Stimulation and inhibition of FVIII-specific memory B-cell responses by CpG-B (ODN 1826), a ligand for Toll-like receptor 9

Blood ◽  
2011 ◽  
Vol 117 (1) ◽  
pp. 259-267 ◽  
Author(s):  
Peter Allacher ◽  
Christina K. Baumgartner ◽  
Aniko G. Pordes ◽  
Rafi U. Ahmad ◽  
Hans Peter Schwarz ◽  
...  
Keyword(s):  
B Cells ◽  
Memory B Cells ◽  
Cpg Odn ◽  
Specific Memory ◽  
Cpg Oligodeoxynucleotide ◽  
Low Concentrations ◽  
Essential Components ◽  
High Concentrations

Abstract Factor VIII (FVIII)–specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138− spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.

Agonist for Toll-Like Receptor (TLR) 9 Amplifies as Well as Inhibits the Differentiation of FVIII-Specific Memory B Cells into Antibody-Producing Plasma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3382-3382
Author(s):  
Peter Allacher ◽  
Christina Hausl ◽  
Aniko Ginta Pordes ◽  
Rafi Uddin Ahmad ◽  
Hartmut J Ehrlich ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Cpg Dna ◽  
Cell Responses ◽  
Specific Memory ◽  
B Cell Responses

Abstract Memory B cells are essential for maintaining long-term antibody responses. They can persist for years even in the absence of antigen and are rapidly re-stimulated to differentiate into antibody-producing plasma cells when they encounter their specific antigen. Previously we demonstrated that ligands for TLR 7 and 9 amplify the differentiation of FVIII-specific memory B cells into anti-FVIII antibody-producing plasma cells at low concentrations of FVIII and prevent the inhibition of memory-B-cell differentiation at high concentrations of FVIII. The modulation of FVIII-specific memory-B-cell responses by agonists for TLR is highly relevant for the design of new immunotherapeutic approaches in patients with FVIII inhibitors because TLR are activated by a range of different viral and bacterial components. Specifically, TLR 7 is triggered by single-stranded RNA derived from viruses and TLR 9 is triggered by bacterial DNA containing unmethylated CpG motifs. We further explored the modulation of FVIII-specific memory-B-cell responses by agonists for TLRs by studying a broad range of concentrations of CpG DNA, a ligand for TLR 9, both in vitro and in vivo using the murine E17 model of hemophilia A. We used CpG-DNA in concentrations ranging from 0.1 to 10,000 ng/ml to study the modulation of FVIII-specific memory-B-cell responses in vitro and verified the specificity of the effects observed by including a blocking agent for TLR 9 and GpC-DNA, a non-stimulating negative control for CpG DNA. Furthermore, we used doses of CpG DNA ranging from 10 to 50,000 ng per dose to study the modulation of FVIII-specific memory-B-cell responses in vivo. E17 hemophilic mice were treated with a single intravenous dose of 200 ng FVIII to stimulate the generation of FVIII-specific memory B cells and were subsequently treated with another dose of FVIII that was given together with CpG DNA. We analyzed titers of anti-FVIII antibodies in the circulation of these mice one week after the second dose of FVIII. Previously we had shown that a single dose of 200 ng FVIII, given intravenously to E17 hemophilic mice, stimulates the formation of FVIII-specific memory B cells but is not sufficient to induce anti-FVIII antibodies that would be detectable in the circulation. Our results demonstrate a biphasic effect of CpG DNA on the re-stimulation of FVIII-specific memory B cells and their differentiation into antibody-producing plasma cells. Both in vitro and in vivo studies show that CpG DNA at high doses inhibits the re-stimulation and differentiation of FVIII-specific memory B cells. However, CpG DNA at low doses amplifies these processes. Amplification and inhibition of memory-B-cell responses are due to specific interactions of CpG DNA with TLR 9. Both effects are blocked by addition of a blocking agent for TLR 9 in vitro. We conclude that triggering of TLR 9 by bacterial DNA has a substantial influence on FVIII-specific memory-B-cell responses. The consequence of TLR 9 triggering can be inhibitory or stimulatory, depending on the actual concentration of the bacterial DNA. Our findings demonstrate the potential modulatory effects of bacterial infections on the regulation of FVIII inhibitor development.

