NY-ESO-1 regions recognized by CD4 and CD8 T cells. CD4- and CD8-enriched populations (2 x 10⁶) from PBMCs of patients were stimulated two or three times with irradiated autologous CD4- and CD8-depleted PBMCs (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. The cells (1x10⁵) from the culture were then assayed for IFN-γ secretion in response to PFA-treated autologous CD4- and CD8-depleted PBMCs (1 x 10⁵) pre-pulsed with individual peptides for 4 hr using FACS.