Cell-mediated response was assessed by detecting intracellular interferon-gamma (IFN-γ) production by effector T cells, after antigen-specifi c stimulation in vitro to generate effector cells from memory cells (18) . Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Hypaque, Pharmacia Biotech, Uppsala, Sweden) and frozen at -150°C. Thawed PBMC in culture medium (RPMI 1640, 10% FCS, 2 mmol/L L-glutamine) were stimulated with the infl uenza vaccine preparation (1.5 μg/mL), UV-inactivated infl uenza (H5N1) (MOI 0.1), or synthetic infl uenza (H5N1) peptides (NA and HA) (1 μg peptide/mL) for 3 days and expanded for 6 additional days in the presence of recombinant interleukin-2 (IL-2) (5 IU/mL, Boehringer-Mannheim, Mannheim, Germany). On day 9, cells were restimulated with the same antigens in the presence of 1 μg/mL αCD28 and αCD49d (immunoglobulin G1 [IgG 1 ], K clones CD28.2 and 9f10, respectively; Becton Dickinson, Mountain View, CA, USA) and of Brefeldin-A (10 μg/mL, Sigma, St. Louis, MO, USA). As a negative control, a mock virus preparation, obtained with uninfected MDCK cells, or irrelevant peptides were used. To control the spontaneous cytokine production, cells incubated with only αCD28 and αCD49d were included.