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High-throughput profiling of antigen-specific antibody secreting cells using an antibody secretion trap (TRAPnSeq)

ABSTRACT: Following activation by cognate antigen, B cells undergo fine-tuning of their antigen receptors and may ultimately differentiate into antibody-secreting cells (ASCs). While antigen-specific antibodies from B cell receptor (BCR) expressing B cells can be readily cloned and sequenced following flow sorting, antigen-specific plasma cells that lack surface BCR cannot be easily profiled in a high-throughput way. Here, we report an approach, TRAPnSeq (antigen specificity mapping through Ig secretionTRAPandSequencing), that allows capture of secreted antibodies on the surface of ASCs, which in turn enables high-throughput screening of single ASCs against large antigen panels and recovery of paired VH:VL antibody sequences. This approach incorporates flow cytometry, standard microfluidic platforms and DNA barcoding technologies to isolate and characterize antigen-specific ASCs through single cell V(D)J, RNA and antigen barcode sequencing. We show the utility of TRAPnSeq by profiling antigen-specific IgG and IgE plasma cells from mouse and humans and validate antigen binding by ELISA. TRAPnSeq can easily be combined with existing B cell platforms to accelerate antibody discovery from ASCs and can further be expanded to any protein secreting-cells.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE233507 | GEO | 2023/07/07

REPOSITORIES: GEO

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Transcriptomic analysis of human B cell responses to vaccination

Project description:Analysis of the gene expression profiles of naÃ¯ve B cells, resting memory B cells (MBCs), activated B cells (ABCs) and antibody-secreting cells (ASCs) isolated from peripheral blood one week following influenza vaccination. Upon antigen exposure B cells eventually bifurcate into two distinct lineages, plasmablasts and memory B cells. We previously reported that plasmablasts or antibody-secreting cells (ASCs) could be transiently detected in blood shortly after infection or vaccination of humans. Here, we define the phenotype and the transcriptional program of a novel human antigen-specific B cell subset, referred to as activated B cells (ABCs). ABCs do not spontaneously secrete antibodies and possess a unique transcriptional profile that distinguishes them from ASCs and resting memory B cells. Clonal lineages present among day 7 ABCs persisted in blood for up to three months post-influenza immunization indicating that ABCs may be destined to join the long-term memory B cell pool. ABCs and ASCs can be clearly distinguished in blood following influenza and Ebola virus infections. Interrogating ABCs will expand our understanding of the differentiation, maturation and longevity of human B cell responses. Total RNA was isolated from 10,000 cells of each population.

2015-12-31 | E-GEOD-68245 | biostudies-arrayexpress

Transcriptomic analysis of human B cell responses to vaccination

Project description:Analysis of the gene expression profiles of naïve B cells, resting memory B cells (MBCs), activated B cells (ABCs) and antibody-secreting cells (ASCs) isolated from peripheral blood one week following influenza vaccination. Upon antigen exposure B cells eventually bifurcate into two distinct lineages, plasmablasts and memory B cells. We previously reported that plasmablasts or antibody-secreting cells (ASCs) could be transiently detected in blood shortly after infection or vaccination of humans. Here, we define the phenotype and the transcriptional program of a novel human antigen-specific B cell subset, referred to as activated B cells (ABCs). ABCs do not spontaneously secrete antibodies and possess a unique transcriptional profile that distinguishes them from ASCs and resting memory B cells. Clonal lineages present among day 7 ABCs persisted in blood for up to three months post-influenza immunization indicating that ABCs may be destined to join the long-term memory B cell pool. ABCs and ASCs can be clearly distinguished in blood following influenza and Ebola virus infections. Interrogating ABCs will expand our understanding of the differentiation, maturation and longevity of human B cell responses.

