Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq analysis of quiescent human splenic B cell populations

ABSTRACT: Memory B cells represent one of the pillar of humoral protection and are found in the circulation and secondary lymphoid organs several decades after initial pathogen or vaccine encounter. The exact cellular and biological mechanisms allowing long-term survival of quiescent cells in such a fluctuating microenvironment remain poorly understood. scRNA-seq analysis of long-lived Vaccinia-specific and total IgG+ memory B cells had helped us identify two subsets of quiescent switched memory B cells in the spleen of adult donors, separated by the expression of CR2(CD21). To better understand the relationship between these two resting splenic memory B cell subsets, we sorted CD21hiCD20hi and CD21intCD20int IgG+ memory B cells from the live IgD-CD27+IgG+CD20+CD3-CD14-CD16- compartment of the spleen of four organ donors, collected between 2015 and 2019. For all donors, both IgG+ memory B cell subsets were sorted alongside naïve B cells (IgD+CD27-CD38-CD24-CD20+CD3-CD14-CD16-). Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed on 50000 cells of each sorted population, according to published methods (Buenrostro, Nature Methods, 2013, PMID: 24097267) and sent for library sequencing by Integragen (Evry, France) (See linked experiment). RNA was extracted from remaining cells and sent for library preparation and sequencing by Integragen (Evry, France).

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Pascal Chappert

PROVIDER: E-MTAB-11622 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Memory B cells represent one of the pillar of humoral protection and are found in the circulation and secondary lymphoid organs several decades after initial pathogen or vaccine encounter. The exact cellular and biological mechanisms allowing long-term survival of quiescent cells in such a fluctuating microenvironment remain poorly understood. Assessing the true longevity of human memory B cells against common pathogens is, in most settings, hindered by the possibility for any given individual to re-encounter them during his lifetime. Smallpox, officially declared eradicated in 1980 (Fenner WHO 1988), represents one notable exception. Alongside smallpox eradication, anti-smallpox vaccination, based on the live Vaccinia virus, was progressively discontinued during the 1970s. As such, any detectable anti-vaccinia memory B cell in an individual post 2010 would likely have been generated more than 40 years ago, without any re-stimulation with the immunizing antigen during that timeframe. Anti-vaccinia antibodies serum titers remain stable in human for more than 80 years after first vaccination (Amanna NEJM 2007, PMID: 17989383), with several identified immunodominant epitopes including the envelope glycoprotein B5R (Lantto J Virol 2011, PMID: 21147924). Anti-vaccinia memory B cells have similarly been followed longitudinally in the blood for up to 65 years post vaccination (Amanna NEJM 2007, PMID: 17989383 ; Crotty JI 2003, PMID : 14607890) and, following a contraction phase taking place in the first couple of years after immunization, reach a remarkably stable plateau that last for decades at around 0.1% of total circulating IgG+ memory B cells. Functional analysis of such long-lived memory B cells, however, is still lacking. As a proxy to study long-lived memory B cells, IgG+ switched memory B cells specific for the immunodominant Vaccinia antigen B5R (B5R+ IgG+ memory B cells) were sorted and analyzed by scRNAseq from the spleen of six organ donors, collected between 2015 and 2018. For all donors, B5R+ IgG+ memory B cells were sorted alongside total IgG+ memory B cells (live IgD-CD27+IgG+CD20+CD3-CD14-CD16-) and naïve B cells (live IgD+CD27-CD38-CD24-CD20+CD3-CD14-CD16-). Single-cell mRNA sequencing was performed according to an adapted version of the SORT-seq protocol (Muraro et al., 2016, PMID: 27693023), with cDNA libraries generation, sequencing and reads alignment performed at Single Cell Discoveries (Utrecht, Netherlands).

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RNA-seq analysis of quiescent human splenic B cell populations

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