In vivo activated allogeneic CTLs require target hepatocyte ASMase for efficient killing ex vivo. Hepatocytes, isolated as described in “Hepatocyte isolation,” were coincubated with splenic T cells harvested from lethally irradiated wild-type C57BL/6 recipients 10 to 14 days after transplantation of LP BM⁺ T cells. (A) A total of 2 × 10⁶ GVH-activated splenic CTLs were coincubated with 0.5 × 10⁶ wild-type C57BL/6 or B6.MRL.lpr (FasR^−/−) hepatocytes (left panel) in complete medium for 16 hours. Alternatively, dimethyl sulfoxide– or concanamycin A–pretreated (100 ng/mL, 30 minutes) GVH-activated splenic CTLs were coincubated with 0.5 × 10⁶ wild-type C57BL/6 hepatocytes for 16 hours (right panel). Apoptosis was quantified after fixation by nuclear bisbenzimide staining. (B) asmase^−/− hepatocytes are resistant to apoptosis induced by GVH-activated splenic CTLs. CTL coincubation was performed as in panel A, and apoptosis was quantified 16 hours thereafter. (C) Representative images of asmase^+/+ (top left panel) and asmase^−/− (bottom left panel) C57BL/6 hepatocytes after 10 minutes of coincubation in suspension with 2 × 10⁶ GVH-activated splenic T cells. Hepatocytes were fixed and stained with 4,6-diamidino-2-phenylindole (DAPI) and Cy-3-labeled anticeramide mAb as described in “Hepatocyte apoptosis and platform detection assays.” Arrows indicate ceramide-rich platform generation on the outer leaflet of the plasma membrane. Note that, after incubation, cells were centrifuged at 50g for 4 minutes at 4°C before staining and imaging. Hence, CTLs (small blue nuclei) distributed with hepatocytes (large blue nuclei) do not reflect biologic association. Images were acquired using Zeiss Plan-NEOFLUAR 40×/1.3 NA oil DIC lens and Zeiss AxioCam MRm camera, and were processed with Zeiss AxioVision software and Adobe Photoshop Version 7.0 software. (D) Quantification of ceramide-rich platforms in asmase^+/+ and asmase^−/− hepatocytes after incubation with 2 × 10⁶ GVH-activated splenic CTLs. A total of 0.5 × 10⁶ hepatocytes were coincubated for the indicated times, fixed, and stained as in panel C. Exogenous C₁₆-ceramide bypasses the requirement for target cell ASMase, restoring platform generation (E) and conferring apoptosis (F) onto GVH-activated CTL-stimulated asmase^−/− hepatocytes. Platform generation and apoptosis were quantified as in panels D and A, respectively. (G) Disruption of membrane GEMs with nystatin inhibits CTL-induced hepatocyte apoptosis. A total of 0.5 × 10⁶ wild-type hepatocytes, preincubated with 50 μg/mL nystatin for 30 minutes and resuspended in RPMI containing 1% lipid-free FBS, were coincubated with 2 × 10⁶ GVH-activated splenic T cells, and apoptosis was quantified as in panel A. Data (mean ± SEM) represent triplicate determinations from 3 independent experiments each for panels A, B, D, E, F, and G.