. Peptides from each protein were pooled into their respective mega-pool and used for subsequent experiments.Freshly collected nasal cells were stimulated with peptide pools in an IFN-γ ELISpot assay.
Cs or nasal lymphocytes were seeded per well into ELISpot plates precoated with human IFN-γ antibody overnight at 4°C. Next, the cells were stimulated for 18 h with the peptide pools at 1 μg/ml. The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase and developed using the KPL BCIP/NBT phosphatase substrate .
Cs using EasySep Positive Selection Kits II . Flowthroughs consisting of CD3cells were collected. The cells were then pulsed with 5 μg/ml of peptides or DMSO for 1 h in 37°C. After incubation, pulsed cells were washed twice before addition of autologous nasal cells together with anti-CD40L-PE and anti-CD107a-APC . After 3 h of incubation at 37°C, the cells were washed in PBS stained with Zombie NIR Fixable Viability Kit to exclude dead cells in subsequent analysis.