HD preculture enhances the recall responses of PBMCs to WT1-derived antigens. (A-B) IFN-γ T-cell responses from fresh or precultured PBMCs of 1 representative HLA–A0201-positive HSCT patient to titrated amounts of the peptide WT1_HUMAN 126-134 or WT1_HUMAN 356-364, respectively. Unpaired Student t test: ***P < .0005. Data represent mean ± SD for triplicate samples. The experiment presented in (A) was repeated for 2 different patients. (C) Compiled data from Figure 4B from 11 patients. Average time between stem cell transplantation and sampling: 814 days; from 166 to 2372 days. Data are presented as in Figure 2B. Raw data: supplemental Figure 5D. Mann-Whitney U test: ***P < .0005; ****P < .0001. Data represent median with IQR. (D) Improved monitoring of anti–WT1-directed T-cell responses from an HLA–A0201-positive patient 4 months (top), 1 year (middle), and 5 years (bottom) after HSCT upon HD preculture of PBMCs. Unpaired Student t test: **P < .005; ***P < .0005; ****P < .0001. Data represent mean ± SD for triplicate samples. n.s., not significant.