Regulation of bifurcating B cell trajectories by mutual antagonism between IRF4 and IRF8
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Harinder Singh
PROVIDER: E-GEOD-70712 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Upon antigen recognition B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GC). We uncover a double negative feedback loop between interferon regulatory factors IRF4 and IRF8, which regulates the initial bifurcation of activated B cells as well as the GC response. IRF8 dampens BCR signaling, facilitates antigen specific interaction with helper T cells, and promotes selection of high affinity clones while antagonizing IRF4 driven plasmablast differentiation. Genomic analysis reveals concentration dependent action of IRF4 and IRF8 in regulating distinctive gene expression programs. Stochastic modeling suggests that the double negative feedback is sufficient to initiate bifurcating B cell developmental trajectories. Naïve B cells were isolated from Irf8+/+ (wild type, WT) mice spleen and activated in vitro with 10μg/ml LPS (Sigma). CD138hi IgMhi (IRF4hi*) cells and CD138lo IgMlo (IRF8hi*) cells were sorted by flow cytometry at 72 hours. Total RNA was prepared by using Rneasy Mini kit (Qiagen) and sequenced with Illumina HiSeq2500. Alignment was performed with Taphat2, and transcript abundance quantification using Cuffdiff function from Cufflinks. GC B cells were sorted from CD19cre/+ Irf8+/+ (Ctrl) or CD19cre/+ Irf8flox/flox (Irf8 cKO) mice on dpi 13 post NP-KLH immunization. Total RNA was prepared by using Rneasy Mini kit (Qiagen) and amplified with with Ovation RNA-Seq System v2 (NuGEN). The data were analyzed by Wardrobe. More details are provided in the manuscript.
2015-08-25 | E-GEOD-70711 | biostudies-arrayexpress
Project description:Upon antigen recognition B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GC). We uncover a double negative feedback loop between interferon regulatory factors IRF4 and IRF8, which regulates the initial bifurcation of activated B cells as well as the GC response. IRF8 dampens BCR signaling, facilitates antigen specific interaction with helper T cells, and promotes selection of high affinity clones while antagonizing IRF4 driven plasmablast differentiation. Genomic analysis reveals concentration dependent action of IRF4 and IRF8 in regulating distinctive gene expression programs. Stochastic modeling suggests that the double negative feedback is sufficient to initiate bifurcating B cell developmental trajectories. Naïve B cells were isolated from wild type (WT) mice spleen and activated in vitro with 10μg/ml LPS (Sigma). ChIP was performed by using anti-IRF4, -IRF8 antibodies (Santa Cruz Biotech). For massively parallel sequencing, 10-20 μg of chromatin fragments from indicated samples were immunoprecipitated by using anti-IRF-4 and anti-IRF8 antibodies, and DNA libraries were prepared with Illumina Kit. DNA was sequenced by using the Illumina HiSeq2500. Reads were aligned to the mouse genome (mm9) by using Taphat2 and peak calling were performed by homer 2. GC B cells were sorted from WT mice on dpi 13 post NP-KLH immunization. Cells were flash frozen immediately and process by Active Motif for IRF8 ChIP-Seq. Reads were aligned to mm9 by using BAM and peak calling were performed by using MACS. More details are provided in the manuscript.
2015-08-25 | E-GEOD-70710 | biostudies-arrayexpress
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2013-06-07 | E-GEOD-47703 | biostudies-arrayexpress
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Project description:In immune responses, activated T cells migrate to B cell follicles and develop to T follicular helper (Tfh) cells, a new subset of CD4+ T cells specialized in providing help to B lymphocytes in the induction of germinal centers 1-3. Although Bcl6 has been shown to be essential in Tfh cell function, it may not regulate the initial migration of T cells 4 or the induction of Tfh program as exampled by CXCR5 upregulation 5. Here, we show that the Achaete-Scute homologue 2 (Ascl2) gene that encodes a basic helix-loop-helix (bHLH) transcription factor 6, is selectively upregulated in its expression in Tfh cells. Ectopic expression of Ascl2 uniquely upregulates CXCR5 but not Bcl6 and downregulates CCR7 expression in T cells in vitro and accelerates T cell migration to the follicles and Tfh cell development in vivo. Combined transcriptome profiling and genome-wide occupancy analysis indicate that Ascl2 directly regulates Tfh-related genes while inhibits expression of Th1 and Th17 genes. Acute deletion of Ascl2 as well as blockade of its function with the Id3 protein in peripheral CD4+ T cells results in a failure in Tfh cell development and the germinal center response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances Tfh cell generation. Thus, Ascl2 critically and directly initiates Tfh cell development. Decide Ascl2 binding sites in CD4+ T cells
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Project description:The advances in chemical proteomics have significantly expanded our understanding of the diversity and abundance of fatty-acylated proteins in eukaryotes, and reveal novel functions for these lipid protein modifications. Nonetheless, quantitative comparative proteomic analysis of fatty-acylated proteins in different cellular states is still challenging. To address these limitations, we systematically evaluated different proteomic methods (alk-16 chemical reporter and acyl-RAC) and established robust conditions to selectively and quantitatively profile fatty-acylated proteins in mammalian cells. Using a combination of metabolic labeling with fatty acid chemical reporters, selective chemical enrichment and label-free proteomics, we performed a quantitative analysis of fatty-acylated proteins in naïve and activated macrophages. These studies revealed novel fatty-acylated proteins associated with host immunity that are differently expressed and lipid-modified in different cellular states.
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Project description:B cells diversify and affinity-mature their antigen receptor repertoire in germinal centers (GC). GC B cells receive help signals during transient interaction with T cells, yet it remains unknown how these transient T-B interactions sustain the subsequent proliferative program of selected B cells. Here we show that the transcription factor AP4 is required for sustained GC B cell proliferation and subsequent establishment of a diverse and protective antibody repertoire. AP4 is induced by c-MYC during the T-B interactions and maintained by T cell-derived IL-21. B cell-specific deletion of AP4 resulted in reduced GC sizes and somatic hypermutation and a failure to control chronic viral infection. These results indicate that AP4 integrates T cell-mediated selection and sustained expansion of GC B cells for humoral immunity. Splenic B cells activated with anti-IgM and CD40L in vitro and germinal center B cells sorted from C57BL/6 mice eight days after immunization with sheep red blood cells (SRBCs) were obtained and genome-wide occupancy of c-MYC, AP4 and active histone marks was profiled by chromatin immunoprecipitation and high-throughput sequencing. Gene expression in MYCâAP4â LZ, MYC+AP4+ LZ, AP4+ DZ, and APâ DZ GC B cells from SRBC-immunized AP4-mCherry / c-MYC-GFP dual reporter mice was profiled by RNA-seq.
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