Preventing restimulation of memory B cells in hemophilia A: a potential new strategy for the treatment of antibody-dependent immune disorders

Blood ◽  
2004 ◽  
Vol 104 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Christina Hausl ◽  
Rafi U. Ahmad ◽  
Hans Peter Schwarz ◽  
Eva M. Muchitsch ◽  
Peter L. Turecek ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Hemophilia A ◽  
Memory B Cells ◽  
Third Party ◽  
Activated T Cells ◽  
Specific Memory ◽  
Inducible Costimulator

Abstract Memory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti–factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the restimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator–inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.

scholarly journals Hypogonadism associated withCyp19a1 (Aromatase) post-transcriptional upregulation inCelf1-KO mice

2015 ◽  
pp. MCB.00074-15 ◽  
Author(s):  
Gaella Boulanger ◽  
Marie Cibois ◽  
Justine Viet ◽  
Alexis Fostier ◽  
Stéphane Deschamps ◽  
...  
Keyword(s):  
Rna Binding ◽  
Hypogonadotropic Hypogonadism ◽  
Type I ◽  
Male Gametes ◽  
Low Concentrations ◽  
Reporter Assays ◽  
High Concentrations ◽  
In Vitro Interaction

CELF1 is a multifunctional RNA-binding protein that controls several aspects of RNA fate. The targeted disruption of theCelf1gene in mice causes male infertility due to impaired spermiogenesis, the post-meiotic differentiation of male gametes. Here, we investigated the molecular reasons that underlie this testicular phenotype. By measuring sex hormone levels, we detected low concentrations of testosterone inCelf1-null mice. We investigated the effect ofCelf1disruption on the expression levels of steroidogenic enzyme genes, and we observed thatCyp19a1was upregulated.Cyp19a1encodes aromatase, which transforms testosterone into estradiol. Administration of testosterone or the aromatase inhibitor Letrozole partly rescued the spermiogenesis defects, indicating that a lack of testosterone associated with excessive aromatase contributes to the testicular phenotype. In vivo and in vitro interaction assays demonstrated that CELF1 binds toCyp19a1mRNA, and reporter assays supported the conclusion that CELF1 directly repressesCyp19a1translation. We conclude that CELF1 downregulatesCyp19a1/Aromatasepost-transcriptionally to achieve high concentrations of testosterone compatible with spermiogenesis completion. We discuss the implications of these findings with respect to reproductive defects in men, including patients suffering from isolated hypogonadotropic hypogonadism and myotonic dystrophy type I.

scholarly journals Antigen-Specific B Cell Memory

2000 ◽  
Vol 191 (7) ◽  
pp. 1149-1166 ◽  
Author(s):  
Louise J. McHeyzer-Williams ◽  
Melinda Cool ◽  
Michael G. McHeyzer-Williams
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Memory B Cells ◽  
Cell Memory ◽  
Specific Memory ◽  
B Cell Memory ◽  
Cellular Components

The mechanisms that regulate B cell memory and the rapid recall response to antigen remain poorly defined. This study focuses on the rapid expression of B cell memory upon antigen recall in vivo, and the replenishment of quiescent B cell memory that follows. Based on expression of CD138 and B220, we reveal a unique and major subtype of antigen-specific memory B cells (B220−CD138−) that are distinct from antibody-secreting B cells (B220+/−CD138+) and B220+CD138− memory B cells. These nonsecreting somatically mutated B220− memory responders rapidly dominate the splenic response and comprise >95% of antigen-specific memory B cells that migrate to the bone marrow. By day 42 after recall, the predominant quiescent memory B cell population in the spleen (75–85%) and the bone marrow (>95%) expresses the B220− phenotype. Upon adoptive transfer, B220− memory B cells proliferate to a lesser degree but produce greater amounts of antibody than their B220+ counterparts. The pattern of cellular differentiation after transfer indicates that B220− memory B cells act as stable self-replenishing intermediates that arise from B220+ memory B cells and produce antibody-secreting cells on rechallenge with antigen. Cell surface phenotype and Ig isotype expression divide the B220− compartment into two main subsets with distinct patterns of integrin and coreceptor expression. Thus, we identify new cellular components of B cell memory and propose a model for long-term protective immunity that is regulated by a complex balance of committed memory B cells with subspecialized immune function.

Effects of polymethoxylated flavone metabolites on apoB100 secretion and MTP activity in Huh7.5 cells

2021 ◽  
Vol 18 ◽  
Author(s):  
Danielle R. Gonçalves ◽  
Thais B. Cesar ◽  
John A. Manthey ◽  
Paulo I. Costa
Keyword(s):  
Lipid Synthesis ◽  
Dependent Manner ◽  
Transfer Protein ◽  
Low Concentrations ◽  
Wide Range ◽  
Cell Assays ◽  
Polymethoxylated Flavones ◽  
High Concentrations

