Enumeration of frequency of CD8⁺ T cells responding to CGAg peptides by FACS analysis. PBMCs were stimulated with CGAg peptides for 3 to 6 hours prior to labeling with antibodies to PE-labeled anti-IFN-γ monoclonal antibody and anti-PE magnetic beads. Enrichment of IFN-γ-secreting cells was performed by magnetic separation. PBMCs stimulated with DMSO alone prior to (A) and following (C) magnetic selection. IFN-γ production in response to MAGE-A1-_289-298 peptide prior to (B) and following (D) magnetic selection. The illustrated value refers to the percentage of CD8⁺ T cells responding to either DMSO alone (A,C) or MAGE-A1-289-298 peptide (B,D). The CGAg-specific T-cell response in the positively selected fraction was calculated as a percentage of the total CD8⁺ T-cell pool (see “Materials and methods”). In this example 4.5 × 10⁶ PBMCs were stimulated with peptide of which flow cytometric analysis revealed 24% to be CD3⁺CD8⁺. From these 1 080 000 CD8⁺ T cells, 47 cells were isolated in the positively selected fraction, indicating that the percentage of the CGAg-specific T-cell response in PBMCs was at least 0.004% (47/1 080 000 × 100) of the CD8⁺ T-cell pool.