Kinetics of T-bet, perforin, and Grz B up-regulation are tightly linked during an antigen-specific recall response. (A) The degree of Grz B, perforin, and T-bet^bright positivity was determined at baseline for TPR- and RAK-specific CD8⁺ T cells (labeled by the peptide: MHC I tetramer) from 2 HIV-negative donors, ND172 and ND232, respectively. Percentages represent the fraction of tetramer⁺ cells that fell within each gate. (B-C) PBMCs from ND232 were incubated for either 2 (B) or 3 (C) days in the presence of RAK peptide. The levels of Grz B, perforin, and T-bet are shown within RAK-specific cells, which were identified either by peptide restimulation for 6 hours or by tetramer staining. Events shown have been gated on CD8⁺ T cells. (D) CFSE-labeled PBMCs from ND232 were incubated for either 3 or 5 days in the presence of RAK peptide, and the expression of Grz B, perforin, and T-bet was subsequently determined. All flow cytometric plots show only RAK-specific cells, as determined by tetramer staining. Percentages represent the fraction of tetramer⁺ cells that fell within each quadrant. (E) CFSE-labeled PBMCs from ND172 were incubated for 5 days in the presence of the TPR peptide. The MFI of Grz B, perforin, and T-bet within TPR-specific cells was plotted against each generation number, which was established with FlowJo software Version 8.8.4.