NY-ESO-1–specific CD4⁺ T cells are derived from distinct CD4⁺ T-cell populations showing different sensitivity to CD4⁺ CD25⁺ regulatory T cells between patients with and without NY-ESO-1 antibody. (A) CD4⁺CD25^- T cells isolated from PBMCs were further separated into CD45RA⁺ or CD45RO⁺ using magnetic beads as described in “Study design.” (B) These T cells were cultured with APCs pulsed with appropriate NY-ESO-1 peptides according to respective HLA haplotype, namely NY-ESO-1_157-170 for NC155, NY-ESO-1_87-98 for NC159, NY-ESO-1_143-154 for NW681, NY-ESO-1_121-132 for NW792, NY-ESO-1_87-98 for NW2010, and NY-ESO-1_157-170 for NW2457, and tested for specific T-cell induction using cognate peptide or control HIV peptide by ELISPOT and proliferation assays. (C) Graded amounts of CD4⁺CD25⁺ T cells were added to cultures during in vitro peptide stimulation of NW681 and NW2010 and specific T-cell induction was examined using cognate peptides by ELISPOT and proliferation assays. These experiments were performed independently at least twice with similar results. Data are expressed as means +SD. CPM indicates counts per minute.