Assessment of Antigen-Specific T Cell Response

The antigen-specific T cell response was assessed as described [18 (link)]. Blood was collected from the caudal blood vessels into commercially available 10-mL evacuated tubes coated with lithium-heparin as anticoagulant (Venosafe™, Terumo® Europe). Samples were used within 4 h after collection. Stimulations were performed in triplicate by mixing 100 μL of blood with either 100 μL of culture medium (RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, penicillin-streptomycin, fungizone and 0.05 mM 2-mercaptoethanol) as negative control, 100 μL of pokeweed mitogen (2 μg/mL) as positive control, or 100 μL of ovalbumin or HEL (50 μg/mL) in 96-well microplates (Falcon Microtest™, Becton Dickinson). The culture was incubated at 37°C in a humidified atmosphere with 5% CO₂ for 3 days. Supernatant were then harvested and stored at -20°C in 96-well plastic storage plates (Greiner™ bio-one) until assayed for cytokine content.