CD45RA/CCR7 phenotype and functional properties of anti–MAGE-3.A1 CTL clones. (A) The CTL clones from cancer patients were labeled in the same conditions as the CD8⁺ PBLs from donors. Cells were labeled for 15 minutes at room temperature with an FITC-conjugated anti-CCR7 antibody, a PE-conjugated anti-CD45RA antibody, and an APC-conjugated anti-CD8 antibody. Only the living CD8⁺ PBMCs are shown. Quadrant limits were positioned so as to include in the lower left quadrant 99% of the cells labeled with a control isotype-matched antibody. Lysis was tested by flow cytometry with CTL clones that had been stimulated 15 to 47 days before. The targets were EBV-B cells pulsed with the MAGE-3 peptide. Target cells were mixed with the CTL at an effector-target ratio of 3 and the number of living CD19⁺ target cells was estimated after a 3-hour coculture. Expansion and IL-2 release were estimated after antigenic stimulation. Twenty thousand CTLs were cocultured with 20 000 HLA-A1 EBV-B cells pulsed with the MAGE-3 peptide. Considering that up to 75% of the CTLs died within 2 days, the expansion of the clones was evaluated in a flow cytometer by counting the number of CD8⁺ cells at day 2 and at day 5. The presence of IL-2 was evaluated in the supernatant of the coculture using cytometric bead arrays. (B) A total of 28 anti–MAGE-3.A1 CTL clones was tested in the conditions described in panel A. Ninety-nine percent of the cells labeled with the control isotype-matched antibody had a fluorescence lower than the limit indicated by the dashed line. The best correlation curve and the coefficient of determination (R²) are indicated.