Cross-linking Of Surface Immunoglobulin Research Articles

Src-related thrombocytopenia: a fine line between a megakaryocyte dysfunction and an immune-mediated disease

AbstractSrc-related thrombocytopenia (SRC-RT) is a rare autosomal dominant, inherited platelet disorder resulting from the p.E527K heterozygous germline gain-of-function variant of Src. To date, genetic diagnosis of the disease has only been reported in 7 patients from 3 unrelated families. The clinical features ranged from isolated thrombocytopenia to complex syndromic manifestations characterized by thrombocytopenia, bleeding, myelofibrosis, splenomegaly, and bone disease. We report a new 3-generation kindred with the Src p.E527K variant. Patients presented with rather variable platelet counts (38-139 × 109/L), mildly impaired platelet function, >15% immature platelet fraction, and with a significant proportion of large-giant platelets. Four adults from the family were diagnosed with immune thrombocytopenia (ITP) and underwent splenectomy, achieving sustained platelet counts >75 × 109/L for several years; increases in platelet counts were also observed after corticosteroid therapy. Four of 7 Src p.E527K variant carriers showed immune defects and recurrent infections. In addition, a range of neurological symptoms, from specific language impairment to epilepsy, was seen in some family members. Patient platelets exhibited constitutive Src, Bruton tyrosine kinase, and phospholipase Cγ2 activation, and after stimulating CD19 cells by crosslinking surface immunoglobulin M, phosphorylated extracellular signal-regulated kinase (ERK) was significantly increased in B cells from individuals carrying the Src p.E527K substitution. In summary, in addition to causing impaired platelet production, SRC-RT may associate immune dysregulation and increased platelet consumption. In families in whom several members are responsive to ITP-directed therapies, an underlying Src p.E527K variant should be excluded.

B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition.

ABSTRACTAntigen recognition by the B cell receptor (BCR) is a physiological trigger for reactivation of Epstein-Barr virus (EBV) and can be recapitulated in vitro by cross-linking of surface immunoglobulins. Previously, we identified a subset of EBV microRNAs (miRNAs) that attenuate BCR signal transduction and subsequently dampen lytic reactivation in B cells. The roles of host miRNAs in the EBV lytic cycle are not completely understood. Here, we profiled the small RNAs in reactivated Burkitt lymphoma cells and identified several miRNAs, such as miR-141, that are induced upon BCR cross-linking. Notably, EBV encodes a viral miRNA, miR-BART9, with sequence homology to miR-141. To better understand the functions of these two miRNAs, we examined their molecular targets and experimentally validated multiple candidates commonly regulated by both miRNAs. Targets included B cell transcription factors and known regulators of EBV immediate-early genes, leading us to hypothesize that these miRNAs modulate kinetics of the lytic cascade in B cells. Through functional assays, we identified roles for miR-141 and EBV miR-BART9 and one specific target, FOXO3, in progression of the lytic cycle. Our data support a model whereby EBV exploits BCR-responsive miR-141 and further mimics activity of this miRNA family via a viral miRNA to promote productive lytic replication.IMPORTANCE EBV is a human pathogen associated with several malignancies. A key aspect of lifelong virus persistence is the ability to switch between latent and lytic replication modes. The mechanisms governing latency, reactivation, and progression of the lytic cycle are only partly understood. This study reveals that specific miRNAs can act to support the EBV lytic phase following BCR-mediated reactivation triggers. Furthermore, this study identifies a role for FOXO3, commonly suppressed by both host and viral miRNAs, in modulating progression of the EBV lytic cycle.

Abstract 648: Semi-synthetic peptibodies are a novel personalized therapeutic with activity against lymphoma in vitro and in vivo

