PBMC were thawed and plated, at several dilutions, onto 96-well nitrocellulose dishes coated with 1µg/well of recombinant influenza HA antigens, KLH (negative control), or antibody against human IgG. After a 4-hour culture, the cells were washed off and the plates developed with a second anti-human IgG antibody conjugated to alkaline phosphatase, and TCIP/BCP substrate. Spots were counted on an ImmunoSpot automated plate reader. Plates were set up such that PBMC from all the time points from a single subject were on the same plate. Each plot depicts the results from one of the 10 individual subjects tested.