*3.3. Human Monocyte-Dendritic Cell Di*ff*erentation*

For each assay, human peripheral blood mononuclear cells were isolated from two healthy donors by standard Ficoll density gradient centrifugation. Monocytes were purified from human peripheral blood mononuclear cells using MACS CD14 microbeads (Miltenyi Biotech, Auburn, AL, USA) according to the manufacturer's recommendation. Purity was checked by staining with a FITC-conjugated anti-CD14 antibody (Milteny Biotech) and FACS analysis and was routinely found to be greater than 98%. Immature DCs were obtained by incubating monocytes at 1 × 10<sup>6</sup> cells mL−<sup>1</sup> in an RPMI 1640 medium supplemented with 10% fetal calf serum, 1% L-glutamine 2mM, 1% penicillin and streptomycin, human IL-4 (5 ng/mL), and human GM-CSF (100 ng/mL) for five days.

### *3.4. Cells Staining and Stimulation*

After five days in culture, surface staining was performed on monocyte-derived dendritic cells (moDCs) for flow cytometry analysis. moDCs (0.8 × 10<sup>6</sup> cell/well) were then incubated with synthetic compounds in 12 wells at the reported concentrations. Stimulation with PAM2CSK4 1 μg mL−<sup>1</sup> (Invivogen) was used as positive control. After 24 h, expression of all surface markers was estimated by using the following conjugated mAbs from Miltenyi Biotec: HLA-DR FITC, CD83 PE and CD86 APC, and analyzed by a flow cytometer (BD ACCURI, BD Bioscience, Milano, Italy) according to standard protocol.
