Expression level and kinetics of activation markers after antigen-specific activation. To evaluate a panel of activation markers, a T-cell clone specific for the HLA-A*0201–restricted epitope of Melan-A_(26-35L) was stimulated with peptide-pulsed T2 cells, and assessed for (A) antigen-specific up-regulation of denoted markers 24 hours after stimulation (thick lines) compared with control peptide (filled) or isotype-matched antibodies (dotted line); and (B) expression kinetics of the panel of markers in the same experiment (x-fold increase: MFI of the specifically stimulated sample/MFI of the sample stimulated with the irrelevant control peptide). (C) A polyclonal Melan-A–specific T-cell line generated by 2 in vitro stimulations was assessed for baseline expression and specific up-regulation 24 hours after stimulation of the 3 most informative activation markers (numbers indicate the percentage of viable CD8⁺ T cells in each quadrant).