scholarly journals Single-cell repertoire tracing identifies rituximab refractory B cells during myasthenia gravis relapses

2019 ◽  
Author(s):  
Ruoyi Jiang ◽  
Miriam L. Fichtner ◽  
Kenneth B. Hoehn ◽  
Panos Stathopoulos ◽  
Richard J. Nowak ◽  
...  
Keyword(s):  
B Cells ◽  
Myasthenia Gravis ◽  
B Cell ◽  
Single Cell ◽  
De Novo ◽  
Cell Receptor ◽  
Memory B Cells ◽  
Cell Repertoire ◽  
B Cell Subsets ◽  
Antibody Secreting Cells

AbstractRituximab, a B cell-depleting therapy, is indicated for treating a growing number of autoantibody-mediated autoimmune disorders. However, relapses can occur after treatment and autoantibody-producing B cell subsets may be found during relapses. It is not understood if these autoantibody-producing B cell subsets emerge from the failed depletion of pre-existing B cells or are re-generated de novo. To further define the mechanisms that cause post-rituximab relapse, we studied patients with autoantibody-mediated muscle-specific kinase (MuSK) myasthenia gravis (MG) who relapsed after treatment. We carried out single-cell transcriptional and B cell receptor (BCR) profiling on longitudinal B cell samples. We identified clones present prior to therapy that continued to persist during relapse. Persistent B cell clones included both antibody-secreting cells and memory B cells characterized by gene expression signatures associated with B cell survival. A subset of persistent antibody-secreting cells and memory B cells were specific for the MuSK autoantigen. These results demonstrate that rituximab is not fully effective at eliminating autoantibody-producing B cells and provide a mechanistic understanding of post-rituximab relapse in MuSK MG.

scholarly journals Ex vivo characterization and isolation of rare memory B cells with antigen tetramers

Blood ◽  
2011 ◽  
Vol 118 (2) ◽  
pp. 348-357 ◽  
Author(s):  
Bettina Franz ◽  
Kenneth F. May ◽  
Glenn Dranoff ◽  
Kai Wucherpfennig
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Memory B Cells ◽  
Cell Repertoire ◽  
Low Frequencies

Abstract Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer+ B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.

A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4500-4504 ◽  
Author(s):  
Nadia L. Bernasconi ◽  
Nobuyuki Onai ◽  
Antonio Lanzavecchia
Keyword(s):  
B Cells ◽  
B Cell ◽  
Constitutive Expression ◽  
Human Memory ◽  
Cell Receptor ◽  
Primary Response ◽  
Memory B Cells ◽  
Toll Like Receptors ◽  
Polyclonal Activation ◽  
B Cell Subsets

Abstract Toll-like receptors (TLRs) are pattern recognition receptors that trigger innate immunity. In this study we investigated the expression of 10 TLRs in human naive and memory B-cell subsets. We report that in human naive B cells most TLRs are expressed at low to undetectable levels, but the expression of TLR9 and TLR10 is rapidly induced following B-cell-receptor (BCR) triggering. In contrast, memory B cells express several TLRs at constitutively high levels. The differential expression of TLR9 correlates with responsiveness to its agonist, CpG DNA. Thus, human memory B cells proliferate and differentiate to immunoglobulin (Ig)–secreting cells in response to CpG, while naive B do so only if simultaneously triggered through the BCR. The BCR-induced expression of TLRs in human naive B cells prevents polyclonal activation in a primary response, because it restricts stimulation to antigen-specific B cells. In contrast, the constitutive expression of TLRs in memory B cells allows polyclonal activation of the entire memory pool. Thus, in human B cells TLRs are downstream of BCR and play a role both in the primary response and in the memory phase.

The Chemokine Receptor Profile As Distinctive Criterion Between Normal B-Cell Subsets and As Potential Discriminative Marker to Identify the Cell of Origin in Patients with Chronic Lymphocytic Leukemia and Richter Syndrome

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3890-3890
Author(s):  
Katharina Troppan ◽  
Kerstin Wenzl ◽  
Peter Neumeister ◽  
Christine Beham-Schmid ◽  
Martina Przekopowitz ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
De Novo ◽  
Memory B Cells ◽  
Cell Of Origin ◽  
Richter Syndrome ◽  
Receptor Profile ◽  
B Cell Subsets

