Strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT-1 protein and epitope mapping. (A) The sequence of the WT-1 protein consisting of 575 amino acids and the principle of 11 amino acid–overlapping pentadecapeptides are illustrated. A total of 141 pentadecapeptides are required to span the entire protein. The sequence of 575 amino acids published by Gessler et al was used.³⁷  This sequence includes an additional 126 amino acids in the N-terminus. To match the sequential numbers of amino acids within the WT-1 sequence used with the longest, most frequently described WT-1 isoform D, we numbered the first 126 amino acids with negative values and used the positive values to number the subsequent 449 amino acids described in the longest isoform D. (B) Mapping grid consisting of 24 subpools each containing up to 12 WT-1–derived pentadecapeptides. Each peptide is uniquely contained within 2 intersecting subpools: for example, peptide 75 is uniquely shared by subpools 3 and 19. (C) IFNγ production by WT-1–sensitized CTLs in response to secondary overnight stimulation with the subpools of WT-1 pentadecapeptides loaded on autologous PBMCs. Dominant responses are observed for the subpools no. 3 and 19, both containing 1 common pentadecapeptide no. 75. (D) IFNγ production by the WT-1 CTLs in response to secondary overnight stimulation with the single pentadecapeptide contained within the subpools eliciting the highest responses as per the analysis determined in panel C confirms that the dominant immunogenic sequence is contained within pentadecapeptide no. 75. (E) HLA restriction of the WT-1–specific T cells responding to peptide no. 75 identified by ⁵¹Cr-release assay against a panel of allogeneic CAMs or PHA blasts matching single HLA alleles expressed by the WT-1 CTL donors. These are presented along the x-axis of the graph. The CAMs or PHA blasts used in the assay are unmodified (gray bars) or loaded with the WT-1–dominant epitope (black bars). The WT-1–specific cytotoxic activity of the WT-1 CTLs is restricted by the B3501 HLA allele.