Abstract The complementarity determining region, or idiotype, of the surface immunoglobulin receptor is a true tumor-specific marker on B-cell lymphomas unique to each patient. Antibodies against idiotype can induce complete regression of lymphoma in patients, but since this requires the generation of a custom monoclonal antibody for each patient, it has not been practical. We have developed a method for targeting idiotype using a novel construct that we refer to as a “semi-synthetic peptibody”. It consists of a synthetic peptide ligand for idiotype that is covalently linked to the amino terminus of a recombinant IgG Fc domain. Peptide ligands for idiotype can be identified by high throughput screens of random peptide libraries and produced inexpensively by solid-phase synthesis. Linkage of these idiotype ligands to the Fc domain enhances their pharmacokinetics and augments their anti-tumor effect by activating innate immune effectors. Since each patient-specific synthetic peptide can be chemically linked to a common IgG Fc domain, this modular design yields a custom therapeutic that may be more practical to produce than a unique biologic monoclonal antibody. We demonstrate that semi-synthetic peptibodies bind specifically to the idiotype of tumor cells and induce apoptosis by crosslinking surface immunoglobulin. Additionally, they trigger antibody dependent cellular cytotoxicity, antibody mediated phagocytosis, and complement-mediated lysis of opsonized lymphoma cells in vitro. They possess a favorable pharmacokinetic profile and are sufficient to clear tumor in SCID mice challenged intravenously with a luciferase-labeled human lymphoma cell line. Tumor clearance in vitro and in vivo mediated by peptibody was superior to that achievable with an anti-idiotype monoclonal antibody. Thus, semi-synthetic anti-idiotype peptibodies demonstrate multimodal activity against lymphoma cells in vitro and clear human lymphoma in a disseminated xenograft model. This modular, semi-synthetic design may enable a personalized and targeted therapy that is feasible to produce for patients with B-cell lymphoma. Citation Format: James Torchia, Patrick Ng, Homer Chen, Kipp Weiskopf, Ronald Levy. Semi-synthetic peptibodies are a novel personalized therapeutic with activity against lymphoma in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2014-648

Targeting B-Cell Lymphoma with Idiotype-Specific Peptibodies: Toward a Personalized and Tumor-Specific Therapy

Abstract 3713 Background:The complementarity determining region, or idiotype, of the surface immunoglobulin receptor is a tumor-specific marker on B-cell lymphomas that is unique to each patient. Antibodies against idiotype can induce complete regression of lymphoma in patients, but since this therapeutic approach requires the generation of a custom monoclonal antibody for each patient, it has not been practical. Objective:Here we describe a method for targeting the idiotype on the surface of a B-cell lymphoma by using synthetic idiotype-ligands covalently linked to a recombinant IgG Fc domain (Figure 1A). These peptide idiotype-ligands can be identified through oligopeptide library screens and produced inexpensively by automated solid-phase synthesis. Linkage of idiotype-ligands to the Fc domain serves two purposes: to enhance their pharmacokinetics and to augment their anti-tumor effect by activating immune effector functions. Since each patient-specific peptide can be chemically linked to a common IgG Fc domain, this modular construct design yields a patient-specific therapeutic that does not require the production of a custom biologic macromolecule for each patient. Results:Idiotype peptide-ligands were produced by solid-phase synthesis and covalently linked to the amino-terminus of a recombinant mouse IgG2a Fc domain by native chemical ligation, a method for site-selective polypeptide ligation. The resulting peptibody demonstrated targeted killing of a human lymphoma cell line in vitro by crosslinking surface immunoglobulin and triggering activation-induced death. Additionally, the peptibody triggered complement-mediated cytotoxicity of opsonized lymphoma cells in vitro and activated natural killer cells co-cultured with opsonized lymphoma cells. The peptibody exhibited a favorable pharmacokinetic profile and peptibody treatment was sufficient to clear tumor in SCID mice challenged intravenously with a luciferase-labeled human lymphoma cell line (p = 0.0018; Figure 1B-D). Conclusions:Idiotype-specific peptibodies demonstrate multimodal activity against lymphoma cells in vitro and clear human lymphoma in a disseminated xenograft model. The modular design of this therapeutic may enable a personalized and targeted therapy that is feasible to produce for patients with B-cell lymphoma. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Target silencing of disease-associated B-lymphocytes by chimeric molecules in SCID model of pristane-induced autoimmunity

