Figure 5.

In vitro–activated CTLs require target splenocyte ASMase for efficient killing. (A) Representative images and (B) quantification of ceramide-rich platforms (indicated by arrows in panel A) formed on the surface of Mitotracker Red–labeled, conA-activated (5 μg/mL for 24 hours) target C57BL/6^asmase+/+ and C57BL/6^{asmase−/−} splenocytes, on coincubation for 20 minutes with effector Balb/c splenic T cells that had been activated in vitro with 2 × 10⁶ irradiated (20 Gy) C57BL/6 splenocytes/mL media for 5 days at a target/effector ratio of 2:1. Target splenocytes were fixed with 4% formalin-buffered phosphate, and stained with DAPI and FITC-labeled anticeramide mAb. Images were acquired as in Figure 4C. Platforms were identified as previously described.²⁸ (C) Lysis of ⁵¹Cr-labeled target C57BL/6^asmase+/+ and C57BL/6^{asmase−/−} splenocytes after coincubation with effector Balb/c splenic T cells for 6 hours measured by the chromium-release assay. (D) Cytolytic response of ⁵¹Cr-labeled target C57BL/6^{asmase−/−} splenocytes to activated effector Balb/c splenic T cells as in panel B, in the presence of 500 nM C₁₆-ceramide or C₁₆-dihydroceramide (DCer). Data (mean ± SEM) represent triplicate samples from 3 independent experiments for panels B through D.