Enumeration and characterization of circulating anti–MAGE-A3 T cells in patient CIP-19 and analysis of anti–MAGE-A3 T cells infiltrating the DTH. (A) Clinical evolution of patient CIP-19 and timing of treatments. (B) Anti–MAGE-A3 T-cell frequency and DTH reactivity after indicated treatments are shown. The frequency of total anti–MAGE-A3 T cells was measured by IFN-γ release assay performed on multiple cultures of PBMCs (10⁵) stimulated and then tested against M3-GMLs and UT-GML. After 12 infusions, we detected a strong increase of circulating anti–MAGE-A3 T cells (1.73 × 10⁻⁵). This increase paralleled the development of a strong MAGE-A3–specific DTH reaction. (C) Antigen specificity of a selected CD4⁺ microculture. Microculture no. 8 was tested against MAGE-A3–transduced (M3) or untransduced (UT) autologous EBV in the presence or in the absence of anti-DR, anti-DP, and anti-NGFr (used as control antibody) mAbs. Anti–MAGE-A3 T cells were also tested against autologous EBV pulsed with the M3.DP*0401 peptide²⁶  and against allogeneic MAGE-A3–transduced or untransduced EBV (BM21-EBV) sharing the HLA-DP*1001 allele. Microculture no. 8 specifically recognized the allogeneic HLA-DP*1001 EBV expressing MAGE-3 but not the M3.DP*0401 peptide. Error bars represent SD of experimental replicates. (D,E) MAGE-A3–specific long-term T-cell memory. (D) CD3⁺CD4⁺ T-cell clones from a punch biopsy of an anti–MAGE-A3 DTH performed 9 months after the 14th infusion, proliferated in the presence of MAGE-A3–transduced autologous EBV cells. (E) Upon in vitro expansion, clones no. 30 and no. 45 specifically released IFN-γ in response to MAGE-A3–expressing target cells.