2015-12-31 | GSE68245 | GEO

Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells

Project description:Upon encounter with antigen, mature B cells undergo complex changes in their physiological state and anatomical localization, and initiate a differentiation process that ultimately produces antibody-secreting cells (ASCs), a major effector population of the immune system. To better characterize this process we have defined the transcriptome of all stages of B cell terminal differentiation by performing a large scale RNA-seq experiment. This analysis provides a molecular signature of ASC populations that highlights the stark transcriptional divide between B cells and plasma cells, and also enables the demarcation ASCs based on location, cell cycle status and maturity. The gene expression changes strongly correlated with cell division history and the acquisition of permissive histone modifications and were clearly evident in the small subset of regulatory genes containing super-enhancers. These findings highlight the core transcriptional program that guides B cell terminal differentiation and the production of antibody, essential processes in the immune response. Transcriptional profiling of ex vivo and in vitro B cell and plasma cell populations in mice using RNA sequencing

2015-04-01 | E-GEOD-60927 | biostudies-arrayexpress

Transcriptomic diversity and overlapping clonality across subsets of antibody-secreting and memory B cells from spontaneous germinal centers

Project description:Affinity matured self-reactive antibodies are found in autoimmune diseases like systemic lupus erythematous. Here we used fate-mapping reporter mice and single cell transcriptomics coupled to antibody repertoire analysis to characterize the post-germinal center (GC) B cell compartment in a new mouse model of autoimmunity. Antibody secreting cells (ASCs) and memory B cells (MemBs) from spontaneous GCs grouped into multiple subclusters. ASCs matured into two terminal clusters, with distinct secretion, antibody repertoire and metabolic profiles. MemBs contained FCRL5+ and CD23+ subsets, with different in vivo localization in the spleen. GC-derived FCRL5+ MemBs share transcriptomic and repertoire properties with atypical B cells found in aging and infection and localize to the marginal zone, suggesting a similar contribution to rapid recall responses. While transcriptomically diverse, ASC and MemB subsets maintained an underlying clonal redundancy. Therefore, self-reactive clones could escape subset-targeting therapy by perpetuation of self-reactivity in distinct subsets.

2023-06-22 | GSE203132 | GEO

BLIMP-1 is insufficient to induce antibody secretion in the absence of IRF4

Project description:Differentiation of B cells into antibody secreting cells (ASCs), plasmablasts and plasma cells, is regulated by a network of transcription factors. Within this network factors including PAX5 and BCL6 prevent ASC differentiation and maintain the B cell phenotype whereas BLIMP-1 and high IRF4 expression promote plasmacytic differentiation. BLIMP-1 is thought to induce immunoglobulin secretion. While IRF4 is needed for the survival of ASCs, its role in the regulation of antibody secretion has been controversial. BCL6-deficient DT40 B cell line has upregulated BLIMP-1 expression and secrete antibodies. In order to study the role of IRF4 in regulation of antibody secretion we have created a double knockout (DKO) DT40 B cell line deficient in both IRF4 and BCL6. This DKO cell line did not upregulate PRDM1 (the gene encoding for BLIMP-1) expression or secrete IgM. Even enforced BLIMP-1 expression in DKO cells or IRF4-deficient cells could not induce IgM secretion while it did in WT cells. However, enforced IRF4 expression in DKO cells induced strong IgM secretion. Our findings support a model where IRF4 expression in addition to BLIMP-1 expression is required to induce antibody secretion.

2018-03-05 | GSE94836 | GEO

Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells

2015-04-01 | GSE60927 | GEO

Project description:We analyzed age-related defects in B cell populations from young and aged mice. Microarray analysis of bone marrow resident antibody secreting cells (ASCs) showed significant changes upon aging, affecting multiple genes, pathways and functions including those that play a role in immune regulation, humoral immune responses, chromatin structure and assembly, cell metabolism and the endoplasmic reticulum (ER) stress response. Aged ASCs expressed higher levels of immunoregulators including PD-1. Increased PD-1 expression was also seen on mature aged B cells indicating early defects in the lineage. High PD-1 expression on the aged ASCs correlated with high cellular reactive oxygen species levels. Mitochondrial defects in aged B cells were further confirmed by the absence of an increases in basal respiration observed upon in vitro stimulation of young B cells

