Tetramer assays. CD3⁺ T cells from patient 1 (A) and patient 14 (B) were stained with WT1-A/HLA-A*02:01 tetramer with CD8⁺ and other T-cell markers. Percentage of tetramer-positive CD8⁺ T cells (number shown in the top right of each histogram and highlighted in bold print) were gated on CD3⁺ events after establishing a lymphocyte gate. Cells from before vaccine (time 0 [T0]), after 6 GPS vaccinations (T6), and after 12 GPS vaccinations (T12) are shown. (A) CD8⁺ cells only from patient 1 are shown. Cultures were preincubated with native (WT1-A) and heteroclitic (WT1-A1) peptides. The data are controls (no peptide [−]) on the left followed by representative staining from triplicate cultures following antigen exposure (+) on the right. Before vaccination, there were low percentages of tetramer-positive cells in the CD8⁺ population. (B) Both CD8⁺ and CD8⁻ cells from patient 14 are shown. Cultures are incubated with WT1-A peptides. Bottom row of histograms show concurrent testing with irrelevant tetramers. After vaccination, a large increase in the percentage of WT1-A–specific CD8⁺ T cells was noted in primed cultures indicating that vaccination with the heteroclitic peptide can induce T cells that recognize the native sequence.