobulin types remain to be elucidated. Previous studies in our laboratory have demonstrated that protein kinase R (PKR) plays a crucial role in IgE responses to double-stranded RNA (dsRNA)in vitro. In this study, we show that PKR plays a critical role in IgG expression bothin vivoandin vitro. PKR−/−mice show significantly altered serum IgG levels during respiratory syncytial virus (RSV) infection. IgG2a expression is particularly sensitive to a lack of PKR and is below the detection level in mock- or RSV-infected PKR−/−mice. Interestingly, we show that upon activation by anti-CD40 and gamma interferon (IFN-γ), B cells from PKR−/−mice show diminished major histocompatibility complex class II (MHC II), CD80, and CD86 levels on the cell surface compared to wild-type (WT) mice. Our data also show that PKR is necessary for optimal expression of adhesion molecules, such as CD11a and ICAM-1, that are necessary for homotypic aggregation of B cells. Furthermore, in this report we demonstrate for the first time that upon CD40 ligation, PKR is rapidly phosphorylated and activated, indicating that PKR is an early and novel downstream mediator of CD40 signaling pathways.

scholarly journals Germinal center B cells lack homing receptors necessary for normal lymphocyte recirculation.

1983 ◽  
Vol 157 (3) ◽  
pp. 813-827 ◽  
Author(s):  
R A Reichert ◽  
W M Gallatin ◽  
I L Weissman ◽  
E C Butcher
Keyword(s):  
T Cells ◽  
B Cells ◽  
Germinal Center ◽  
Plasma Cells ◽  
Lymphoid Organs ◽  
Vitro Assay ◽  
Peripheral Node ◽  
Germinal Center B Cells

Germinal center B cells (GCLC) are a discrete population of antigen-activated lymphoblasts that lack surface IgD and express abundant cell surface binding sites for peanut agglutinin (PNA). These phenotypic features render GCLC easily distinguishable from nearly all plasma cells, T cells, and unstimulated B cells, and have enabled us to identify and isolate GCLC from antigen-stimulated murine lymphoid organs. We have examined the migratory properties of these lymphoblasts in (a) short-term in vivo homing studies, and (b) an in vitro assay of lymphocyte binding to post-capillary, high endothelial venules (HEV) in frozen sections of Peyer's patches and peripheral lymph nodes. In the in vivo experiments, intravenously injected GCLC failed to migrate in significant numbers to peripheral lymphoid organs in comparison with T cells or IgD+ B cells. In the in vitro binding assay, GCLC did not adhere to HEV in either Peyer's patch or peripheral node sections. A variety of factors, such as preferential sequestration in the liver, may operate in vivo to influence the localization of these cells. However, their nearly total failure to migrate into lymphoid organs can best be explained by their inability to recognize and adhere to the specialized HEV which normally mediate the emigration of recirculating lymphocytes from the blood into these sites. The concept that GCLC fail to express functional homing receptors for HEV has been further supported by studies using MEL-14, a monoclonal antibody that appears to recognize the lymphocyte surface receptor for peripheral node HEV: In contrast to most peripheral lymphocytes, GCLC fail to bind MEL-14. These migratory and endothelial-recognition properties of GCLC, when viewed in the context of the possible role of these cells as precursors of plasma cells and/or memory B cells, have led us to propose that the inability of GCLC to recognize HEV may be transient and related to a phase of sessile B cell differentiation.

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