scholarly journals Persistence of functional memory B cells recognizing SARS-CoV-2 variants despite loss of specific IgG

2021 ◽  
Author(s):  
Stephan Winklmeier ◽  
Katharina Eisenhut ◽  
Damla Taskin ◽  
Heike Ruebsamen ◽  
Celine Schneider ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Post Infection ◽  
Neutralizing Antibodies ◽  
Memory B Cells ◽  
B Cell Differentiation ◽  
Memory B Cell ◽  
Specific Serum ◽  
Serum Igg

While some COVID-19 patients maintain SARS-CoV-2-specific serum IgGs for more than 6 months post-infection, others, especially mild cases, eventually lose IgG levels. We aimed to assess the persistence of SARS-CoV-2-specific B cells in patients who have lost specific IgGs and analyzed the reactivity of the immunoglobulins produced by these B cells. Circulating IgG memory B cells specific for SARS-CoV-2 were detected in all 16 patients 1-8 months post-infection, and 11 participants had specific IgA B cells. Four patients lost specific serum IgG after 5-8 months but had SARS-CoV-2-specific-B-cell levels comparable to those of seropositive donors. Immunoglobulins produced after in vitro differentiation blocked receptor-binding domain (RBD) binding to the cellular receptor ACE-2, indicating neutralizing activity. Memory-B-cell-derived IgGs recognized the RBD of B.1.1.7 similarly to the wild-type, while reactivity to B.1.351 and P.1. decreased by 30% and 50%, respectively. Memory-B-cell differentiation into antibody-producing cells is a more sensitive method for detecting previous infection than measuring serum antibodies. Circulating SARS-CoV-2 IgG memory B cells persist, even in the absence of specific serum IgG; produce neutralizing antibodies; and show differential cross-reactivity to emerging variants of concern. These features of SARS-CoV-2-specific memory B cells will help to understand and promote long-term protection.

scholarly journals Pregnancy-induced effects on memory B-cell development in multiple sclerosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Malou Janssen ◽  
Liza Rijvers ◽  
Steven C. Koetzier ◽  
Annet F. Wierenga-Wolf ◽  
Marie-José Melief ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Memory B Cells ◽  
B Cell Differentiation ◽  
Third Trimester ◽  
Memory B Cell ◽  
Relapse Risk ◽  
The Third

AbstractIn MS, pathogenic memory B cells infiltrate the brain and develop into antibody-secreting cells. Chemokine receptors not only define their brain-infiltrating capacity, but also assist in their maturation in germinal centers. How this corresponds to pregnancy, as a naturally occurring modifier of MS, is underexplored. Here, we aimed to study the impact of pregnancy on both ex vivo and in vitro B-cell differentiation in MS. The composition and outgrowth of peripheral B cells were compared between 19 MS pregnant patients and 12 healthy controls during the third trimester of pregnancy (low relapse risk) and postpartum (high relapse risk). Transitional, and not naive mature, B-cell frequencies were found to drop in the third trimester, which was most prominent in patients who experienced a pre-pregnancy relapse. Early after delivery, these frequencies raised again, while memory B -cell frequencies modestly declined. CXCR4 was downregulated and CXCR5, CXCR3 and CCR6 were upregulated on postpartum memory B cells, implying enhanced recruitment into germinal center light zones for interaction with T follicular helper (TFH) cells. Postpartum memory B cells of MS patients expressed higher levels of CCR6 and preferentially developed into plasma cells under TFH-like in vitro conditions. These findings imply that memory B- cell differentiation contributes to postpartum relapse risk in MS.

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.

scholarly journals Memory B Cells Are Biased Towards Terminal Differentiation: A Strategy That May Prevent Repertoire Freezing

1997 ◽  
Vol 186 (6) ◽  
pp. 931-940 ◽  
Author(s):  
Christophe Arpin ◽  
Jacques Banchereau ◽  
Yong-Jun Liu
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Clone ◽  
Terminal Differentiation ◽  
Memory B Cells ◽  
B Cell Differentiation

Isolation of large numbers of surface IgD+CD38− naive and surface IgD−CD38− memory B cells allowed us to study the intrinsic differences between these two populations. Upon in vitro culture with IL-2 and IL-10, human CD40–activated memory B cells undergo terminal differentiation into plasma cells more readily than do naive B cells, as they give rise to five- to eightfold more plasma cells and three- to fourfold more secreted immunoglobulins. By contrast, naive B cells give rise to a larger number of nondifferentiated B blasts. Saturating concentrations of CD40 ligand, which fully inhibit naive B cell differentiation, only partially affect that of memory B cells. The propensity of memory B cells to undergo terminal plasma cell differentiation may explain the extensive extra follicular plasma cell reaction and the limited germinal center reaction observed in vivo after secondary immunizations, which contrast with primary responses in carrier-primed animals. This unique feature of memory B cells may confer two important capacities to the immune system: (a) the rapid generation of a large number of effector cells to efficiently eliminate the pathogens; and (b) the prevention of the overexpansion and chronic accumulation of one particular memory B cell clone that would freeze the available peripheral repertoire.

scholarly journals Atypical and classical memory B cells produce Plasmodium falciparum neutralizing antibodies

