WT1-CTL frequencies increase after DLI. Ten patients received 2 or more DLI after transplantation, after which WT1-CTL responses continued to be monitored. (A) Percentages of CD8 and CD4 T cells producing IFN-γ in response to WT1 peptides were determined after transplantation, before administration of DLI (pre-DLI; □), and at multiple time points after DLI, by intracellular IFN-γ analyses. ■ indicates the maximum IFN-γ response observed post-DLI. (B) Percentages of CD8-positive WT1-CTL were also quantified in pre-DLI samples and post-DLI by MHC-tetramer analyses in HLA-A*0201/0301–expressing patients, by staining with the HLA-A2/A3-RMF tetramers. (C) Absolute numbers of WT1-CTL present pre-DLI and post-DLI were quantified by multiplying the percentages of WT1-CTL determined by MHC-tetramer or IFN-γ analyses (presented in panels A and B), by each patient's ALC. (D) Comparing the absolute number of WT1-CTL pre- and post-DLI reveals a 6.6-fold increase in WT1-CTL. (E) Comparison of each patient's ALC recorded pre-DLI and post-DLI reveals no significant difference between the 2 time points. *P < .04 by Wilcoxon matched-pairs signed rank test. Δ P = .0011 by 2-tailed Mann-Whitney test.