Transcriptome analysis of primary human CD4+ T-cell activation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Zoltan Maliga
PROVIDER: E-GEOD-33272 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Ascertain the effects of disease-causing gene mutations on the differentiation status of human naïve CD4+ T cells in the setting of primary immunodeficiencies. Thus, do CD4+ T cells isolated according to a naïve surface phenotype (ie CD4+CD45RA+CCR7+) from healthy donors exhibit a similar gene expression profile as phenotpyically-matched cells isolated from individuals with defined primary immunodeficiencies caused by specific monogenic mutations. Naïve CD4+ T cells were isolated from PBMCs of healthy controls or from different PID patients by sort-purifying CD4+CD45RA+CD127+CD25lo cells. Memory CD4+ T cells from healthy controls were isolated as CD4+CD45RA- cells. RNA was extracted, transcribed to cDNA and then gene expression analysis determined using Affymetrix Human Gene 2.0 or Human Gene 2.1 ST microarray chips. Normalisation (robust multichip average), log2 transformation and probe set summarisation were performed for each dataset using Bioconductor packages implemented in the R statistical computing environment, version 3.1.1. Subsequent processing and analyses were carried out using GenePattern modules (available at http://pwbc.garvan.unsw.edu.au/gp/). The datasets were merged, quantile normalised, and batch corrected using the MergeColumns, NormaliseColumns and ComBat modules. Differential gene expression analysis was assessed using LimmaGP. The top 100 differentially expressed genes between normal healthy naïve and normal healthy memory T cells, as determined by LimmaGP analysis, were used to filter the combined and batch corrected dataset of naïve CD4+ T cells from 7 healthy controls and 19 PID patients, and memory CD4+ T cells from 3 healthy controls. Unsupervised clustering was performed using NMFConsensus and NMF (Brunet et al., 2004).
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