WT-1–specific responses of CTLs generated from PBMCs of healthy donors (n = 56) by stimulation with autologous APCs loaded with total pool of WT-1–derived pentadecapeptides. (A) production of IFNγ in PBMCs alone (as a background), PBMCs coincubated overnight with the total pool of pentadecapeptides spanning the whole sequence of WT-1 protein (PBMC + WT-1 pool), and pregenerated WT-1–specific T cells coincubated overnight with WT-1 peptide–loaded PBMCs. (B) Cytotoxic activity of the WT-1–specific CTLs generated in vitro by stimulation with WT-1 total pool against WT-1⁻ (autologous PHA-stimulated blasts) and WT-1⁺ (autologous PHA-stimulated blasts loaded with the total pool of WT-1 pentadecapeptides) targets at a 50:1 effector: stimulator ratio. (C) IFNγ response measured by FACS staining in different responder cell populations (PBMCs, pregenerated CTLs sensitized in vitro with the RMF peptide loaded on autologous CAM and pregenerated CTLs sensitized with the total pool of WT-1 15-mers) after secondary overnight stimulation with autologous PBMCs either unmodified or loaded with one of the following: RMF peptide, dominant epitopes of WT-1 identified by the epitope-mapping approach in the WT-1–total pool sensitized CTLs, or the WT-1 total pool of the 141 pentadecapeptides. (D) Cytotoxic activity of the WT-1–specific T cells generated in vitro by sensitization with autologous CAMs loaded with the RMF 9-mer or with the total pool of the WT-1 15-mers. The cytotoxicity of the T cells was assessed against autologous WT-1⁻ targets (PHA-activated blasts) and the same targets loaded with RMF peptide, the total pool of WT-1 15-mers, or the dominant WT-1 epitope identified for the same T-cell line.