2019-11-21 | GSE72224 | GEO

Single-cell analysis reveals dynamics of human B cell differentiation and identifies novel B and antibody-secreting cell intermediates

Project description:Differentiation of B cells into antibody-secreting cells (ASCs) is a key process to generate protective humoral immunity. A detailed understanding of the cues controlling ASC differentiation is important to devise strategies to modulate antibody formation. Here, we dissected differentiation trajectories of human naive B cells into ASCs using single-cell RNA sequencing. By comparing transcriptomes of B cells at different stages of differentiation from an in vitro model with ex vivo B cells and ASCs, we uncovered a novel pre-ASC population present ex vivo in lymphoid tissues. For the first time, a germinal-center-like population is identified in vitro from human naive B cells and possibly progresses into a memory B cell population through an alternative route of differentiation, thus recapitulating in vivo human GC reactions. Our work allows further detailed characterization of human B cell differentiation into ASCs or memory B cells in both healthy and diseased conditions.

2022-09-27 | GSE214265 | GEO

ATAC-seq analysis of human blood and bone marrow antibody-secreting cell subsets

Project description:Antibody-secreting cells (ASC) circulate after vaccination and infection and migrate to the bone marrow (BM) where a subset known as long-lived plasma cells (LLPC) persist and secrete antibodies for a lifetime. The mechanisms of how circulating ASC become LLPC is not well elucidated. Here, we show that human early-minted blood ASCs have distinct morphology, transcriptomes, and epigenetics compared to human BM LLPC. Compared to blood ASC, BM LLPC have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. Additionally, LLPC acquire transcriptional and epigenetic differences in multiple cellular pathways that include apoptosis. Upregulation of the anti-apoptotic genes MCL1, BCL2, BCL-XL while simultaneously downregulation of pro-apoptotic genes HRK1, CASP3, and CASP8 occurs in LLPC. Consistent with the decrease in gene expression, pro-apoptotic gene loci are less accessible in LLPC. In contrast, anti-apoptotic gene expression is increased but is not always accompanied by changes in accessibility. Importantly, we show that early-minted blood ASCs undergo morphological and transcriptional changes that make these cells resemble ex vivo BM ASCs, suggesting that the BM microniche at least in part promotes LLPC maturation. Overall, our study demonstrates that early-minted blood ASC in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPC.

2022-01-14 | GSE193642 | GEO

RNA-Seq analysis of human blood and bone marrow antibody secreting cell subsets

Project description:Antibody secreting cells (ASC) circulate after vaccination and infection and migrate to the bone marrow (BM) where a subset known as long-lived plasma cells (LLPC) persist and secrete antibodies for a lifetime. The mechanisms of how circulating ASC become LLPC is not well elucidated. Here, we show that human early-minted blood ASCs have distinct morphology, transcriptomes, and epigenetics compared to human BM LLPC. Compared to blood ASC, BM LLPC have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. Additionally, LLPC acquire transcriptional and epigenetic differences in multiple cellular pathways that include apoptosis. Upregulation of the anti-apoptotic genes MCL1, BCL2, BCL-XL while simultaneously downregulation of pro-apoptotic genes HRK1, CASP3, and CASP8 occurs in LLPC. Consistent with the decrease in gene expression, pro-apoptotic gene loci are less accessible in LLPC. In contrast, anti-apoptotic gene expression is increased but is not always accompanied by changes in accessibility. Importantly, we show that early-minted blood ASCs undergo morphological and transcriptional changes that make these cells resemble ex vivo BM ASCs, suggesting that the BM microniche at least in part promotes LLPC maturation. Overall, our study demonstrates that early-minted blood ASC in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPC.

2021-12-17 | GSE178788 | GEO

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