Expression of transduced WT1-specific TCR. PBMCs were activated by PHA and plate-bound anti-CD3 mAb in the presence of type-1–inducing conditions and infected with a lentivirus carrying WT1-specific TCR α and β chain genes or mock virus. Ten days after infection, expression of the transduced TCR (Vα30 and Vβ5.1) was examined. (A) Cells were stained with FITC-conjugated anti-TCR Vβ5.1 mAb and either PE-conjugated anti-CD4 or anti-CD8 mAb. Fluorescence intensity of the cells was measured by a FACSCalibur instrument and analyzed by CellQuest software. (B) After isolation of CD4⁺ and CD8⁺ T cells by a FACSVantage instrument, mRNA was extracted and converted to cDNA by reverse transcription. TCR Vα30 and Vβ5.1 cDNA were amplified by PCR, separated on 1% agarose gel, and visualized with ethidium bromide. (C) TCR gene–transduced Tc1 cells were stained with PE-conjugated HLA-A24 tetramer loaded with WT1 peptide or HIV envelope peptide. Then, cells were stained with FITC-conjugated anti-CD8 mAb. Staining profile of the cells was measured by a FACSCalibur instrument and analyzed by CellQuest software. In panels A and C, percentage of cells in each quadrant is indicated in cytometer plots.