Identification of pan-A2-alloreactive T cells and tetramer sorting of allo-restricted, WT1-specific CTLs. (A-H) PBMCs of an HLA-A2^- donor were stimulated 6 times in vitro with autologous B lymphocytes coated with A2/pWT126 monomers and stained with FITC-labeled antihuman CD8 antibodies and PE-labeled A2 tetramers containing the peptide epitopes indicated in each panel. (F) The FACS profile of a sample stained simultaneously with all 5 A2 tetramers is shown. (G, H) The FACS profile after staining with HLA-A1 and HLA-A3 tetramers, respectively, is shown. (I-K) FACS profile of a bulk CTL line established from an HLA-A2^- donor after 7 rounds of stimulation with A2/pWT126 monomers followed by nonspecific expansion with CD3/CD28 beads and IL-2. Cells were stained with APC-labeled antihuman CD8 antibodies and PE-labeled A2/pWT126 tetramers before (I) and after (J) FACS sorting of CD8⁺ tetramer-positive T cells. The tetramer-positive cells did not bind A2 tetramers containing control peptides. (K) Killing activity of sorted CD8⁺ tetramer-positive CTLs against A2⁺ WT1⁺ leukemia cells (BV173), A2⁺ WT1^- breast cancer cells (ZR571), and EBV-transformed C1R-A2 cells coated with pWT126 peptides. Uncoated cells and cells coated with the A2-binding pWT235 peptide were used as control.