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.