HLA-A24–restricted antitumor activity of TCR gene–transduced Th1 and Tc1 cells against WT1 peptide–loaded LCLs. (A) WT1-specific TCR α and β chain genes were lentivirally transduced to nonspecific Th1 and Tc1 cells obtained from an HLA-A24⁺ healthy donor as described in “Materials and methods.” Ten days after infection, TCR gene–transduced and control T cells were stained with FITC-labeled anti-CD4 mAb and PE-labeled anti-CD8 mAb and sorted to CD4⁺ and CD8⁺ T cells by a FACSVantage instrument. After isolation, staining profile of Th1 and Tc1 cells was analyzed by FACSCalibur instrument and Cell Quest software. Percentage of cells in each quadrant is indicated in flow cytometer plots. (B-D) Cytotoxic activity of TCR gene–transduced and control Th1 and Tc1 cells against WT1 peptide–pulsed (B), CMV peptide–pulsed (C), or unloaded (D) LCLs (TAK-LCL, which are derived from an HLA-A24⁺ patient with leukemia) was evaluated by 4-hour ⁵¹Cr-release assay. Nonspecific cytotoxicity of Th1 and Tc1 cells against HLA-A24^– LCLs was less than 10%. (E) Blocking of cytotoxicity against WT1 peptide–pulsed HLA-A24⁺ LCLs by anti–HLA-A2 and anti–HLA-A24 mAbs was evaluated at an effector-to-target (E/T) ratio of 20 for Tc1 cells and E/T of 40 for Th1 cells. The percentage of inhibition was calculated by the following formula; % Inhibition = (% cytotoxicity with mAb) / (% cytotoxicity without mAb) × 100. Percentage of cytotoxicity of TCR gene–transduced Tc1 and Th1 against WT1 peptide–loaded LCL was 77% and 57%, respectively. Background cytotoxicity of Tc1 and Th1 against unloaded LCLs was 9% and 13%, respectively. (F-G) Cytotoxic activity of TCR gene–transduced and control Th1 and Tc1 cells against HLA-A24⁺ LCLs derived from healthy donors and retrovirally transduced with WT1 gene (F) and their parental A24⁺ LCLs (G) was evaluated by 4-hour ⁵¹Cr-release assay. Similar results were obtained using TCR gene–transduced Th1 and Tc1 cells derived from 2 other HLA-A24⁺ and 2 HLA-A24^– healthy volunteers. Error bars indicate standard error (SE) in triplicate samples.