Functional analysis of short-term T-cell cultures. PBMCs were isolated and subjected to CSA and staining with HLA-peptide tetramer. ELISPOT analysis on freshly isolated PBMCs was unreliable due to the low frequency of responding cells (data not shown) and so T cells were cultured with peptide for 14 days prior to an ELISPOT assay. Staining with HLA-peptide tetramer was performed at the same time. (A) Patient 21. (Ai) CSA of PBMCs illustrating 0.03% CD8 specific to CGAg peptide MAGE-A1_289-298. (Aii) MAGE-A1-_289-298 tetramer stain of PBMCs also stained 0.03% of the CD8 T-cell pool. (Aiii) MAGE-A1-_289-298 tetramer stain of T-cell line after 14 days of culture showed 0.09% of CD8 T cell-binding tetramer. (Aiv) IFN-γ ELISPOT analysis at day 14 T-cell line (TCL) revealed that 0.04% of T cells responded to MAGE-A1-_289-298 peptide. Similar analysis for peripheral blood in patient 22 revealed 0.04% of CD8⁺ T cells specific to MAGE-A2-_157-166 peptide(Bi) and 0.04% of CD8 staining with MAGE-A2-_157-166 tetramer (Bii). At day 14, 0.26% of CD8 stained with tetramer (Biii) and 0.05% of T cells responded to MAGE-A2-_157-166 peptide in ELISPOT assay (Biv). (C) Similar analysis for peripheral blood in patient 7 illustrated 0.02% of CD8⁺ T cells specific to MAGE-A1-_289-298 (Ci) and 0.03% of CD8 staining with MAGE-A1-_289-298 tetramer (Cii). At day 14, 0.05% of CD8 stained with tetramer (Ciii) and 0.04% of T cells responded to MAGE-A1-_289-298 peptide in ELISPOT assay (Civ). (D) A tetramer for the peptide MAGE-A3-_195-203 restricted to HLA-A^*2401 was not available; however, analysis of peripheral blood from patient 23 revealed 0.05% of CD8⁺ T cells specific to MAGE-A3-_195-203 peptide by IFN-γ CSA. At day 14, 0.05% of T cells responded to the peptide in ELISPOT assay (Dii). (Note: To maximize the number of events analyzed by FACS, PBMCs were gated on CD8 only when tetramer staining was performed on peripheral blood.)