Human CD8⁺ Melan-A–specific T cells expose elevated amounts of PS after Ag recognition. Flow cytometric analysis of annV-FITC binding on the cell surface of Melan-A–specific CTLs. (A) CTLs were labeled with membrane dye PKH-26 and stimulated for 4 hours either with unloaded, Melan-A–loaded, or gp100-loaded T2 cells at a T2/CTL ratio of 1:2.5 and stained with annV-FITC. Unstimulated CTLs (0 and 4 hours) served as a control. Shown is one representative flow cytometric analysis of 5. (B) Melan-A–specific CTLs were activated with different types of stimuli for 4 hours to induce PS exposure: T2 cells loaded with 5μg/mL Melan-A peptide, 200 U/mL IL-2, 2000 U/mL IFN-γ, anti-CD3/CD28–coated beads, or complete medium as a control. Cells were stained with annV-FITC and dead/apoptotic cells were excluded via PI staining. One representative experiment of 5 is shown. Numbers in panels A and B represent percentages of annV⁺/PKH26⁺ T cells. (C) Melan-A–specific CTLs were stimulated with 2.5 μM ionomycin (▴) or left unstimulated (○) at 37°C in medium containing 1.5 mM CaCl₂ and annV-FITC. Flow cytometric analysis was performed at indicated time points. Mean values ± SEM from 4 independent experiments are shown. (D) CTLs were labeled with PKH26 and coincubated for 4 hours with HLA-A2⁺ Melan-A–expressing (MeI493) or Melan-A^– (Na8) melanoma cell lines at different tumor cell/CTL ratios. Preincubation of MeI493 with an anti–MHC-I–blocking mAb (clone W6/32) was performed to demonstrate MHC class I-restricted Ag recognition. Results represent percent annV-FITC⁺ cells of total PKH-26⁺ CTLs. Data show one representative experiment of 2.