CD137 up-regulation as a surrogate marker for specifically activated CD8⁺ T cells in vitro and ex vivo. The activation threshold for several markers of response was compared in responding T-cell clones (A), T-cell lines (B), or memory T cells directly ex vivo (C). (A) Two HLA-A*0201–restricted T-cell clones, specific for either Melan-A_(26-35L) or WT1_(126-134), were stimulated with titrating amounts of peptides. IFNγ (squares) and TNFα (triangles) production was assessed after 5 hours of stimulation in the presence of brefeldin A, and CD137 expression (circles) was determined 24 hours after stimulation (note: these reflect optimal time points for detecting each of these events). (B) Six separate T-cell lines specific for the NS3_(1406-1415)-epitope of HCV were generated, and assessed for cytokine production and degranulation after 5 hours of stimulation, and CD137 expression after 24 hours and pMHC-multimer staining of the unstimulated sample. Pearson correlation (2-tailed) was calculated, and the P value and the coefficient of determination (r²) appear in each plot. (C) PBMCs from 7 CMV⁺/HLA-A*0201⁺ donors were thawed, rested overnight, and analyzed for pMHC multimer staining, cytokine production, and degranulation after 5 hours of stimulation and CD137 expression at 24 hours. P value; r² appear in each plot.