Suppression of the polyclonal B-cell responses by concanavalin A-treated bone marrow B cells in vitro

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.

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Direct Infection of B Cells by Dengue Virus Modulates B Cell Responses in a Cambodian Pediatric Cohort

Frontiers in Immunology ◽

10.3389/fimmu.2020.594813 ◽

2021 ◽

Vol 11 ◽

Author(s):

Vinit Upasani ◽

Hoa Thi My Vo ◽

Heidi Auerswald ◽

Denis Laurent ◽

Sothy Heng ◽

...

Keyword(s):

B Cells ◽

Dengue Virus ◽

B Cell ◽

Immune Cells ◽

Cell Formation ◽

Denv Infection ◽

Cell Responses ◽

B Cell Responses

Dengue is an acute viral disease caused by dengue virus (DENV), which is transmitted by Aedes mosquitoes. Symptoms of DENV infection range from inapparent to severe and can be life-threatening. DENV replicates in primary immune cells such as dendritic cells and macrophages, which contribute to the dissemination of the virus. Susceptibility of other immune cells such as B cells to direct infection by DENV and their subsequent response to infection is not well defined. In a cohort of 60 Cambodian children, we showed that B cells are susceptible to DENV infection. Moreover, we show that B cells can support viral replication of laboratory adapted and patient-derived DENV strains. B cells were permissive to DENV infection albeit low titers of infectious virions were released in cell supernatants CD300a, a phosphatidylserine receptor, was identified as a potential attachment factor or receptor for entry of DENV into B cells. In spite of expressing Fcγ-receptors, antibody-mediated enhancement of DENV infection was not observed in B cells in an in vitro model. Direct infection by DENV induced proliferation of B cells in dengue patients in vivo and plasmablast/plasma cell formation in vitro. To summarize, our results show that B cells are susceptible to direct infection by DENV via CD300a and the subsequent B cell responses could contribute to dengue pathogenesis.

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CNS Autoimmune Responses in BCMA-Deficient Mice Provide Insight for the Failure of Atacicept in MS

Neurology Neuroimmunology & Neuroinflammation ◽

10.1212/nxi.0000000000000973 ◽

2021 ◽

Vol 8 (3) ◽

pp. e973

Author(s):

Gaurav Kumar ◽

Zahra Maria ◽

Uday Kohli ◽

Agnieshka Agasing ◽

James L. Quinn ◽

...

Keyword(s):

Cell Culture ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

Inflammatory Responses ◽

Cell Responses ◽

Cd20 Antibody ◽

B Cell Responses ◽

Anti Cd20 ◽

Deficient Mice

ObjectiveB cells have emerged as a therapeutic target for MS. Anti-CD20 antibodies, which deplete B cells, are effective therapies for MS. However, atacicept (TACI-Fc), which blocks BAFF and APRIL and reduces B cells, unexpectedly exacerbates MS. We tested the hypothesis that B cell maturation antigen (BCMA), a receptor for BAFF and APRIL, plays a role in the paradoxical effects of anti-CD20 antibody and TACI-Fc using experimental autoimmune encephalomyelitis (EAE).MethodsEAE was induced in wild-type (BCMA+/+) and BCMA-deficient (BCMA−/−) mice with an immunization of rodent myelin oligodendrocyte glycoprotein (MOG)35–55 peptide. Treatment with anti-CD20 antibody, TACI-Fc, and isotype controls was administered by intraperitoneal injections. CNS infiltration was evaluated by histology; immune cell phenotypes were evaluated by flow cytometry; MOG-specific antibodies were determined by ELISA. Mixed bone marrow chimeras and cell culture assays were used to identify the specific subsets of immune cells affected by BCMA deficiency.ResultsFirst, we found that BCMA−/− mice had more severe EAE compared with BCMA+/+ mice and the increased disease was associated with elevated anti-MOG B-cell responses. Second, we found that anti-CD20 therapy attenuated EAE in BCMA−/− mice but not in BCMA+/+ mice. Third, TACI-Fc attenuated EAE in BCMA+/+ mice but not in BCMA−/− mice. Mixed bone marrow chimeric and cell culture experiments demonstrated that BCMA deficiency elevates inflammatory B-cell responses but inhibits inflammatory responses in macrophages.ConclusionsBCMA has multifaceted roles during inflammation that affects therapeutic efficacies of anti-CD20 and TACI-Fc in EAE. Our results from BCMA-deficient mice provide insights into the failure of atacicept in MS.

