Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression in healthy and gene deficient human naÃ¯ve CD4+ T cells

ABSTRACT: Ascertain the effects of disease-causing gene mutations on the differentiation status of human naÃ¯ve CD4+ T cells in the setting of primary immunodeficiencies. Thus, do CD4+ T cells isolated according to a naÃ¯ve 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. NaÃ¯ve 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 naÃ¯ve and normal healthy memory T cells, as determined by LimmaGP analysis, were used to filter the combined and batch corrected dataset of naÃ¯ve 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).

ORGANISM(S): Homo sapiens

SUBMITTER: Stuart Tangye

PROVIDER: E-GEOD-81408 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:CD8+ and CD4+ T cells from HIV infected patients with HIV-RNA viremia of >50 copies/ml and CD8+ and CD4+ T cells from healthy controls were isolated by negative selection (Miltenyi Biotech, Auburn, CA). Cell sorting of the CD4 and CD8 T cell subsets were performed based on surface staining of CD3+CD8+ or CD3+CD4+ and na ve CD45RA+CD27+CD127highHLA-DRlow, and CD3+CD8+ or CD3+CD4+ and memory CD45RA-CD27+CD127highHLA-DRlow. Sorted cell populations were spun down and stored as dry pellets at -80M-BM-0C. Samples analyzed by transcript levels of genes related to cytokine signaling were determined by the JAK/STAT Signaling Pathway microarray. CD8+ and CD4+ T cells from HIV infected patients with HIV-RNA viremia of >50 copies/ml and CD8+ and CD4+ T cells from healthy controls were isolated by negative selection (Miltenyi Biotech, Auburn, CA). Cell sorting of the CD4 and CD8 T cell subsets were performed based on surface staining of CD3+CD8+ or CD3+CD4+ and naM-CM-/ve CD45RA+CD27+CD127highHLA-DRlow, and CD3+CD8+ or CD3+CD4+ and memory CD45RA-CD27+CD127highHLA-DRlow. Sorted cell populations were spun down and stored as dry pellets at -80M-BM-0C. Samples analyzed by transcript levels of genes related to cytokine signaling were determined by the JAK/STAT Signaling Pathway microarray (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-039A.html, SABiosciences Frederick, MD). Briefly, total RNA was harvested from each individual T cell subset from each patient or healthy control and contaminating DNA was digested with DNase. Messenger RNA was converted to cDNA and loaded onto PCR array plates for quantitative real-time PCR. Quantification of transcript levels was determined by normalizing to 5 housekeeping genes from each individual sample. Relative gene expression levels for each subset were averaged and compared between cell populations from the patient group and healthy controls. Because of the multiple comparisons only p values M-bM-^IM-$ 0.01 were considered significant.

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Gene expression in healthy and gene deficient human naÃ¯ve CD4+ T cells

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