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Physiopathology
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User name	Lars Rogge
Email	lrogge@pasteur.fr
Phone / Fax number	+33 1 40 61 38 2 / +33 1 45 68 89 2
Location	Institut Pasteur (Department of Immunology) - 25, rue du Dr. Roux - 75724 Paris, France

Scientific description

Natural CD4+CD25+ regulatory T cells (Treg) act in a dominant way to actively suppress immune activation and are essential in the maintenance of peripheral tolerance to self-antigens. These cells were initially characterized by high-level expression of the alpha chain of the IL-2 receptor (CD25). More recently, the identification of Foxp3 as a transcription factor essential for the development and function of Treg has provided an important breakthrough for the analysis of this CD4+ T cell sub-population. Foxp3 mutations are responsible for the pathogenesis of IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), a fatal human X-linked disorder characterized by extensive multiorgan lymphocyte infiltration and abnormal activation of effector CD4+ T cells. At a very young age, IPEX patients present with massive lymphoproliferation, early-onset insulin-dependent diabetes mellitus, severe enteropathy, and additional autoimmune pathologies resulting from the absence of Treg.
Despite of the pivotal role of Treg in the control of autoimmunity and their potential medical applications, such as the induction of transplantation tolerance or reduction of tumor immunity, the molecular basis of the immunosuppressive activity of this cell population remains unknown. We have attempted to harness the unique features of human Treg by analyzing the transcriptome of highly-purified Treg and â€œconventionalâ€ CD4+CD25- T cells.

Technical description

Gene expression profiling of enriched CD4+CD25+HLADR+ and CD4+CD25- T cells was performed using Applied Biosystems 1700 microarray platform following protocols provided by the supplier. CD4+CD25+HLADR+ and CD4+CD25- T cells were stimulated for 24h with anti-CD3 (1XE, Sanquin, 1Î¼g/ml) and anti-CD28 (CD28.2, BD, 1Î¼g/ml) and total RNA was purified using Trizol followed by RNA cleanup using Ribopure columns (Ambion). 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. 10 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.