Spleen cells from 1 to 2 individual mice were pooled, and seeded in triplicates in 96-well plates at 2 × 105 cells/well in RPMI 1640 medium (200 μl volume) containing heat-inactivated fetal calf serum (10%), penicillin/streptomycin, glutamine, and HEPES (25 mM), at 37 °C and 5% CO2 (all from Sigma-Aldrich). Cells were stimulated for 6 days with 15-mer peptides (10 μM; resulting in DMSO concentration 0.1%), a combination of anti-CD3 and anti-CD28 antibodies (positive control, 3 μg and 2 μg/ml; clones 145–2C11 and 37.51; eBioscience, San Diego, CA), endotoxin-free ovalbumin (negative control), recombinant influenza A hemagglutinin, neuraminidase or nucleoprotein (all 10 μg/ml). For each stimulation, two plates were run in parallel (from two different spleen cell pools).

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