Expansion of NY-ESO-1–specific CTLs. (A) NY-ESO-1–specific T cells are spontaneously present and can be progressively expanded by stimulation with APCs pulsed with NY-ESO-1_157-C165V analog peptide. Note that there is equivalent staining with NY-ESO-1_157-165 wild-type and NY-ESO-1_157-C165V analog peptide-loaded A2 tetramers. (B) NY-ESO-1–specific CTLs kill both primary, the HLA-A2⁺ U266 myeloma cell line, and autologous PHA-blasts pulsed with NY-ESO-1_157-C165V, and not control PHA-blasts pulsed with a MAGE-3_112-120 A2-binding peptide or K562 cells. (C) NY-ESO-1–specific CTLs produce IFN-γ and not IL-4 (Tc1 type), contain cytolytic granules, and are of memory-effector type. VLA indicates very late antigen; FITC, fluorescein isothiocyanate.