Isolation of allorestricted FMNL1-specific T cells. (A) FMNL1-PP2–specific CD8⁺ T cells in bulk cultures after 2 stimulations with peptide-pulsed T2 cells, in sorted T-cell lines and in T cells cloned by limiting dilution, were detected by flow cytometry using anti-CD8 monoclonal antibodies and FMNL1-PP2–specific HLA-A2 tetramers. The numbers in the fluorescence-activated cell sorting (FACS) plot represent percentage of cells in that quadrant. As control, staining with HLA-A2 and HLA-B7 pp65-specific tetramers was performed. (B) Peptide recognition of cloned T cells was investigated using T2 cells pulsed with FMNL1-PP2 (■) and T2 cells pulsed with Flu (□) by ⁵¹Cr-release assay at an effector-target ratio of 7.5:1. Error bars indicate standard deviation of tested duplicates. (C) Cytokine secretion of the FMNL1-PP2–specific T-cell clone SK22 was investigated in response to T2 cells pulsed with the peptide FMNL1-PP2 (■) and pulsed with the Flu peptide (□) at an effector-target ratio of 1:5. Culture supernatants were harvested after 24 hours and assessed by BioPlex multicytokine analysis.