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the generation of autoantibodies against a diverse array of self-antigens. The B cells producing immunoglobulin G (IgG) antibodies to double-stranded DNA appear to play a main role in the disease progression. Their specific elimination is a reasonable mechanism for effective therapy of SLE. The presently used approaches for silencing autoreactive disease-associated B cells are nonspecific and more precise therapies are needed. We have previously constructed a chimeric protein molecule consisting of several DNA-mimotope peptides coupled to a rat monoclonal anti-mouse CD32 (FcγRIIb) antibody. The mineral oil pristane induces a lupus-like syndrome in non-autoimmune mice leading to the development of glomerulonephritis and lupus-associated autoantibodies. In the present paper, using a pristane-induced autoimmune model in SCID mice, we analyzed the ability of the chimeric antibody to suppress selectively the autoreactive B lymphocytes by cross-linking B-cell surface immunoglobulin receptors with the inhibitory IgG FcγRIIb receptors. Treatment with DNA-like chimeric molecules inhibited B- and T-cell proliferation, restricted the number of anti-DNA antibody-producing cells and suppressed the generation of IgG anti-DNA antibodies. In contrast, phosphate buffered saline (PBS)-injected control mice experienced an increase of disease-associated antibody levels and developed glomerulonephritis similar to pristane-treated donor Balb/c mice.

Constitutive activation of distinct BCR-signaling pathways in a subset of CLL patients: a molecular signature of anergy

AbstractStimulation through the B-cell antigen receptor (BCR) is believed to be involved in the natural history of chronic lymphocytic leukemia (CLL). Some cases respond to the in vitro cross-linking of surface immunoglobulin (sIg) with effective activation. In contrast, the remaining cases do not respond to such stimulation, thereby resembling B cells anergized after antigen encounter in vivo. However the biochemical differences between the 2 groups are ill defined, and in humans the term B-cell anergy lacks a molecular definition. We examined the expression and activation of key molecules involved in signaling pathways originating from the BCR, and we report that a proportion of CLL patients (a) expresses constitutively phosphorylated extracellular signal-regulated kinase (ERK)1/2 in the absence of AKT activation; (b) displays constitutive phosphorylation of MEK1/2 and increased nuclear factor of activated T cells (NF-AT) transactivation; and (c) is characterized by cellular unresponsiveness to sIg ligation. This molecular profile recapitulates the signaling pattern of anergic murine B cells. Our data indicate that constitutive activation of mitogen activated protein (MAP) kinase signaling pathway along with NF-AT transactivation in the absence of AKT activation may also represent the molecular signature of anergic human B lymphocytes. CLL cases with this signature may be taken as a human model of anergic B cells aberrantly expanded.

High CD69 Protein Expression Predicts a Poor Prognosis in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

CD69 membrane protein is expressed early in the activation of lymphoid cells and is inducible by B-cells through cross-linking of surface immunoglobulins. Moreover, B-CLL cells exhibit features of activated and of antigen-experienced B lymphocytes overexpressing activation markers such as CD23, CD25, CD69 and CD71 (Damle, 2002). Furthermore, we have demonstrated that ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). The generation of antimurine CD69 monoclonal antibodies able to induce down-modulation or partial depletion of CD69+ cells (Sancho, 2006), prompted us to evaluate the real impact of CD69 expression on B-CLL prognosis. The primary aims of our study were:1.to determine progression-free survival (PFS) and overall survival (OS) upon CD69 expression;2.whether CD69 could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally3.whether CD69 was an independent prognostic factor. Therefore, we investigated 247 pts, median age 65 years, 131 males and 116 females.With regard to modified Rai stages, 69 pts had a low stage, 167 an intermediate stage and 11 a high stage. CD69 was determined by multicolor flow cytometry fixing a cut-off value of 30%. CD69+ B-CLL pts were 73/247 (30%). CD69 >30% was significantly associated with an intermediate/high Rai stage (p=0.001), lymphocyte doubling time (LDT) <12 months (p=0.00005) and beta-2 microglobulin >2.2 mg/dl (p=0.002). Lower CD69 expression and IgVH mutated status (>2%) were significantly correlated (73/92; p=0.0003). Furthermore, we found significant associations between lower CD69 and lower CD38 (137/184; p=0.02) or lower ZAP-70 (110/144; p=0.01). Lower levels of soluble CD23 (sCD23) were strongly associated with lower CD69 (127/158; p<0.00001). With regard to clinical outcome, both a significant shorter PFS (Figure) and OS were observed in CD69+ pts (5% vs 56% at 14 years; p<0.00001 and 44% vs 66% at 14 years; p=0.00001) as well as in ZAP-70+ pts (7% vs 62% at 12 years; p<0.00001 and 26% vs 90% at 14 years; p<0.00001). To further explore the prognostic impact of CD69, we investigated its expression within ZAP-70+ (103 pts) and ZAP-70 negative (144 pts) subsets. CD69+ pts showed a shorter PFS both within the ZAP-70+ subset (11% vs 27% at 8 years; p=0.004) and within the ZAP-70 negative subset (21% vs 79% at 12 years, p<0.00001). In multivariate analysis of PFS and OS, in which Rai modified stages, CD38, sCD23, LDT, CD69 and ZAP-70 entered, both ZAP-70 (p=0.0003 and p=0.0002) and CD69 (p=0.005 and p=0.0004) resulted to be independent prognostic factors. Therefore, CD69, determined by flow cytometry, could be considered as a new promising immunologic prognostic parameter in B-CLL. Furthermore, since the ZAP-70 negative subgroup consists of a heterogeneous population presenting variable outcome, CD69 might better stratify B-CLL subsets and early identify progressive pts in order to take timely therapeutic decisions. [Display omitted]

