Human serum

Dendritic cells were generated from peripheral blood monocytes as described before. At day 5 iDC were stimulated with LPS (10 ng/ml; Sigma-Aldrich) and pulsed with tetanus toxoid (10 μg/ml; Sigma-Aldrich). On day 6, when DC were harvested, fresh CD3⁺ lymphocytes were obtained from buffy coats by density gradient centrifugation and separation via AutoMACS device (Miltenyi Biotech) using CD3⁺ microbeads according to the manufacturer’s instructions. Purified CD3⁺ T cells were suspended in 0.5 µM CFSE (Invitrogen) diluted in RPMI 1640, and incubated at 37°C in the dark for 10 min. An equal volume of RPMI 1640 containing 10% human serum (type AB, PAA Laboratories GmbH) was added to inactivate the extracellular CFSE. The cells were washed three times in PBS and then resuspended in the proliferation medium consisting of RPMI 1640 medium enriched with 10% human serum, IL-2 (10 U/ml; Immunotools), IL-4 (145 U/ml; Miltenyi Biotech), and GM-CSF (140 U/ml). Mature dendritic cells (mDC) were harvested at day 6, washed with PBS, and resuspended in proliferation medium as well. CFSE CD3⁺ T cells were mixed at a ratio of 10:1 (10⁵ T cells to 10⁴ dendritic cells per well) in a volume of 1 ml. CFSE-labeled T cells alone were used as a negative control. After day 4, cells were harvested, and CFSE fluorescence was analyzed via flow cytometry.