Aeb071

All drugs used in this study (AEB071, CGM097, RAD001, MEK162, LEE011) were obtained from Novartis Institutes for Biomedical Research (NIBR, Cambridge, USA). AEB071, MEK162, RAD001, CGM097 and LEE011 are selective inhibitors of PKC, MEK1/2, mTORC1, MDM2 and CDK4/6 respectively. For in vivo administration, compounds were diluted in 20% propylene glycol + 50% solutol + 30% PBS. The control groups were treated with this solution (vehicle). Each compound was administrated 5 days a week at the dose indicated in each figure legend (see Supplementary Materials for further details).
For in vitro experiments, compound powders were dissolved in DMSO for a stock concentration of 10mM final, aliquoted and stored at −20°C. Further dilutions were made according to each experiment design.

Different subsets of Tregs were treated or not, washed three times, and added at ratio 1:3 (1.25 × 10⁵ Tregs: 5 × 10⁵ CD4⁺CD25⁻ T cells) to CD4⁺CD25⁻ T cells at final concentration 2 × 10⁶/ml. The cells were co-cultured on anti-CD3 mAb (5 μg/ml) pre-coated 24-well plates for 48 hr. The PKC-θ inhibitor, AEB071, was provided by Novartis (Ridgefield, CT) and dissolved in DMSO. T cells were pretreated for 30 min with 10 μM at 37 °C and washed three times. Cytokine secretion was determined by ELISA as previously described^{20 (link)}, using Human IL-17 (R&D Systems Inc, Minneapolis, MN) and IFN-γ Cytoset^tm (Biosource; Camarillo, CA). The capacity of expanded Tregs to suppress target cell proliferation in vitro was assessed by using 5-carboxyfluorescein-diacetate-succinimide ester (CFSE) inhibition assay^{31 (link)}. Peripheral blood mononuclear cells (PBMCs) were purified, labeled with CFSE (InVitrogen), and stimulated with anti-CD3 mAb-coated beads (Dynal) in 96-well round-bottom plates with or without expanded cells (Treg:PBMC at ratios of 1:4-1:64). Assays were harvested on day 4. Cells were stained with anti-CD4 and/or −8 mAb and data acquired by LSRII. Data was analyzed using the proliferation platform in FlowJo (Treestar, Ashland, OR), and suppression determined from the Division Index.