Frequencies of memory CD8⁺ T cells that bind the WT1_126-134/HLA-A*0201 tetramer are greater in IST responders compared withnonresponders. (A) Gating strategy and an example of tetramer binding are shown (patient 4; supplemental Table 1). Live T cells were separated from dead cells, B cells, and monocytes in a CD14/CD19/ViViD (dump channel) versus CD3 bivariate plot, and single cells were identified in a FSC-A versus FSC-H plot. Intact lymphocytes were next identified in an FSC-A versus SSC-A plot. Fluorochrome aggregates were subsequently excluded from the analysis in a CD45RO versus CD8 plot. Naive cells were identified within the CD8⁺ T-cell pool as CD27⁺CD45RO⁻CD57⁻ cells (bottom panels); memory CD8⁺ T cells were identified within the L-gate shown in the CD27 versus CD45RO plot. Binding of the WT1_126-134/HLA-A*0201 tetramer was observed in the memory CD8⁺ T-cell compartment but not in naive or CD8⁻ memory T-cell populations (top right panels). The HIV p17 Gag_77-85/HLA-A*0201 and CMV pp65_495-503/HLA-A*0201 tetramers were used as negative and positive controls, respectively. (B) The mean frequency of memory CD8⁺ T cells binding to the WT1_126-134/HLA-A*0201 tetramer in IST responders was significantly higher compared with IST nonresponders (P < .0001) and healthy controls (P < .0001). R, responders; NR, nonresponders; Normal, healthy controls.