Background: Citrus polymethoxylated flavones (PMFs) reduce the synthesis of liver lipoproteins in animal and in vitro cell assays, but few studies have evaluated the direct effects of their metabolites on this highly regulated process. Objective: To investigate the effects of representative metabolites of PMF on the secretion of liver lipoproteins using the mammalian cell Huh7.5. Method: In this study, the influences of three PMFs and five previously isolated PMF metabolites on hepatic apoB-100 secretion and microsomal transfer protein (MTP) activity were evaluated. Tangeretin (TAN), nobiletin (NOB) and 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), and their glucuronides (TAN-Gluc, NOB-Gluc and HMF-Gluc) and oxidatively demethylated metabolites (TAN-OH, NOB-OH, HMF-OH) were incubated with Huh7.5 cells to measure their inhibitory effects on lipid synthesis. Results: The results showed that TAN, HMF and TAN-OH reduced the secretion of apoB-100 in a dose-dependent manner, while NOB and the other tested metabolites showed no inhibition. MTP activity in the Huh7.5 cells was significantly reduced in the presence of low concentrations of TAN, and in high concentrations of NOB-OH. This study also showed that PMFs and PMF metabolites produced a wide range of effects on apoB-100 secretion and MTP activity. Conclusion: The results suggest that while PMFs and their metabolites control dyslipidemia in vivo, the inhibition of MTP activity cannot be the only pathway influenced by these compounds.

scholarly journals Virus-Encoded MicroRNAs Facilitate Gammaherpesvirus Latency and Pathogenesis In Vivo

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Emily R. Feldman ◽  
Mehmet Kara ◽  
Carrie B. Coleman ◽  
Katrina R. Grau ◽  
Lauren M. Oko ◽  
...  
Keyword(s):  
B Cells ◽  
Memory B Cells ◽  
Mutant Virus ◽  
Murine Gammaherpesvirus ◽  
Wide Range ◽  
Cellular Pathways ◽  
Regulatory Rnas ◽  
Host Target

ABSTRACTGammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesisin vivois unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis ofin vitroandin vivoMHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virusin vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infectedin vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replicationin vitroandin vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease.IMPORTANCEGammaherpesviruses such as EBV and KSHV are widespread pathogens that establish lifelong infections and are associated with the development of numerous types of diseases, including cancer. Gammaherpesviruses encode many small noncoding RNAs called microRNAs (miRNAs). It is predicted that gammaherpesvirus miRNAs facilitate infection and disease by suppressing host target transcripts involved in a wide range of key cellular pathways; however, the precise contribution that these regulatory RNAs make toin vivovirus infection and pathogenesis is unknown. Here, we generated a mutated form of murine gammaherpesvirus (MHV68) to dissect the function of gammaherpesvirus miRNAsin vivo. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. Moreover, the MHV68 miRNAs were essential for the development of virus-associated pneumonia, implicating them as a critical component of gammaherpesvirus-associated disease.

Tracking Factor VIII-Specific Memory B Cells in Hemophilia A Patients with Factor VIII Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1016-1016 ◽  
Author(s):  
Irene Lang ◽  
Jerzy Windyga ◽  
Anna Klukowska ◽  
Josenato Ilas ◽  
Hans Peter Schwarz ◽  
...  
Keyword(s):  
B Cells ◽  
Tetanus Toxin ◽  
Plasma Cells ◽  
Hemophilia A ◽  
Positive Control ◽  
Negative Control ◽  
Memory B Cells ◽  
Healthy Individuals ◽  
Specific Memory

