Project description:Transcriptomic profiling and unsupervised clustering of cMyc+ GC-B cells identify the four subpopulations representing positive-selection stages.
Project description:To investigate the impact caused by miR-155 ablation, positively selected light zone (LZ) GC B cells were sorted by flow cytometry and used to analyse transcriptomic profiles.
Project description:The pathways regulating the formation of the germinal center (GC) dark- (DZ) and light- (LZ) zones are unknown. We show that FOXO1 expression is restricted to the GC DZ and is required for DZ formation, since its absence in mice leads to the complete loss of DZ gene programs and the formation of LZ-only GCs. FOXO1-negative GC B-cells display normal somatic hypermutation, but defective affinity maturation and class switch recombination. The function of FOXO1 in sustaining the DZ program involves the transactivation of the chemokine receptor CXCR4, and the cooperation with BCL6 in the trans-repression of genes involved in immune activation, DNA-repair and plasma cell differentiation. These results have also implications for understanding the role of FOXO1 mutations in lymphomagenesis. We used microarrays to determine the consequences of FOXO1 deletion in the GC B cell comparment, and correlate these data with phenotypic changes GC B cell subpopulations were collected by Fluorescence Activated Cell Sorting (FACS) from B cell enriched fractions of splenic mononuclear cell pools (12 days after SRBC immunization). 20ng of total RNA (RIN>9) for each sample was used as a template for linear cDNA amplification (Ovation RNA amplification Kit, NuGen). cDNA was labeled using the Encore Biotin Labeling Kit (NuGen) and hybridized to Affymetrix Mouse 430.2 gene expression arrays
Project description:DZ (B220+ IgDlo GL-7+ CD95+CXCR4+CD86lo) or LZ (B220+ IgDlo GL-7+ CD95+CXCR4loCD86+) GC B cells were sorted from male Sle1.yaa lupus mice at 2,4,6 months of age. RNA-seq was employed to assess transcriptional changes in the different GC B cell subsets throughout course of disease.
Project description:To investigate the impact caused by miR-155 ablation, GC B cells were sorted by flow cytometry and used to analyze transcriptomic profiles.