Abstract Chemokine receptors are G-protein-coupled cell surface receptors, which dissociate upon activation by their ligands and cause downstream signaling. Several studies have revealed the crucial contribution of chemokine receptors and their ligands in normal B-cell differentiation and development of hematopoietic malignancies. The Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Due to the lack of knowledge on the chemokine receptor, we aimed to investigate their expression profile in patients with CLL and Richter syndrome. Therefore, we investigated the mRNA expression levels of 18 known chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, XCR1, CX3CR1) by using semi-quantitative real-time PCR on seven samples of paired (CLL and transformed DLBCL) RS samples, additionally four CLL samples -all of them subsequently transformed into DLBCL-, and eight transformed DLBCL samples originating from CLL. Additionally, 30 samples of de-novo DLBCL, including 10 germinal center B-cell (GCB) lymphomas, 12 non-germinal center B-cell lymphomas (non-GCB), and 8 unclassified DLBCL were included. Four samples of naïve B-cells (CD5 neg), CD5+ naïve B-cells and CD27+ memory B-cells (n=12) served as non-neoplastic controls. No differences in the chemokine receptor profile were detected between CD5+ and negative naïve B-cells. When comparing CD27+ memory B-cells to naïve B-cells a significant lower expression level was found for CCR7 (7-fold), CXCR4 (4-fold), and CXCR5 (1.5 fold). CCR7 (5-fold) and CXCR4 (5-fold) were also lower expressed in CD27+ memory B-cells compared to CD5+ naïve B-cells. Five out of 18 chemokine receptors were differentially expressed comparing the distinct normal B-cell subsets with RS samples. Comparing CLL samples and RS samples to CD5+ naïve B-cells, CXCR4 (12-fold for CLLs and 10-fold for RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for RS samples) were lower expressed, whereas CXCR3 (10-fold for CLLs and 8.5-fold for the transformed samples) was higher expressed and CCR5 de-novo expressed. Compared to naïve B-cells, the same chemokine receptors were deregulated: CXCR4 (10-fold for CLLs and 8.5-fold for the RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for the transformed samples) were lower expressed, CXCR3 (45-fold for CLLs and 30-fold for the transformed samples) was higher expressed and CCR5 was de-novo expressed. Comparing CLL samples and transformed RS samples to CD27+ memory B-cells, CCR5 (5.1-fold for CLLs and 4.3-fold for the RS samples) and CCR7 (8.7-fold for CLLs and 10-fold for the transformed samples) were higher expressed in both malignancies. Only one chemokine receptor was found to be differentially expressed in our seven paired RS samples: CCR6 showed a trend of a higher expression (1.4-fold) in CLL components. Considering RS and GCB DLBCL, CCR1, CCR5, and CXCR6 were found to be significantly down-regulated in RS (at least 4-fold), in contrast to CCR7 and CXCR4, which showed higher expression levels in RS (6-fold). CCR1 and CCR5 were lower expressed comparing RS and non-GCB DLBCL (25-fold and 8-fold), whereas CCR7 again, together with CXCR7, was higher expressed (3- fold and 6-fold respectively). Our data indicate a difference in the chemokine receptor profile within normal B-cell subsets. These differences are also reflected in the different expression profile of low and high aggressive component of CLL/RS compared to the distinct B cell subtypes. Hence, in future these multiple deregulated CC and CXC receptors might serve as a further hint in identifying the cell of origin of different B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-cell transcriptomic analyses define distinct peripheral B cell subsets and discrete development pathways

2020 ◽  
Author(s):  
Alexander Stewart ◽  
Joseph Ng ◽  
Gillian Wallis ◽  
Vasiliki Tsioligka ◽  
Franca Fraternali ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Heterogeneous Population ◽  
Memory B Cells ◽  
Double Negative ◽  
Memory Cells ◽  
Relative Paucity ◽  
Age Related ◽  
B Cell Subsets

AbstractSeparation of B cells into different subsets has been useful to understand their different functions in various immune scenarios. In some instances, the subsets defined by phenotypic FACS separation are relatively homogeneous and so establishing the functions associated with them is straightforward. Other subsets, such as the “Double negative” (DN, CD19+CD27-IgD-) population, are more complex with reports of differing functionality which could indicate a heterogeneous population. Recent advances in single-cell techniques enable an alternative route to characterise cells based on their transcriptome. To maximise immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamical transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. One involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and on its own developmental branch but with links to the classical memory branch.

scholarly journals Characterising the Phenotypic Diversity of Antigen-Specific Memory B Cells Before and After Vaccination

