Foxp3, a transcription factor of the forkhead/winged helix family, plays a unique role in the transcriptional control of regulatory CD4+CD25+ T (Treg) cells. The identification of potential target genes associated with the regulatory phenotype and the characterization of combinatorial interactions of transcription factors (TF) in promoter regions in genes regulated by Foxp3 has become important to understand the transcriptional control of Treg cells.

Transcription profiling of human regulatory T cells to identify genes under Foxp3 control

Foxp3-dependent transcriptional control of human regulatory T cells

Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

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Foxp3, a transcription factor of the forkhead/winged helix family, plays a unique role in the transcriptional control of regulatory CD4+CD25+ T (Treg) cells. The identification of potential target genes associated with the regulatory phenotype and the characterization of combinatorial interactions of transcription factors (TF) in promoter regions in genes regulated by Foxp3 has become important to understand the transcriptional control of Treg cells.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v3

Title: Affymetrix EukGE-WS2v3 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-4610

Culture medium was IMDM, supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, and non essential amino acids (PAA Laboratories, Linz, Austria).

P-MEXP-4611

CD4+ T cells were prepared from human peripheral blood of normal healthy donors by centrifugation over Ficoll-Hypaque (Biochrom AG, Berlin, Germany) gradients followed by MACS isolation using the CD4+ no-touch T cell isolation kit and AutoMACS technology (Miltenyi Biotech, Bergisch Gladbach, Germany). CD4+ T cells were labelled with FITC-conjugated anti-CD4 (RPA-T4, BD Bioscience, San Jose, CA) and PE-conjugated anti-CD25 (M-A251; BD) and sorted with a MoFlo cell sorter (DakoCytomation, Fort Collins, CO). The purity of each cell population was always higher than 98%. CD4+CD25+ T cells were stimulated once with plate-bound anti-CD3 (TR66, 1 µg/ml), soluble anti-CD28 (CD28.2, BD), and 100 U/ml recombinant human IL2 (Proleukin, kindly provided by P. Wagner, Chiron Corporation, Emeryville, CA). Thereafter, cells were stimulated weekly with irradiated allogeneic EBV-transformed B cells (LG2-EBV, kindly provided by T. Boon, LICR, Brussels) and IL2. CD4+CD25- T cells were stimulated immediately after cell sorting with irradiated allogeneic LG2-EBV cells.

P-MEXP-4892

For biotin-labeled target synthesis starting from 3 µg of total RNA, reactions were performed using standard protocols supplied by the manufacturer (Affymetrix; Santa Clara, CA). Briefly, 5 µg total RNA were converted to dsDNA using 100 pmol of a T7T23V primer (Eurogentec; Seraing, Belgium) containing a T7 promotor. The cDNA was then used directly in an in-vitro transcription reaction in the presence of biotinylated nucleotides (ENZO BioArray HighYield RNA Transcript Labeling Kit). 

P-MEXP-4894

For normalization all array experiments were scaled to a target intensity of 150 (for scaling 4% capped median is used), otherwise using the default values of the Microarray suite (MAS5). Quantitation types are described elsewere (Affymetrix Technical Note: “New Statistical Algorithms for Monitoring Gene Expression on GeneChip® Probe Arrays.” (2001))

P-MEXP-6625

RNA was isolated using RNAeasy (Qiagen; Hilden, Germany) according to the standard protocol provided by the manufacturer.

Transcription profiling of human regulatory T cells to identify genes under Foxp3 control

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