Peripheral blood mononuclear cells (PBMC) were isolated using standard Ficoll density gradient centrifugation and resupsended in complete RPMI containing 10% human AB serum, 100 units/ml of Penicillin, 100 mg/ml of Streptomycin, 2 mM L-Glutamine, 1 mM Sodium Pyruvate and 25 mM Hepes. Cells were cultured at a concentration of 1610 5 cells/well in 96-wells round-bottomed plates at 37uC in an atmosphere of 5% CO 2 . The cells were stimulated with IVTT-produced FluM, FluHA, CMVpp65 and EBNA3A antigens, either unpurified; associated to Polybeads or ProteinG beads; or purified using NI-NTA resin or MagneHis Niparticles. Unpurified and purified IVTT products were used at a dilution of 1:100, 1:1000 or 1:10000 for evaluation of cytokines in the supernatant using the Cytometric Bead Array assay, or at a dilution of 1:1000 (unpurified) or 1:100 (purified) for evaluation of IFN-c secreting cells via ELISpot. Negative controls included empty pIVEX HisHA IVTT reaction alone or associated to Polybeads or ProteinG beads, or medium only. A CEF peptide pool consisting of 32 synthetic peptides representing defined CD8 + T cell epitopes from human Cytomegalovirus, Epstein-Barr Virus and Influenza Virus (Anaspec, San Jose, CA) at 5 mg/ml total was used as a positive control [44] . Phytohemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO) at 2 mg/ml was used as a mitogen control.