2021 ◽  
Vol 12 ◽  
Author(s):  
M. Christian Tjiam ◽  
Sonia Fernandez ◽  
Martyn A. French
Keyword(s):  
B Cells ◽  
B Cell ◽  
Phenotypic Diversity ◽  
Binding Capacity ◽  
Booster Vaccination ◽  
Cell Receptor ◽  
Memory B Cells ◽  
Cell Phenotype ◽  
Antigen Binding ◽  
B Cell Subsets

The diversity of B cell subsets and their contribution to vaccine-induced immunity in humans are not well elucidated but hold important implications for rational vaccine design. Prior studies demonstrate that B cell subsets distinguished by immunoglobulin (Ig) isotype expression exhibit divergent activation-induced fates. Here, the antigen-specific B cell response to tetanus toxoid (TTd) booster vaccination was examined in healthy adults, using a dual-TTd tetramer staining flow cytometry protocol. Unsupervised analyses of the data revealed that prior to vaccination, IgM-expressing CD27+ B cells accounted for the majority of TTd-binding B cells. 7 days following vaccination, there was an acute expansion of TTd-binding plasmablasts (PB) predominantly expressing IgG, and a minority expressing IgA or IgM. Frequencies of all PB subsets returned to baseline at days 14 and 21. TTd-binding IgG+ and IgA+ memory B cells (MBC) exhibited a steady and delayed maximal expansion compared to PB, peaking in frequencies at day 14. In contrast, the number of TTd-binding IgM+IgD+CD27+ B cells and IgM-only CD27+ B cells remain unchanged following vaccination. To examine TTd-binding capacity of IgG+ MBC and IgM+IgD+CD27+ B cells, surface TTd-tetramer was normalised to expression of the B cell receptor-associated CD79b subunit. CD79b-normalised TTd binding increased in IgG+ MBC, but remained unchanged in IgM+IgD+CD27+ B cells, and correlated with the functional affinity index of plasma TTd-specific IgG antibodies, following vaccination. Finally, frequencies of activated (PD-1+ICOS+) circulating follicular helper T cells (cTFH), particularly of the CXCR3-CCR6- cTFH2 cell phenotype, at their peak expansion, strongly predicted antigen-binding capacity of IgG+ MBC. These data highlight the phenotypic and functional diversity of the B cell memory compartment, in their temporal kinetics, antigen-binding capacities and association with cTFH cells, and are important parameters for consideration in assessing vaccine-induced immune responses.

scholarly journals Multimodal repertoire analysis unveils B cell biology in health and immune-mediated diseases

2022 ◽  
Author(s):  
Mineto Ota ◽  
Masahiro Nakano ◽  
Yasuo Nagafuchi ◽  
Satomi Kobayashi ◽  
Hiroaki Hatano ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Cell Biology ◽  
Memory B Cells ◽  
Dependent Manner ◽  
Cell Repertoire ◽  
Repertoire Analysis ◽  
Immune Mediated ◽  
B Cell Subsets

Despite involvement of B cells in the pathogenesis of immune-mediated diseases, biological mechanisms underlying their function are scarcely understood. To overcome this gap, comprehensive analysis of the B cell repertoire is essential. Here, we cataloged and investigated the repertoire of five B cell subsets from 595 cases under immune-mediated diseases and health. CDR-H3 length among naive B cells was shortened among autoimmune diseases in an interferon signature-dependent manner. VDJ gene usage was skewed especially in plasmablasts and unswitched-memory B cells of systemic lupus erythematosus patients with frequent usage of VDJ genes used mainly in naive B cells and not unswitched-memory B cells of healthy controls. We developed a scoring system for this skewing, and it correlated with peripheral helper T cell transcriptomic signatures and disease activity and decreased after belimumab treatment. Moreover, genetic association analysis identified three molecules possibly involved in somatic hyper-mutation processes in humans. Our multimodal repertoire analysis brings new insights to B cell biology.

scholarly journals A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike

2021 ◽  
Author(s):  
Mathieu Claireaux ◽  
Tom G Caniels ◽  
Marlon de Gast ◽  
Julianna Han ◽  
Denise Guerra ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Cell Repertoire ◽  
Fold Enrichment ◽  
B Cell Repertoire ◽  
High Fraction ◽  
Antibody Class

AbstractDelineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that ∼82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched (∼0.05%) and unswitched B cells (∼0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

scholarly journals Longitudinal analysis of FcRL5 expression and clonal relationships among classical and atypical memory B cells following malaria

