Lymphocyte Transformation Test:
Conventional LTT (C-LTT) . This was performed with PBMCs directly labelled with CFSE (Thermo Fisher Scientific, Waltham, USA). PBMCs were cultured in plates at 1.5x105 cells per well in complete RPMI medium (supplemented with 10% FBS, 2mM L-Glutamine, 50ng/mL Streptomycin and 5mg/mL Gentamycin (Normon, Madrid, Spain)) and with the culprit drug at different concentrations (Table S1) for 6 days at 37°C and <5% of CO2.34PBMCs without stimulus, only culture media and phytohemaglutinin (PHA) (Sigma, St. Louis, USA) at 20µg/mL were used as negative and positive control respectively.
Drug-primed-moDCs LTT (dDC-LTT) . Immature moDCs were transformed from monocytes (CD14+ cells) isolated from PBMCs by positive selection (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured for 5 days in complete RPMI medium supplemented with 100ng/mL of IL-4 and 200ng/mL of GM-CSF (both from R&D Systems Inc, Minneapolis, USA). These immature moDCs were cultured with the culprit drug for 3 days at different concentrations (Table S1). After this, 1.5x104 drug-primed-moDCs were co-cultured with 1.5x105 autologous CFSE-labelled-lymphocytes for 6 days at 37°C and 5% of CO2.