Ag-primed annV^int CTLs are activated but nonapoptotic T cells. (A) Phenotypic analysis of annV^int cells by flow cytometry. Melan-A–specific CTLs were stimulated for 4 hours with peptide-loaded T2 cells and subsequently stained with the following mAbs: CD69, HLA-DR, CD71, CD95, and TCRαβ. FITC-conjugated mAbs were combined with annV-PE and PE-conjugated mAbs with annV-FITC. Fluorescence intensity on gated CD8⁺, annV^int CTLs are presented by the solid line. As a control annV^neg CTLs from an unstimulated control are shown (dashed line). Gray histograms show isotype controls. (B) PI staining of annV^int and annV^high CTLs. The Melan-A–specific CTL clone (S2Cl14) was labeled with the membrane dye PKH-26 and stimulated with peptide-loaded T2 cells for 24, 48, 72, and 96 hours. PI staining was performed in parallel. Dot plots show annV-FITC and PI expression on gated annV^int (middle column) and annV^high (right column) T cells. The left column shows gating of cells. (C) Melan-A–specific CTLs were stimulated overnight with Melan-A–loaded T2 cells at a ratio of 3:1. The TUNEL assay was performed with FACS-sorted annV^int and annV^high CTLs. Representative experiments are shown.