Here, we have evaluated the effects of simvastatin blockade of the mevalonate pathway on the induction of Foxp3-expressing iTregs in vitro. We demonstrate BI 6727 that simvastatin itself can mediate induction of Foxp3+ T cells and can also synergize with low levels of TGF-β in the induction of functional Foxp3+ Tregs. The effects of simvastatin are secondary to a blockade of protein

geranylgeranylation, are mediated 24 hr after TCR stimulation, and are associated with TCR-specific DNA demethylation of the Foxp3 promoter and TCR-specific induction of Smad6 and Smad7 proteins. The implications of these results for the use of simvastatin as an immunosuppressive drug will be discussed.

DO11.10 TCR transgenic RAG2 deficient (−/−), 5CC7 TCR transgenic RAG2−/−, and B10.A mice were obtained from Taconic Farms (Germantown, NY). The Foxp3-GFP-Knock-in (Foxp3gfp) mice were provided by Dr V. Kuchroo (Harvard Medical School, Boston, MA). All the mice were maintained under pathogen-free conditions in the National Institute of Allergy and Infectious Disease animal facility. Mice were used between 4 and 8 weeks of age. Recombinant human IL-2 and recombinant mouse TGF-β were purchased from Peprotech (Rocky Hill, NJ). Simvastatin, geranylgeranyl pyrophosphate and farnesyl pyrophosphate were purchased from AZD1152HQPA Alexis Biochemicals (Plymouth Meeting, PA) and mevalonate, FTI-276 (farnesyl transferase inhibitor), and GGTI-2133 (geranylgeranyltransferase I inhibitor) were purchased from Sigma (St Louis, MO). Allophycocyanin-conjugated anti-Foxp3 (FJK-16s), fluorescein isothiocyanate-conjugated

anti-CD4 (L3T4), anti-CD3ε antibody (145-2C11) and anti-CD28 antibody were purchased from eBioscience, Inc. (San Diego, CA). Anti-phospho-Smad3 antibody and anti-Smad3 antibody were purchased from Cell Signaling Technology (Danvers, MA). Anti-Smad6/7 (N-19) antibody was purchased from Santacruz Biotechnology (Santa Cruz, CA). For neutralization of TGF-β, anti-TGF antibody (1D11) was obtained from R&D Systems (Minneapolis, MN). CD4+ T cells were purified from mouse lymph nodes or spleen using magnetic beads (Miltenyi Biotec, Auburn, CA). Foxp3gfp CD4 T cells were isolated by fluorescence-activated Calpain cell sorting (FACSAria). Foxp3+ Tregs were induced by stimulating CD4+ Foxp3− T cells (1 × 106) with plate-bound anti-CD3 (1–2 μg, 145-2C11) and plate-bound anti-CD28 antibody (1–2 μg) in the presence of a given concentration of TGF-β1 and/or simvastatin for 72 hr in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml), l-glutamine (2 mm), HEPES (10 mm), non-essential amino acids (0.1 mm), sodium pyruvate (1 mm) and 2-mercaptoethanol (50 μm).