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B Cell Responses to CpG Correlate with CXCL16 Expression Levels in Common Variable Immunodeficiency

The Scientific World JOURNAL ◽

10.1100/2012/960219 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Vassilios Lougaris ◽

Manuela Baronio ◽

Massimiliano Vitali ◽

Giacomo Tampella ◽

Annarosa Soresina ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Common Variable Immunodeficiency ◽

Expression Levels ◽

Cell Responses ◽

Surface Receptor ◽

B Cell Responses ◽

And Function ◽

Common Variable

Broad Toll-like receptor 9 (TLR9) signalling defects after CpGin vitrostimulation have been described in common variable immunodeficiency (CVID). CXCL16, a surface receptor, was recently shown to influence cell responses to CpG. We evaluated the expression and function of CXCL16 on B cells from healthy controls and CVID patients. We report that CXCL16 is normally expressed on B cells throughout peripheral maturation. Decreased B cell expression of CXCL16 was observed in a subgroup of CVID patients that correlated with defectivein vitroresponses to CpG (such as upregulation of CD69, CD86, AICDA, IL-6, and TLR9). Our data suggest that expression levels of a surface receptor, namely, CXCL16, correlate with B cell responses mediated by TLR9 in common variable immunodeficiency.

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Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients

Arthritis Research & Therapy ◽

10.1186/s13075-020-02372-z ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

John Marken ◽

Sujatha Muralidharan ◽

Natalia V. Giltiay

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Cell Activation ◽

Cytokine Production ◽

B Cell Activation ◽

Cell Responses ◽

Healthy Donors ◽

B Cell Responses

Abstract Background CD40-CD40L is a key co-stimulatory pathway for B cell activation. As such, its blockade can inhibit pathogenic B cell responses in autoimmune diseases, such as Sjogren’s syndrome (SjS) and systemic lupus erythematosus (SLE). In this study, we examined the in vitro effects of KPL-404, a humanized anti-CD40 monoclonal antibody (Ab), on primary human B cells derived from either healthy donors (HD) or autoimmune patients and compared them to the effects of G28-5, a partially antagonistic anti-CD40 antibody. Methods PBMCs from HD or SjS and SLE patients were cultured in high-density cell cultures in the presence of IgG4 isotype control or anti-CD40 Abs KPL-404 or G28-5. Cells were stimulated with anti-CD3/CD28 cross-linking reagent ImmunoCult (IC) to induce CD40L-CD40-mediated B cell responses. B cell proliferation and activation, measured by dilution of proliferation tracker dye and the upregulation of CD69 and CD86, respectively, were assessed by flow cytometry. Anti-CD40 Ab cell-internalization was examined by imaging flow cytometry. Cytokine release in the PBMC cultures was quantified by bead-based multiplex assay. Results KPL-404 binds to CD40 expressed on different subsets of B cells without inducing cell depletion, or B cell proliferation and activation in in vitro culture. Under the same conditions, G28-5 promoted proliferation of and increased CD69 expression on otherwise unstimulated B cells. KPL-404 efficiently blocked the CD40L-CD40-mediated activation of B cells from HD at concentrations between 1 and 10 μg/ml. Treatment with KPL-404 alone did not promote cytokine production and blocked the production of IFNβ in healthy PBMC cultures. KPL-404 efficiently blocked CD40L-CD40-mediated activation of B cells from patients with SjS and SLE, without affecting their anti-IgM responses or affecting their cytokine production. Consistent with the differences of their effects on B cell responses, KPL-404 was not internalized by cells, whereas G28-5 showed partial internalization upon CD40 binding. Conclusions Anti-CD40 mAb KPL-404 showed purely antagonistic effects on B cells and total PBMCs. KPL-404 inhibited CD40L-CD40-mediated B cell activation in PBMC cultures from both healthy controls and autoimmune patients. These data support the therapeutic potential of CD40 targeting by KPL-404 Ab for inhibiting B cell responses in SjS and SLE.

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