Evaluation of the cytotoxic activity of HIV-1-specific CD8⁺T cells by use of fluorogenic caspase-3 substrates. (A) Untreated or peptide-pulsed autologous BCLs were coincubated for 60 minutes with freshly isolated PBMCs at an effector-to-target (E/T) ratio of 50:1 and subsequently stained with FITC-labeled caspase-3 substrates. Gating was performed according to fluorescence signals of the target cell fluorophore and FSC/SSC characteristics. Percentages indicate the proportion of caspase-3-positive cells. Only HLA class I-matched BCLs labeled with the specific peptide were lysed by CD8⁺ T cells. (B) Kinetic analysis of B8-EI8-labeled target cell apoptosis after addition of PBMCs at an E/T ratio of 50:1. The assay was run in triplicate; results indicate the mean and standard deviation (error bars), demonstrating a low (< 10%) intra-assay variability. (C) Evolution of B8-EI8-labeled target cell apoptosis following 60 minutes of coincubation with freshly isolated PBMCs from an additional study person at various E/T ratios. (D) Simultaneous assessment of caspase-3 activation and 7-aminoactinomycin uptake in B8-EI8-labeled target cells at various time points after exposure to PBMCs at an E/T ratio of 50:1.