Germinal Center and Cultured B cell expression libraries

Comparison of gene expression profiles between in vitro mitogen-activated spleen B lymphocytes and FACS-sorted Germinal Center B lymphocytes isolated from intestinal PeyerPatches of C57BL/6 mice. In order to explore gene expression signatures that may be orchestrating somatic hypermutation (SHM, also known as affinity maturation) during antigen-induced B lymphocyte differentiation in Germinal Centers (GC), we profiled whole transcriptome gene expression of ex vivo, commensal microflora-activated GC B-cells that exhibit efficient SHM, with in vitro activated B-cell blasts, which fail to undergo SHM. In In particular, we examine our hypothesis that differential expression or the DNA endonucleases apex1 and apex2 regulate SHM. Using Templated Oligo Sequencing (TempO-Seq®) in conjunction with conventional intracellular FACS staining and B lymphocyte cell sorting, we compare gene expression profiles of in vitro mitogen-activated (LPS and anti-IgD-dextran), short-term (48h) cultured B cell blasts with Germinal Center B-cells isolated from intestinal Peyer Patches (PP, identified by surface staining B220+, CD95+, GL7+), compared to each other and to naive PP B-cells (B220+, CD95-, GL7-). We find that unique, distinct transcriptional signatures dominate the DNA repair processes in GC B-cells or in vitro B-cell blast, shedding light on genomic processes that orchestrate efficient affinity maturation of B-cells during GC differentiation. We use Templated Oligo Sequencing (TempO-Seq), a RNA motif-specific short tandem Oligo DNA hybridization technique to quantitatively measure whole transcriptome mRNA in conjunction with conventional surface membrane, fixable live/dead discriminator dye, and intracytoplasmic fluorescence antibody staining followed by FACS to isolate B cell populations of interest.