WT1 epitope spreading occurs after DLI. Immunogenic epitopes were identified in 3 healthy donor lymphocyte products and in the 3 patients who received those DLI, at varying time points after T-cell infusion. Epitopes inducing IFN-γ responses were mapped using the grid shown, whereby subpools inducing dominant responses intersect to reveal the single common peptide containing the immunodominant epitope. (A) CD8⁺ T-cell production of IFN-γ in response to WT1 peptide subpools, in DLI products for, and PBMC isolated from UPN1, UPN7, and UPN9 post-DLI. (B) CD4⁺ T-cell production of IFN-γ in response to WT1 peptide subpools, in DLI products for, and PBMC isolated from UPN1, UPN7, and UPN9 post-DLI. (C) Epitope mapping grid outlining subpool peptide compositions. The mapping grid consisting of 24 subpools each containing up to 12 WT1-derived pentadecapeptides. Each peptide is uniquely contained within 2 intersecting subpools. For example, subpools 6 and 24, to which CD8⁺ and CD4⁺ T cells from the DLI product for UPN1 both react, uniquely share peptide 138.