Abstract The development of neutralizing anti-FVIII antibodies (FVIII inhibitors) in about 30% of patients with severe hemophilia A is the most serious complication in the treatment of hemophilia patients with FVIII products. Little information is available on the immunological mechanisms that regulate the development and maintenance of FVIII inhibitors. Memory B cells are a central component of humoral immunity. They drive the rapid anamnestic antibody response that occurs after re-exposure to antigen and seem to be important for replenishing the pool of long-lived plasma cells to maintain long-term antibody levels in the absence of antigen. Nothing is known about the dynamics of FVIII-specific memory B cells in patients with hemophilia A who develop FVIII inhibitors. Recently, Crotty et al. (J Immunol Methods, 2004) described an in vitro assay to quantify antigen-specific memory B cells in human blood. This assay utilizes a 6-day polyclonal stimulation of peripheral blood mononuclear cells (PBMC) followed by an antigen-specific ELISPOT for the detection of memory B cells that have differentiated into antibody-secreting plasma cells in vitro. We adapted this assay to human FVIII and used it to track FVIII-specific memory B cells in the blood of hemophilia A patients with and without FVIII inhibitors. Human serum albumin was used as a negative control and tetanus toxin as a positive control. The numbers of FVIII-specific, tetanus toxin-specific (positive control) and albumin-specific (negative control) memory B cells were calculated as percentage of total IgG memory B cells. So far, we have analyzed 14 patients with hemophilia A (age: 6–65 years). 8 were positive and 6 were negative for FVIII inhibitors. For comparison, we have analyzed 20 healthy individuals (age: 19–48 years). 2 out of 8 patients with inhibitors had detectable FVIII-specific memory B cells in their peripheral blood cells. However, none of the patients without inhibitors and none of the healthy individuals had any detectable FVIII-specific memory B cells in their circulation. The detection limit for FVIII-specific memory B cells in patients with inhibitors was about 0.2 % (percent of total IgG memory B cells). Current activities focus on further advancing the method with the aim to improve the detection limit for the detection of FVIII-specific memory B cells. All samples analyzed (including patients and healthy individuals) were negative for human serum albumin-specific memory B cells (negative control). Tetanus toxin-specific memory B cells (positive control) were found in both patients and healthy blood donors. The percentage of tetanus toxin-specific memory B cells in individuals who were vaccinated with tetanus toxoid was in the range of 0.25 – 0.58 % (percent of total IgG memory B cells). We conclude that the method described is suitable to track FVIII-specific memory B cells in the circulation. We are currently asking the question whether the presence of FVIII-specific memory B cells in the circulation correlates with the persistence of FVIII inhibitors. Furthermore, we will monitor patients with inhibitors during ITI therapy in order to find out whether the disappearance of FVIII-specific memory B cells in the circulation could be an early predictor of a successful ITI outcome.

Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3224-3231 ◽  
Author(s):  
Hiroshi Kataoka ◽  
Justin R. Hamilton ◽  
David D. McKemy ◽  
Eric Camerer ◽  
Yao-Wu Zheng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Wild Type ◽  
Protease Activated Receptors ◽  
Erk Phosphorylation ◽  
Low Concentrations ◽  
Extracellular Signal ◽  
High Concentrations ◽  
Mouse Aorta

AbstractDefining the relative importance of protease-activated receptors (PARs) for thrombin signaling in mouse endothelial cells is critical for a basic understanding of thrombin signaling in these cells and for the rational use of knockout mice to probe the roles of thrombin's actions on endothelial cells in vivo. We examined thrombin- and PAR agonist–induced increases in cytoplasmic calcium, phosphoinositide hydrolysis, extracellular signal-regulated kinase (ERK) phosphorylation, and gene expression in endothelial cells from wild-type and PAR-deficient mice. PAR1 and PAR4 agonists triggered responses in wild-type but not in Par1–/– and Par4–/– endothelial cells, respectively. Calcium imaging confirmed that a substantial fraction of individual endothelial cells responded to both agonists. Compared with wild-type cells, Par1–/– endothelial cells showed markedly decreased responses to low concentrations of thrombin, and cells that lacked both PAR1 and PAR4 showed no responses to even high concentrations of thrombin. Similar results were obtained when endothelial-dependent vasorelaxation of freshly isolated mouse aorta was used as an index of signaling in native endothelial cells. Thus PAR1 is the major thrombin receptor in mouse endothelial cells, but PAR4 also contributes. These receptors serve at least partially redundant roles in endothelial cells in vitro and in vivo and together are necessary for the thrombin responses measured.

scholarly journals SARS-CoV-2 specific memory B-cells from individuals with diverse disease severities recognize SARS-CoV-2 variants of concern.

2021 ◽  
Author(s):  
Zoe L Lyski ◽  
Amanda E Brunton ◽  
Mathew I Strnad ◽  
Peter E Sullivan ◽  
Sarah A.R. Siegel ◽  
...  
Keyword(s):  
B Cells ◽  
Memory B Cells ◽  
Specific Antibodies ◽  
Mild Disease ◽  
Cell Compartments ◽  
Specific Memory ◽  
Prior Infection ◽  
Single Time Point ◽  
Time Point

In this investigation we examined the magnitude, breadth, and durability of SARS-CoV-2 specific antibodies in two distinct B-cell compartments: long-lived plasma cell-derived antibodies in the plasma, and peripheral memory B-cells along with their associated antibody profiles elicited after in vitro stimulation. We found that magnitude varied amongst individuals, but was the highest in hospitalized subjects. Variants of concern (VoC) -RBD-reactive antibodies were found in the plasma of 72% of samples in this investigation, and VoC-RBD-reactive memory B-cells were found in all but 1 subject at a single time-point. This finding, that VoC-RBD-reactive MBCs are present in the peripheral blood of all subjects including those that experienced asymptomatic or mild disease, provides a reason for optimism regarding the capacity of vaccination, prior infection, and/or both, to limit disease severity and transmission of variants of concern as they continue to arise and circulate.

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