Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human CD4 and CD8 T cells to identify effects of cell selection on gene expression

ABSTRACT: Negative immunomagnetic selection has become the method of choice for isolating T cell subsets for functional studies due to concerns that directly binding antibody to the surface of a cell, as occurs with positive selection, results in cross-linking of surface antigens, altered gene transcription and subsequent cellular activation. However there is little data to support this. We therefore examined the impact of the method of immunomagnetic cell selection on the gene expression profile of healthy human CD4 and CD8 T cells in a total of 21 cases. Experiment Overall Design: This study was composed of 4 groups of samples: CD4 Negative Selection (n=5), CD4 Positive Selection (n=5), CD8 Negative Selection (n=5), CD8 Positive Selection (n=6). Two samples (PS6 CD8 and PS9 CD4) were removed from the analysis due to discrepant normalised unscaled standard errors

ORGANISM(S): Homo sapiens

SUBMITTER: Rifca Le Dieu

PROVIDER: E-GEOD-14926 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background. T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non obese diabetic (NOD) mouse strain. Results. Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data we identify differentially expressed candidate genes including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12. Conclusions. The data provide a molecular map of the negative selection response in vivo, and by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death. Experiment Overall Design: Analysis of two thymic populations - early single positive thymocytes (CD4+CD8lowCD69+TCRhi) and early double positive thymocytes (CD4+CD8+CD69-TCRlow). Samples purified from the 3A9 TCR transgenic (TCR) to characterise positive selection and the 3A9 TCR x insHEL double transgenic (Dbl) to characterise negative selection towards an Aire-dependent organ-specific antigen. Both the non autoimmune C57BL10.H2k (B10) and autoimmune Non obese diabetic (NOD) strains were analysed in this method. Three biological replicates were used for each of these eight different groups.

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Transcription profiling of human CD4 and CD8 T cells to identify effects of cell selection on gene expression

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