Induction of CD40 Expression and Enhancement of Monoclonal Antibody Production on Murine B Cell Hybridomas by Cross‐Linking of IgG Receptors

The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (micromax) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demonstrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.

Hypersensitivity and Loss of Disease Site Targeting Caused by Antibody Responses to PEGylated Liposomes

The systemic application of nucleic acid drugs requires delivery systems that overcome the poor pharmacokinetics, limited biodistribution, and inefficient uptake of nucleic acids. PEGylated liposomes show considerable promise because of their intrinsic ability to accumulate at disease sites and facilitate transfection of target cells. Unlike many viral vectors, PEGylated liposomes are generally considered to be nonimmunogenic. We have developed a PEGylated liposome for the systemic administration of plasmid DNA that achieves high levels of selective gene expression at distal tumor sites. Here we report that the in vivo efficacy and safety of these systems can be severely compromised following repeat administration. This phenomenon is characterized by a loss of disease site targeting, accelerated clearance from the blood, and acute hypersensitivity. These effects are fully attributable to a surprisingly robust, long-lived antibody response generated against polyethylene glycol (PEG) that results from the strong adjuvant effect of the plasmid payload. Importantly, immunogenicity may be substantially reduced by modifying the alkyl chain of the PEG-lipid conjugate, thereby allowing successful repeat dosing of the modified plasmid formulations without adverse side effects. Immunogenicity is a relevant concern for a number of nonviral delivery systems given the potent immunostimulatory properties of many nucleic acid drugs.

Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation

AbstractThe PU.1 transcription factor is a key regulator of hematopoietic development, but its role at each hematopoietic stage remains unclear. In particular, the expression of PU.1 in hematopoietic stem cells (HSCs) could simply represent “priming” of genes related to downstream myelolymphoid lineages. By using a conditional PU.1 knock-out model, we here show that HSCs express PU.1, and its constitutive expression is necessary for maintenance of the HSC pool in the bone marrow. Bone marrow HSCs disrupted with PU.1 in situ could not maintain hematopoiesis and were outcompeted by normal HSCs. PU.1-deficient HSCs also failed to generate the earliest myeloid and lymphoid progenitors. PU.1 disruption in granulocyte/monocyte-committed progenitors blocked their maturation but not proliferation, resulting in myeloblast colony formation. PU.1 disruption in common lymphoid progenitors, however, did not prevent their B-cell maturation. In vivo disruption of PU.1 in mature B cells by the CD19-Cre locus did not affect B-cell maturation, and PU.1-deficient mature B cells displayed normal proliferation in response to mitogenic signals including the cross-linking of surface immunoglobulin M (IgM). Thus, PU.1 plays indispensable and distinct roles in hematopoietic development through supporting HSC self-renewal as well as commitment and maturation of myeloid and lymphoid lineages.

Ex Vivo Stimulation of B Cells Latently Infected with Gammaherpesvirus 68 Triggers Reactivation from Latency

Murine gammaherpesvirus 68 (gammaHV68) infection of mice results in the establishment of a chronic infection, which is largely maintained through latent infection of B lymphocytes. Acute virus replication is almost entirely cleared by 2 weeks postinfection. Spontaneous reactivation of gammaHV68 from latently infected splenocytes upon ex vivo culture can readily be detected at the early stages of infection (e.g., day 16). However, by 6 weeks postinfection, very little spontaneous reactivation is detected upon explant into tissue culture. Here we report that stimulation of latently infected splenic B cells harvested at late times postinfection with cross-linking surface immunoglobulin (Ig), in conjunction with anti-CD40 antibody treatment, triggers virus reactivation. As expected, this treatment resulted in B-cell activation, as assessed by upregulation of CD69 on B cells, and ultimately B-cell proliferation. Since anti-Ig/anti-CD40 stimulation resulted in splenic B-cell proliferation, we assessed whether this reactivation stimulus could overcome the previously characterized defect in virus reactivation of a v-cyclin null gammaHV68 mutant. This analysis demonstrated that anti-Ig/anti-CD40 stimulation could drive reactivation of the v-cyclin null mutant virus in latently infected splenocytes, but not to the levels observed with wild-type gammaHV68. Thus, there appears to be a role for the v-cyclin in B cells following anti-Ig/anti-CD40 stimulation independent of the induction of the cell cycle. Finally, to assess signals that are not mediated through the B-cell receptor, we demonstrate that addition of lipopolysaccharide to explanted splenocyte cultures also enhanced virus reactivation. These studies complement and extend previous analyses of Epstein-Barr virus and Kaposi's sarcoma-associated virus reactivation from latently infected cell lines by investigating reactivation of gammaHV68 from latently infected primary B cells recovered from infected hosts.

IgE crosslinkage of Fcε receptor I induces both production and activation of matrix metalloproteinase-9 in mast cells

Since mast cells play pivotal roles in allergic inflammations, we investigated how IgE-mediated stimulation modulated mast cell matrix metalloproteinase (MMP)-9 production, and its enzymatic activation. In this study, we clearly demonstrated that proMMP-9 released from murine bone marrow-derived cultured mast cells (BMCMC) was activated to its valid form after crosslinking of surface immunoglobulin (Ig)E. Serine protease inhibitors sensitive to chymases inhibited the phenomenon, indicating that certain chymases may be responsible for activation of proMMP-9. Although binding of IgE to its specific receptors did not alter MMP-9 production, the IgE crosslinkage increased both expression of mRNA, and production of MMP-9 in mast cells. Glucocorticoid declined extra cellular processing of proMMP-9 without affecting mRNA expression. These findings give rise to the possibility that production and activation of mast cell MMP-9 may be increased in the affected sites, thereby resulting in an exacerbation of tissue degradation in inflammatory conditions.

H(2)O(2) induces translocation of APE/Ref-1 to mitochondria in the Raji B-cell line.

Reactive oxygen species (ROS) are generated as by-products of respiration and are used as signal transducing intermediates in out-in signaling pathways. ROS are also generated during inflammatory responses and it has been shown that hydrogen peroxide may trigger activation of B-lymphocytes, similar to cross-linking of surface immunoglobulins. On the other hand, both exogenous and endogenous generated ROS are a major source of nuclear and mitochondrial DNA (mtDNA) damage. The base excision repair (BER) enzyme APE/Ref-1 normally repairs small nuclear DNA lesion such as oxidized or alkylated bases. It is not clear though whether DNA repair mechanisms able to abolish oxidative damage from nuclear DNA are present into mitochondria too. Here we show by confocal microscopy and Western blot analysis that in the B-lymphocyte Raji cell line a fraction of APE/Ref-1 rapidly re-localizes into mitochondria following H(2)O(2) activation. Targeting of APE/Ref-1 to mitochondria is not associated with cytochrome-c loss or apoptosis induction. These findings indicate that the APE/Ref-1 translocates to mitochondria in response to oxidative stress and thereby it might exert a protective function.

Distinct patterns of mitogen-activated protein kinase phosphorylation and Epstein-Barr virus gene expression in Burkitt's lymphoma cell lines versus B lymphoblastoid cell lines.

