HLA class I– and class II–restricted, WT-1–specific T cells respond to the same immunodominant peptide 15-mer derived from WT-1 protein in the WT-1 CTL sensitized with the WT-1 total pool of overlapping 15-mers loaded on autologous CAMs. (A) Production of IFNγ by the CD8⁺ and CD4⁺ WT-1–specific T cells in response to secondary overnight stimulation with the same dominant WT-1–derived 15-mer no. 41. (B) Identification of the immunogenic sequence of amino acids within pentadecapeptide no. 41 by IFNγ production after secondary overnight stimulation with autologous PBMCs loaded with a panel of 9-mers either unique for the peptide no. 41 (LDFAAPGAS [LDF]) or contained within the neighboring overlapping 15-mer no. 40 (PVLDFAPPG [PVL] or VLDFAPPGA [VLD]) and no. 42 (DFAPPGASA [DFA]). Only the 9-mer uniquely presented within the 15-mer no. 41, LDF, elicited an IFNγ response. (C) Peptide-specific cytotoxic activity of WT-1 CTLs against the panel of 9-mers and 11-mers contained within peptide no. 41 and loaded on autologous PHA-stimulated blasts is observed against both the 11-mer LDF and 9-mer LDF contained within the 11-mer LDF, as determined in a standard ⁵¹Cr-release assay at a 25:1 effector: stimulator ratio. (D) HLA restriction of the cytotoxic activity of the WT-1 CTLs. T cells restricted by HLA-A0201 lyse targets loaded with either the 11-mer or the 9-mer, whereas those restricted by HLA DRB₁0402 only lysed targets loaded with the 11-mer.