Abstract
Alternative splicing of pre-mRNA is a mechanism that increases the protein diversity of a single gene by differential exon inclusion/exclusion during post-transcriptional processing. While alternative splicing is established to occur during lymphocyte activation, little is known about the role it plays during the immune response. Our study is among the first reports of a systematic genome-wide analysis of activated human T and B lymphocytes using whole exon DNA microarrays integrating alternative splicing and differential gene expression. Purified human CD2+ T or CD19+ B cells were activated using protocols to model the early events in post-transplant allograft immunity and sampled as a function of time during the process of immune activation. Here we show that 3 distinct classes of alternatively spliced and/or differentially expressed genes change in an ordered manner as a function of immune activation. We mapped our results to function-based canonical pathways and demonstrated that some are populated by only one class of genes, like integrin signaling, while other pathways, such as purine metabolism and T cell receptor signaling, are populated by all three classes of genes. Our studies augment the current view of T and B cell activation in immunity that has been based exclusively upon differential gene expression by providing evidence for a large number of molecular networks populated as a function of time and activation by alternatively spliced genes, many of which are constitutively expressed.
Highlights
The technology for investigating gene expression in cells and tissues has developed significantly over the last decade, making global gene expression profiling using microarrays relatively straightforward
T cells were activated using anti-CD3/CD28 beads and B cells were activated with anti-CD40 antibody cross-linked by anti-IgG plus rIL2 and rIL10
A higher fraction of stimulated CD4+ T cells produced IFNc, IL-2, and TNFa as determined by intracellular cytokine staining while no detectable IL-10 or IL-4 producing cells were detected in either control or stimulated CD4+ T cells
Summary
The technology for investigating gene expression in cells and tissues has developed significantly over the last decade, making global gene expression profiling using microarrays relatively straightforward. Until recently, the field has concentrated largely on studies of differential gene expression and discovering signatures that correlate with various biological challenges or disease states. The premise of analyzing differential gene expression is limited by the view that molecular mechanisms or biomarkers are represented by classes of genes either up- or down-regulated in a given situation. Alternative splicing (AS) is a process by which a single pre-mRNA transcript can give rise to multiple protein isoforms through the mechanism of coordinated intron removal and differential exon joining. The advent of highthroughput genomics has dramatically changed the view of alternative splicing from a single gene perspective to the level of genome-wide discovery and quantification
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