Targeted CTL activity is perforin dependent. (A) Perforin and granzyme content of anti–third-party H-2^d CTLs. Wild-type H-2^d CTLs (top row) and perforin^−/− CTLs (bottom row) were fixed and stained intracellularly for perforin (green) and granzyme B (red). 4,6-diamidino-2-phenylindole (DAPI) staining of nuclei may be observed in merge (blue). Scale bar = 15μm. (B) The role of perforin in targeted CTL cytotoxicity. Perforin ^−/− H-2^d CTLs were targeted with 2C CD8⁺ T cells. After 5 hours, 2C cell depletion was evaluated and compared with the depletion induced by wild-type CTLs, as well as by nonspecific CTLs. Error bars represent SD from triplicate samples. Data shown are single experiments representative of 3 experiments. **P = .0015. (C) The role of the granule-mediated cytotoxic pathway in the induction of tolerance toward MHC-mismatched bone marrow by targeted CTLs. In an established graft rejection model,⁶  supralethally irradiated C3H mice were reconstituted with host T cells and transplanted with allogeneic (H-2^d), fully mismatched, T cell–depleted bone marrow either alone (n = 13) or supplemented with donor-type CTLs (H-2^d, n = 6). For evaluation of the role of the granule-mediated pathway, H-2^d perforin ^−/− CTLs were used (n = 17), and their capacity to induce tolerance was compared with that of perforin-competent wild-type CTLs. Data shown are from 2 experiments combined.