Liposaccharide

DC generation was carried out as previously described [19 (link),20 (link)]. Briefly, monocytes were isolated from fresh peripheral blood monononuclear cells from healthy donors using CD14 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) following manufacturer instructions and cultured at 1 × 10⁶ cells/mL in DC Medium (CellGenix, Freiburg, Germany) in the presence of 2 mM glutamine (ThermoFisher Scientific, Waltham, USA), 2% human serum (Bioconcept, Allschwil, Switzerland), 250 IU/mL IL-4 (Miltenyi Biotech, Bergisch Gladbach, Germany), and 500 IU/mL GM-CSF (Miltenyi Biotech, Bergisch Gladbach, Germany). After 4 days, cells were incubated at 37 °C with WTL at a cell ratio of 1:1 or DC Medium (unloaded control) for 24 h, followed by cell maturation with 60EU/mL liposaccharide (Sigma-Aldrich, Buchs, Switzerland) and 2000 IU/mL IFN- (Miltenyi Biotech, Bergisch Gladbach, Germany) for 16 h.

Blood samples were collected, incubated at room temperature and centrifuged at 1,000 × g. Splenic CD11c⁺ DCs were stimulated overnight with 100 ng/ml liposaccharide (Sigma-Aldrich, München, Germany). Cytokines were quantified in the supernatants from stimulated DCs or CD4⁺ T cells, and in sera by using a Luminex Screening assay (R&D Systems, Wiesbaden, Germany) and a Luminex 200 system with Luminex IS software (Luminex Corporation, MV’s-Hertogenbosch, Netherlands) according to the manufacturer’s instructions. The detection limits were 1.32 pg/ml for IL-10, 0.73 pg/ml for IFN-γ, 0.13 pg/ml for TNF-α, and 1.31 pg/ml for IL-12, respectively.