2013 ◽  
Vol 210 (2) ◽  
pp. 389-399 ◽  
Author(s):  
Matthias F. Muellenbeck ◽  
Beatrix Ueberheide ◽  
Borko Amulic ◽  
Alexandra Epp ◽  
David Fenyo ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
B Cells ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Clinical Symptoms ◽  
Memory Function ◽  
Clinical Malaria ◽  
Memory B Cells ◽  
Gene Repertoire ◽  
Memory B Cell

Antibodies can protect from Plasmodium falciparum (Pf) infection and clinical malaria disease. However, in the absence of constant reexposure, serum immunoglobulin (Ig) levels rapidly decline and full protection from clinical symptoms is lost, suggesting that B cell memory is functionally impaired. We show at the single cell level that natural Pf infection induces the development of classical memory B cells (CM) and atypical memory B cells (AtM) that produce broadly neutralizing antibodies against blood stage Pf parasites. CM and AtM contribute to anti-Pf serum IgG production, but only AtM show signs of active antibody secretion. AtM and CM were also different in their IgG gene repertoire, suggesting that they develop from different precursors. The findings provide direct evidence that natural Pf infection leads to the development of protective memory B cell antibody responses and suggest that constant immune activation rather than impaired memory function leads to the accumulation of AtM in malaria. Understanding the memory B cell response to natural Pf infection may be key to the development of a malaria vaccine that induces long-lived protection.

scholarly journals Recovery of Memory B-cell Subsets and Persistence of Antibodies in Convalescent COVID-19 Patients

2021 ◽  
Author(s):  
Anuradha Rajamanickam ◽  
Nathella Pavan Kumar ◽  
Arul Nancy P ◽  
Nandhini Selvaraj ◽  
Saravanan Munisankar ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Cell Subset ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Immature B Cells ◽  
B Cell Subsets

It is essential to examine the longevity of the defensive immune response engendered by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. We examined the SARS-CoV-2-specific antibody responses and ex vivo memory B-cell subsets in seven groups of individuals with COVID-19 classified based on days since reverse-transcription polymerase chain reaction confirmation of SARS-CoV-2 infection. Our data showed that the levels of IgG and neutralizing antibodies started increasing from days 15 to 30 to days 61 to 90, and plateaued thereafter. The frequencies of naive B cells and atypical memory B cells decreased from days 15 to 30 to days 61 to 90, and plateaued thereafter. In contrast, the frequencies of immature B cells, classical memory B cells, activated memory B cells, and plasma cells increased from days 15 to 30 to days 61 to 90, and plateaued thereafter. Patients with severe COVID-19 exhibited increased frequencies of naive cells, atypical memory B cells, and activated memory B cells, and lower frequencies of immature B cells, central memory B cells, and plasma cells when compared with patients with mild COVID-19. Therefore, our data suggest modifications in memory B-cell subset frequencies and persistence of humoral immunity in convalescent individuals with COVID-19.

scholarly journals Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

2021 ◽  
Author(s):  
Casper Marsman ◽  
Dorit Verhoeven
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Formation ◽  
B Cell Differentiation ◽  
Differentiation Potential ◽  
Human B Cell

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

scholarly journals RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1051-1051
Author(s):  
Renee C Tschumper ◽  
Collin A Osborne ◽  
Pritha Chanana ◽  
Jaime I Davila ◽  
Denise K Walters ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
In Vitro Model ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Rna Seq ◽  
Repertoire Analysis ◽  
V Region ◽  
Vitro Model

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kristian Assing ◽  
Christian Nielsen ◽  
Marianne Jakobsen ◽  
Charlotte B. Andersen ◽  
Kristin Skogstrand ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Formation ◽  
Memory B Cells ◽  
Memory B Cell

Abstract Background Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections. Case presentation We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001. Conclusions To the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

scholarly journals Modulation of spontaneous B-cell differentiation in macroglobulinemia by retinoic acid

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2206-2210 ◽  
Author(s):  
Y Levy ◽  
S Labaume ◽  
MC Gendron ◽  
JC Brouet
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Culture Supernatants

Abstract We previously showed that clonal blood B cells from patients with macroglobulinemia spontaneously differentiate in vitro to plasma cells. This process is dependent on an interleukin (IL)-6 autocrine pathway. We investigate here whether all-trans-retinoic acid (RA) interferes with B-cell differentiation either in patients with IgM gammapathy of undetermined significance (MGUS) or Waldenstrom's macroglobulinemia (WM). RA at a concentration of 10(-5) to 10(-8) mol/L inhibited by 50% to 80% the in vitro differentiation of purified B cells from four of five patients with MGUS and from one of five patients with WM as assessed by the IgM content of day 7 culture supernatants. We next determined whether this effect could be related to an inhibition of IL- 6 secretion by cultured B cells and/or a downregulation of the IL-6 receptor (IL-6R), which was constitutively expressed on patients' blood B cells. A 50% to 100% (mean, 80%) inhibition of IL-6 production was found in seven of 10 patients (five with MGUS and two with WM). The IL- 6R was no more detectable on cells from patients with MGUS after 2 days of treatment with RA and slightly downregulated in patients with WM. It was of interest that B cells susceptible to the action of RA belonged mostly to patients with IgM MGUS, which reinforces our previous data showing distinct requirements for IL-6-dependent differentiation of blood B cells from patients with VM or IgM MGUS.

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