Mountain View
biomedical and healthCAre Data Discovery Index Ecosystem
help Advanced Search
Title: Genome Wide Analysis of Immune Activation in Human T and B Cells Reveals Distinct Classes of Alternatively Spliced Genes      
keywords:
Transcriptome or Gene expression
ID:
PRJNA111381
description:
Alternative splicing of pre-mRNA is a mechanism that increases the protein diversity of a single gene by differential exon inclusion 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 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. Keywords: T cells, B cells, alternative splicing, exon Overall design: CD2+ T-lymphocytes and CD19+ B-lymphocytes were purified from peripheral blood mononuclear cells (PBMC) of 10 normal human donors. Freshly isolated, resting CD2+ T-cells were resuspended in RPMI-1640 complete media and activated with CD3/CD28 Dynal beads. Cultures were sampled at 24, 48 and 72hrs. A subset of isolated, resting cells immediately stabilized by RNALater solution were used as the baseline comparison (Time 0). Freshly isolated CD19+ magnetic-bead purified resting B cells were resuspended in RPMI-1640 complete media to which anti-CD40 antibody was added, followed by cross-linking with goat-anti-mouse anti-IgG1. The cells were then cultured with rIL2 and rIL10 (100 ng/ml each) to simulate the T-cell dependent B cell activation of an alloimmune response. RNA was extracted at time 0, 24, 48 and 72 hours post activation using the mirVana miRNA Isolation Kit. The GeneChip® The RNA was converted into labeled cDNA using the GeneChip® WT Sense Target Labeling kit (Affymetrix). Labeled cDNA was hybridized to Affymetrix Human Exon 1.0 ST arrays. Data for mRNA transcript profiles were generated in the form of CEL files using standard protocols (http://www.affymetrix.com/).
accesstypes:
download
landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA111381
authentication:
none
authorization:
none
name:
Homo sapiens
ncbiID:
ncbitax:9606
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject