Frozen PBMCs were thawed and allowed to recover in RPMI 1640 culture medium (Life Technologies, Paisley, United Kingdom) containing 2 mmol L-glutamine, 25 mmol/l HEPES, 25 μg/ml gentamycin (Sigma-Aldrich) and 10% of heat-inactivated AB serum (Innovative Research, Novi, MI, USA), for 1 h in an incubator at 37 °C and 5% CO2. Cells were seeded in duplicates or triplicates in 96-well round-bottom plates (Nunc, Roskilde, Denmark) at 2 × 105 cells/well, using RPMI 1640 culture medium supplemented with 5% of heat-inactivated AB serum, 2 mmol L-glutamine, 25 mmol/lHEPES and 25 μg/ml gentamycin (200 µl/well), and stimulated for 6 days with 15-mer peptides (10 μM; resulting DMSO concentration 0.1%), tetanus toxoid (20 μg/ml), PBS (negative control), recombinant influenza A hemagglutinin, neuraminidase or nucleoprotein (all at 10 μg/ml). Generally, samples were stimulated with different T cell peptides selected at random. Peptide testing was prioritized, and proteins were only tested if enough cells were available. For epitope mapping, peptides were not selected at random. Instead, more samples were used for testing the inner three than the outer two peptide epitopes. No patient or control samples that had passed viability control (trypan blue staining, positive response to stimulation with tetanus control) were excluded from the study.

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