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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

Molecular profiling of CD4+ T cell populations during interleukin-2 therapy of HIV-infected individuals: a longitudinal nested study of the ANRS 118 trial

The goal of this project was to determine the long-term effects of intermittent IL-2 therapy on CD4+ T cells in HIV patients and to characterize at the molecular level a novel subset of CD4+ T cells expressing high-levels of the IL-2 receptor alpha chain (CD25) that appears in the peripheral blood of HIV patients treated with HAART + IL-2. This subset can account for up to 70% of the total CD4+ T cell pool of treated patients and persists for a long time. The phenotype of the IL-2-induced CD4+CD25+ T cell subset in HIV patients is similar to a regulatory T cell subset (â€œnaturalâ€ CD4+CD25+ T cells, Treg) from healthy individuals, which plays a critical role in the control of autoimmunity. However, it is at present not known whether IL-2 therapy affects the generation and/or function of human Treg. 
To assess the molecular characteristics of IL-2-expanded CD4+CD25+ T cells, we performed a longitudinal analysis of the gene expression profiles of enriched CD4+CD25- and CD4+CD25+ T cells from 10 HIV-infected patients before the first (week 0) and eight weeks after the third IL-2 cycle (week 24) using microarrays.

Technical description

Gene expression profiling of enriched CD4+CD25+ and CD4+CD25- T cells was performed using Applied Biosystems 1700 microarray platform following protocols provided by the supplier. CD4+CD25+ 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 RNeasy columns. 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.