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
S. Jake Gonzales ◽  
Sebastiaan Bol ◽  
Ashley E. Braddom ◽  
Richard Sullivan ◽  
Raphael A. Reyes ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Longitudinal Analysis ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Developmental Pathways ◽  
Malaria Episode ◽  
Transmission Region ◽  
B Cell Subsets

Abstract Background Chronic and frequently recurring infectious diseases, such as malaria, are associated with expanded populations of atypical memory B cells (MBCs). These cells are different from classical MBCs by the lack of surface markers CD21 and CD27 and increased expression of inhibitory receptors, such as FcRL5. While the phenotype and conditions leading to neogenesis of atypical MBCs in malaria-experienced individuals have been studied extensively, the origin of these cells remains equivocal. Functional similarities between FcRL5+ atypical MBCs and FcRL5+ classical MBCs have been reported, suggesting that these cells may be developmentally related. Methods Here, a longitudinal analysis of FcRL5 expression in various B cell subsets was performed in two children from a high transmission region in Uganda over a 6-month period in which both children experienced a malaria episode. Using B-cell receptor (BCR)-sequencing to track clonally related cells, the connections between IgM+ and IgG+ atypical MBCs and other B cell subsets were studied. Results The highest expression of FcRL5 was found among IgG+ atypical MBCs, but FcRL5+ cells were present in all MBC subsets. Following malaria, FcRL5 expression increased in all IgM+ MBC subsets analysed here: classical, activated, and atypical MBCs, while results for IgG+ MBC subsets were inconclusive. IgM+ atypical MBCs showed few connections with other B cell subsets, higher turnover than IgG+ atypical MBCs, and were predominantly derived from naïve B cells and FcRL5− IgM+ classical MBCs. In contrast, IgG+ atypical MBCs were clonally expanded and connected with classical MBCs. IgG+ atypical MBCs present after a malaria episode mainly originated from FcRL5+ IgG+ classical MBCs. Conclusions Collectively, these results suggest fundamental differences between unswitched and class-switched B cell populations and provide clues about the primary developmental pathways of atypical MBCs in malaria-experienced individuals.

scholarly journals Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells

2021 ◽  
Author(s):  
Sarah Wilmore ◽  
Karly-Rai Rogers-Broadway ◽  
Joe Taylor ◽  
Elizabeth Lemm ◽  
Rachel Fell ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mrna Translation ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Mrna Stabilization

AbstractSignaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

scholarly journals Characterization of B-cell Receptor Heavy Chain Complementarity-determining Region 3 Repertoire Based on the Differences of Symbiotic Microorganisms in the Gut of Mice

2021 ◽  
Author(s):  
Jun Li ◽  
Yurong Pan ◽  
Qingqing Ma ◽  
Long Ma ◽  
Bin Shi ◽  
...  
Keyword(s):  
Small Intestine ◽  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Intestinal Microflora ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Positive Effects ◽  
Significant Difference

Abstract Background Colonization of gut microorganism is related to maturation of B cells in peripheral immune organs. This study aims to investigate the effect of intestinal microflora in Germ-free (GF), Specific Pathogen-free (SPF) and Clean (CL) BALB/C mice to small intestine total B-cell and memory B-cell receptor (BCR) complementary-determining region 3 (CDR3) repertoire. Results The composition and characteristics of intestinal microflora were analyzed by 16S rDNA sequencing. Genomic DNA extracted from small intestine tissue and memory B-cells of GF, SPF and CL mice were conducted via high-throughput DNA sequencing methods. As expected, significant differences of gut microflora diversity were observed in the three mice groups. CL group showed the most diversity, followed by SPF group, and GF group had the lowest diversity. Moreover, anormogenesis of intestinal lymphoid tissue were observed in GF mice. Diversity of the BCR heavy chain CDR3 repertoire in memory B cells were significant difference among three groups, but not in total B cells. The nucleotide polymorphism, usage frequency of gene segments (V, D, J, V–J gene segments) and amino acid of total B cells and memory B cells CDR3 were comparable among three mice groups, and there was significant difference between CL and GF mice groups. Conclusions The results of this study advocate that the colonization of intestinal microorganisms affect the diversity of B cells CDR3 repertoire. Elucidating mechanism of microbiome participated in the function of intestinal mucosal immune system may have positive effects on human health, and it requires further investigation.

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