Antibody, CD4 and CD8 T cell responses in vaccinated patients. NY-ESO-1 antibody response was determined using recombinant NY-ESO-1 protein by ELISA. CD4 and CD8 T cell responses were determined by IFN-γ secretion assay. CD4- and CD8-enriched populations (2 x 10⁶) obtained from PBMCs of patients using MACS beads were stimulated once (1° IVS) or twice (2° IVS) 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 each 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. Net % of IFN-γ secreting CD4 and CD8 T cells of the total CD4 and CD8 T cells, respectively, in cultures were plotted for (A) prolonged (10-18 vaccinations) and (B) shorter (3-5 vaccinations) time periods after repeated vaccinations. Values >0.1% were considered to be significant.