In order to understand mechanistic relationships between signaling pathways regulating mitogen-activated protein kinase (MAPK) phosphorylation and Epstein-Barr virus (EBV) reactivation, we compared MAPK phosphorylation, and EBV reactivation and latency in Burkitt's lymphoma cell lines (BLCLs) versus B lymphoblastoid cell lines (LCLs). EBV was reactivated in the BLCLs Akata and Raji, and in a LCL OB-R33 cells after cross-linking surface immunoglobulin (sIg) with anti-Ig. After stimulation with anti-Ig, MAPK phosphorylation was strongly induced in all BLCLs and in a few LCLs, but not in other LCLs. MAPK was constitutively phosphorylated in most LCLs but not in BLCLs. Expression of EBNA2 and LMP1, and LMP2A was analyzed with both immunoblotting and RT-PCR. EBNA2 and LMP1 were expressed in most LCLs and in some BLCLs. LMP2A was expressed in all BLCLs and LCLs except Namalwa cells. To test the hypothesis that LMPI induces constitutive MAPK phosphorylation, the LMP1 expression vector was transfected into Akata cells. MAPK phosphorylation was not induced in such transfected cells. Our results indicate that BLCLs and LCLs respectively have distinct MAPK phosphorylation patterns, and that induction of MAPK phosphorylation correlates with EBV reactivation in a few cell lines but not in most of the tested cell lines.

Role of Epstein-Barr virus-encoded latent membrane protein 2A on virus-induced immortalization and virus activation.

To quantitatively evaluate the role of Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in immortalization of peripheral B-lymphocytes, we used the Akata cell system to generate an EBV recombinant in which the first exon of the LMP2A gene was disrupted. The results indicated that deletion of the LMP2A gene did not affect the immortalization efficiency of EBV in B-lymphocytes. Deletion of the LMP2A gene made EBV-transformed lymphocytes more permissive for virus replication in response to surface immunoglobulin cross-linking. On the other hand Akata cells, in which LMP2A expression was much lower than in EBV-transformed lymphocytes, were equally permissive for virus replication whether they were infected with wild EBV or LMP2A-knockout EBV. The results raise a question as to the role of LMP2A in inhibition of disruption of virus latency in vivo, where LMP2A expression has been expected to be low as in Akata cells.

Lymphokine dependence of STAT3 activation produced by surface immunoglobulin cross-linking and by phorbol ester plus calcium ionophore treatment in B cells.

Stimulation of B cells by surface immunoglobulin (sIg) triggering, or through the mitogenic combination of phorbol ester and calcium ionophore, is accompanied by activation of STAT transcription factors. The mechanism responsible for the delayed nuclear accumulation of phosphorylated STAT3 was examined in detail, focusing on the role of B cell-derived lymphokines. sIg-induced activation of STAT3 was partially inhibited in B cells obtained from IL-6- or IL-10-deficient mice, and was partially blocked by neutralizing antibodies directed against either of these lymphokines. sIg-induced STAT3 activation was completely inhibited by combining IL-6- and IL-10-specific neutralizing antibodies, or by adding individual neutralizing antibodies to B cells obtained from lymphokine-deficient animals. In contrast, IL-10 alone appeared to account for STAT3 activation resulting from B cell stimulation with phorbol ester and calcium ionophore. In keeping with these results, soluble IL-6 and IL-10 were found in supernatant fluid obtained from stimulated B cells. This work indicates that a lymphokine pathway is responsible for STAT3 activation that occurs late after B cell stimulation, and points out differences in B cell activation that result from stimulation through the antigen receptor and through pharmacological mimicry of signaling mediators.

CD19-dependent Activation of Akt Kinase in B-lymphocytes

CD19 is rapidly phosphorylated upon B-cell antigen receptor (BCR) cross-linking, leading to the recruitment of downstream signaling intermediates. A prominent feature of CD19 signaling is the binding and activation of phosphoinositide 3-kinase (P13K), which accounts for the majority of PI3K activity induced by BCR ligation. Recent findings have implicated activation of the serine/threonine kinase Akt as imparting survival signals in a PI3K-dependent fashion. Using CD19-deficient B-lymphoma cells and mouse splenic B-cells, we show that CD19 is necessary for efficient activation of Akt following cross-linking of surface immunoglobulin or Igbeta. In the absence of CD19, Akt kinase activity is reduced and transient. In addition, coligation of CD19 with surface immunoglobulin leads to augmented Akt activity in a dose-dependent manner. Thus, CD19 is a key regulator of Akt activity in B-cells; as such it may contribute to pre-BCR or BCR-mediated cell survival in vivo.

