2.7. Hemagglutinin-Specific T Cells by IFN-γ ELISpot

Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by gradient technique using Lymphoprep™ (Axis-Shield, Dundee, UK) within 8 h after venipuncture. After 18 to 72 h at −80 °C, samples were transferred to liquid nitrogen for long-term storage. ELIspot assays were performed using a human IFN-gamma ELISpot Development Module from R&D System (SEL285) following the manufacturer’s recommendations. After thawing, PBMC cell suspensions (150,000 cells/well) were placed in individual wells of PVDF-bottom plates (Millipore, MSIPS4510, Darmstadt, Germany) pre-coated with the human IFN-γ capture antibody. Cells were stimulated with an array of 139 peptides spanning the HA protein of the A/California/04/2009 (H1N1) pdm09 strain of influenza virus (GenPept: {"type":"entrez-protein","attrs":{"text":"ACQ76318","term_id":"229535949"}}ACQ76318) obtained from BEI Resources (NR-15433) and arranged into 7 pools, each containing 19 or 20 peptides, and used at a final concentration of 2 µg/peptide/mL. Following an 18-hour stimulation, plates were incubated with the biotinylated human IFN-γ detection antibody. Signal detection was performed using the ELISpot Blue Color Module from R&D system (SEL 002) following manufacturer’s recommendations. Developed plates were counted with a CTL ImmunoSpot^® S5 Reader and spot-forming units (SFU) were measured in each well automatically by the CTL ImmunoSpot^® software. After background subtraction, the sum of SFU for the seven HA peptide pools was calculated and expressed as SFU per million PBMC.