SFR KO mice germinal center B cell RNA sequencing
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE147990 | GEO | 2020/11/16
REPOSITORIES: GEO
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Project description:Microarrays of gene expression in mouse germinal center light zone and dark zone B cells sorted according to the expression of cell surface molecules CD83 and CXCR4 We used microarray data to identify genes differentially expressed by B cells in the light and dark zones of germinal centers from mouse skin-draining lymph nodes 12 days after subcutaneous immunization with NP-OVA in alum. Germinal center B cells (B220+CD38-CD95+) were sorted from immunized mouse lymph nodes into light zone (CXCR4hi/CD83lo) and dark zone (CXCR4lo/CD83hi) populations, from which RNA was extracted
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Project description:Microarrays of gene expression in mouse germinal center light zone and dark zone B cells sorted according to the expression of cell surface molecules CD83 and CXCR4 We used microarray data to identify genes differentially expressed by B cells in the light and dark zones of germinal centers from mouse skin-draining lymph nodes 12 days after subcutaneous immunization with NP-OVA in alum.
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Project description:We performed RNA-seq (MARS-seq) on germinal center (GC) light zone and dark zone B cells sorted to purity from freshly dissociated mouse popliteal lymph nodes immunized with NP-KLH for 7 to 14 days. Germinal center B cell subsets were gated as follows: Dump (CD8, CD4, F4/80, Gr-1)- B220+ CD38- GL-7+ FAS+. Dark zone GC cells were in addition CD86lo, CXCR4hi; LZ cells were in addition CD86hi, CXCR4lo. Three different mouse models were used: AID-cre Mettl3-flox, AID-cre Ythdf2-flox, Igf2bp3-knockout. We used these data to derive differentially expressed genes between B cell subsets of control and knockout genotypes. We also performed m6A-IP on ex vivo activated wild type B cells and determined the m6A targets in B cells. These studies revealed that the GC response depends on m6A RNA methylation and the functions of specific methyl-readers Ythdf2 and Igf2bp3.
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Project description:A range of cellular interactions and cytokines influence immunoglobulin class switch recombination (CSR), which occurs both in extrafollicular antibody responses and germinal centers. This study addresses where CSR occurs during extrafollicular responses, allowing identification of the cellular interactions and cytokine-producing cells that influence CSR in vivo. The response in mice of B cells specific for 4-hydroxy-3-nitrophenyl-acetyl (NP) to NP-Ficoll was analyzed. Immunohistology and flow cytometry confirm responding NP-binding cells move to the outer T zone of the spleen and start dividing there on the second day. During the third day they move either to follicles as germinal center founding cells, or extrafollicular foci as plasmablasts. Analysis of expression of AID, Bcl-6 and Blimp-1 mRNA in single NP-binding cells indicates a proportion of T zone B blasts express AID transiently without Bcl-6 and these cells are shown to undergo CSR. By contrast Blimp-1+ cells emerging on the third day and were universally AID- and hence unable to initiate CSR. Bcl-6 mRNA without protein, expressed by 40% of naïve NP-binding cells, is rapidly downregulated. Bcl-6 and AID are coexpressed by germinal centre founding cells only on moving to follicles during the third day after immunization. Keywords: TaqMan Low Density Array
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Project description:Previous studies have demonstrated that E-proteins induce AID expression in activated B cells. Here we have examined the role of Id3 in germinal center (GC) cells. We found that Id3 expression is high in follicular B-lineage cells but declines in GC cells. Immunized mice depleted for Id3 expression displayed a block in germinal center B cell maturation, showed reduced numbers of marginal zone B cells and class switched cells, were associated with decreased antibody titers and lower numbers of plasma cells. In vitro Id3-depleted B cells displayed a defect in class switch recombination. Whereas AID levels were not altered in Id3-depleted activated B cells, the expression of a subset of genes encoding for signaling components of antigen receptor, cytokine receptor and chemokine receptor mediated signaling was significantly impaired. We propose that during the GC reaction Id3 levels decline to activate the expression of genes encoding for signaling components that mediate B cell receptor and or cytokine-mediated signaling to promote the differentiation of GC B cells.
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Project description:Productive B cell responses are critical to protect a host from infection. The spleen and lymph nodes are populated by resting follicular B cells, which can enter germinal centers upon antigen encounter. Once in the germinal center, B cells migrate between the dark and light zones, where they undergo somatic hypermutation and selection, respectively. While germinal center B cells have been studied, an intense molecular understanding of these cells/subsets (and the differences between them) is lacking.
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Project description:RNAseq of germinal center B cells with different Blimp-1-GFP reporter level. Sorted germinal center populations were defined by their expression of Blimp-1-GFP reporter and the cell surface marker CD138 before they were further split into dark zone or light zone / dark zone like or light zone like cells based on their expression of the surface marker CXCR4 and CD83. We used RNAseq to find genes differentially regulated in various Blimp-1-GFP expressing germinal center populations from the spleen at day 10 after intraperitoneal immunisation with sheep red blood cells.
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