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 activate the ATF2 transcription factor by increasing the levels of phosphorylated p38 and c-Jun N-terminal kinases.

Expression of either Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) or BRLF1 (R) is sufficient to convert EBV infection from the latent to lytic form. Disruption of viral latency requires transcriptional activation of the Z and R promoters. The Z and R proteins are transcriptional activators, and each immediate-early protein activates expression of the other immediate-early protein. Z activates the R promoter through a direct binding mechanism. However, R does not bind directly to the Z promoter. In this study, we demonstrate that the ZII element (a cyclic AMP response element site) in the Z promoter is required for efficient activation by R. The ZII element has been shown to be important for induction of lytic EBV infection by tetradecanoyl phorbol acetate and surface immunoglobulin cross-linking and is activated by Z through an indirect mechanism. We demonstrate that both R and Z activate the cellular stress mitogen-activated protein (MAP) kinases, p38 and JNK, resulting in phosphorylation (and activation) of the cellular transcription factor ATF2. Furthermore, we show that the ability of R to induce lytic EBV infection in latently infected cells is significantly reduced by inhibition of either the p38 kinase or JNK pathways. In contrast, inhibition of stress MAP kinase pathways does not impair the ability of Z expression vectors to disrupt viral latency, presumably because expression of Z under the control of a strong heterologous promoter bypasses the need to activate Z transcription. Thus, both R and Z can activate the Z promoter indirectly by inducing ATF2 phosphorylation, and this activity appears to be important for R-induced disruption of viral latency.

Differential effects of injections of anti-mu and anti-delta monoclonal antibodies on B-cell populations in adult mice: regulation of xenoreactive natural antibody-producing cells.

The depletion of differential B cell and xenoreactive natural antibodies (XNA) by anti-delta and anti-mu injections was analyzed in adult mice. Sequential treatment with anti-delta and then anti-mu induces a complete depletion of B cells and XNA and represents a potential approach to induce xenograft tolerance. Adult mice were injected with anti-mu, anti-delta, anti-delta then anti-mu, or control isotype monoclonal antibodies from day 0 to day 14. The different B-cell populations were analyzed by FACS and immunohistology. Ig production was tested by ELISA. XNA were analyzed by FACS. Anti-mu injections induced a depletion of IgMhigh, immature B cells, marginal zone B cells, and B1 cells and an increase of IgG-XNA production. Anti-delta injections induced mature conventional IgDhigh B-cell depletion and increased IgM-XNA production. Interestingly, sequential injections of anti-delta then anti-mu induced a depletion of immature B cells, mature B cells (MZ, B2, and B1), and XNA. These results demonstrate that mature B-cell depletion in adult mice can be obtained by mAb injections and depends on the surface immunoglobulin cross-linking threshold. Indeed, anti-mu mAb depleted IgMhigh B cells (MZ and B1) and anti-delta, IgDhigh B cells (B2). The differential B-cell suppression shows that conventional B cells are responsible in the IgG-XNA production and MZ and B1 cells in the IgM-XNA production. Sequential repeated injections of anti-delta then anti-mu mAb depleted all B-cell populations and suppressed the whole XNA production.

Protein Kinase C-α Is Essential for Ramos-BL B Cell Survival

The Ramos-Burkitt lymphoma (BL) B cell line is driven into growth arrest and apoptosis by cross-linking surface immunoglobulin. We demonstrate that protein kinase C (PKC) activity is required for Ramos B cell proliferation and survival. A variety of PKC inhibitors trigger a significant decrease in [3H]thymidine incorporation with a concomitant increase in cell death. Antisense depletion of expression of the PKC-α isoform is sufficient to trigger cell death in the absence of any other signal, demonstrating a requirement for this isoform for survival of Ramos-BL B cells. Cross-linking surface immunoglobulin also leads to depletion of PKC-α levels, suggesting that this may be one mechanism by which this signals for cell death in Ramos-BL B cells.