Enumeration and characterization of circulating anti–MAGE-A3 T cells in patient CIP-5. (A) Clinical evolution of patient CIP-5 and timing of treatments; PD: progressive disease, NED: not evidence of disease. (B) Frequency of circulating anti–MAGE-A3 T cells estimated in patient CIP-5 during the treatment. The frequency of total anti–MAGE-A3 T cells was measured as described in Figure 2. The frequency of anti–MAGE-A3.A26 and anti–MAGE-A3.B44 T cells was measured by tetramer staining or IFN-γ release assay, performed on multiple cultures of PBMCs (10⁵) stimulated with the peptide M3_250-258. After 15 infusions, patient CIP-5 developed a strong increase of circulating anti–MAGE-A3 T cells (3.08 × 10⁻⁵) that paralleled a MAGE-A3–specific DTH reaction; P stands for after infusion. (C) HLA-I restriction of the selected CD8⁺ microculture no. 10. HLA-I restriction was characterized as described in Figure 1. Microculture no. 10 recognized HLA-A26, -B44 and Cw01-restricted MAGE-A3–derived epitopes and autologous tumor cells (CIP-5-mel). Autologous lymphocytes expressing MAGE-A3 (M3-GMLs) were used as positive control.