IFN-γ secretion assay. The CD4-enriched population (2 x 10⁶) obtained from PBMCs of a CHP-NY-ESO-1-vaccinated patient (P-2) (day 155) using MACS CD4 beads was stimulated twice with irradiated autologous CD4- and CD8-depleted cells (2 x 10⁶) in the presence of a mixture of 28 18-mer overlapping peptides and a 30-mer C-terminal peptide spanning the entire NY-ESO-1 protein (1 µg/ml for each peptide) for 12 days. The cells (2 x 10⁵) from the stimulation culture were assayed for IFN-γ secretion in response to PFA-treated CD4- and CD8-depleted PBMCs (2 x 10⁵) pre-pulsed or not pulsed with the peptide mixture for 4 hr using FACS.