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Physiopathology
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User name	Christophe Becavin
Email	becavin@ihes.fr
Phone / Fax number	(+033)0160926683 / (+033)0160926609
Location	Institut des Hautes Etudes Scientifiques () - Le Bois-Marie 35, route de Chartres - 91440 Bures-sur-Yvette, France

Scientific description

CD4+CD25+ regulatory T cells (Treg) play a critical role to maintain self-tolerance and the control of autoimmunity. These cells are characterized by expression of the transcription factor FOXP3 and possess potent immunosuppressive activity. Recent results by our lab and by others have shown that several subpopulations of Treg exist in humans. However, phenotypic, functional and molecular properties of these Treg subpopulations have remained poorly defined. The goal of the project was to investigate the heterogeneity of the human Treg cell compartment in order to better understand the role of these cells in controlling immune responses in health and disease. We have described three different subsets of Treg cells in adult peripheral blood:
CD25hi FOXP3hi CD45RA- HLADR+ (aTreg),
CD25hi FOXP3intm CD45RA- HLADR- (cTreg)
and CD25hi FOXP3intm CD45RA+ HLADR- (nTreg).
To assess the molecular characteristics of these three different subsets and to reliably define them, we have established their gene expression profiles by microarrays analyses.

Technical description

Gene expression profiling of sorted CD4+CD25-CD45RA+ (Tconv) and CD4+CD25+CD127-CD45RA+HLADR- (nTreg),+HLADR-, CD4+CD25+CD127-CD45RA-HLADR- (cTreg) CD4+CD25+CD127-CD45RA-HLADR+ (aTreg) human T cells was performed using Applied Biosystems 1700 microarray platform following protocols provided by the supplier. Cells were stimulated for 24h with anti-CD3/CD28 beads (Invitrogen) and total RNA was extracted from cells using the Ribopure kit (Ambion) following the manufacturerâ€™ s instructions. RNA (0.5 Î¼g) was converted into double-stranded cDNA using the nanoAmp RT-IVT kit (Applied Biosystems). To minimize the bias encountered with PCR amplified RNA, one single round of linear amplification was performed from total RNA according to the AB protocol. 15 Î¼g of Digoxigenin-UTP labeled cRNA was fragmented and hybridized to microarrays for 16h at 55 Â°C. Washing, staining, and scanning were performed using a protocol provided by Applied Biosystems.

Reference

Pubmed : http://www.ncbi.nlm.nih.gov/pubmed/23818545