CTLs kill recognizing CD8+ T cells in rapid, granule-mediated fashion. (A) Targeted CTL cytotoxic response. H-2^d CTLs (directed against a third party) were targeted with 2C CD8⁺ T cells loaded with calcein, a probe that is lost on cell death. After 1, 2, 5, 10, 15, and 20 hours of incubation at 3 CTL-to-2C ratios (1:1, 1:2, and 1:5), depletion of calcein-positive 1B2⁺CD8⁺ 2C T cells was evaluated by FACS. 2C CD8⁺ T cells were incubated with identically treated H-2^s CTLs as control. (B) Depletion of recognizing T cells by targeted H-2^d/b (F1). F1 CTLs were targeted with 2C cells prepared as in A at a 1:1 ratio for 5 hours. Depletion was determined as in panel A and compared with the depletion induced by H-2^d CTLs (anti–third-party) CTLs. (C) Dependence on intracellular Ca²⁺. CTLs were pretreated with BAPTA-AM and targeted with 2C CD8⁺ T cells. After 5 hours, 2C cell depletion was evaluated as in A and compared with the depletion induced by untreated CTLs. (D) Specific granzyme release associated with CTL activity. H-2^d/b (F1) CTLs were targeted with 2C CD8⁺ T cells or incubated with the specific stimulator cells against which they had been generated for 4 hours, and granzyme release was evaluated by the BLT-esterase assay. 2C cells had no detectable granzyme secretion. Similarly treated H-2^s CTLs did not release granzyme. Error bars in panels A-D represent SD from triplicate samples. Data shown are single experiments representative of 3 experiments. ***P < .001.