Project description:Transcriptomic profiling and unsupervised clustering of cMyc+ GC-B cells identify the four subpopulations representing positive-selection stages.
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: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
Project description:Despite the importance of memory B cells for protection from recurrent infection, how these cells are selected during germinal center (GC) reactions remains unclear. We show here that light zone (LZ) GC B cells with lower affinity BCRs express a less CD40 signature and relatively high levels of Bach2, being prone to enter the memory B cell pool. We also find that Bach2 contributes to memory B cell generation in a Blimp-1 independent manner and that its higher expression confers on LZ GC cells a more advantage for entering the memory B cell compartment. Thus, our data support an instructive model in which weak T cell help keeps Bach2 expression relatively high, thereby being predisposed to enter the memory pool.
Project description:To determine functional overlap between cMyc and AP4 in CD8+ T cell priming, we retrovirally expressed cMyc or AP4 in cMyc-deficient CD8+ T cells and examined gene expression after activation.
Project description:Despite the importance of memory B cells for protection from recurrent infection, how these cells are selected during germinal center (GC) reactions remains unclear. We show here that light zone (LZ) GC B cells with lower affinity BCRs express a less CD40 signature and relatively high levels of Bach2, being prone to enter the memory B cell pool. We also find that Bach2 contributes to memory B cell generation in a Blimp-1-independent manner and that its higher expression confers on LZ GC cells a more advantage for entering the memory B cell compartment. Thus, our data support an instructive model in which weak T cell help keep Bach2 expression relatively high, thereby being predisposed to enter the memory pool.
