CTLs cause target cell–mitochondria polarization to point of contact. (A) Representative microscope images of target cell mitochondrial localization in E/T conjugates. Target Jurkat cells (i-iv) or target mouse L1210 (v-vii) were incubated with human CTLs (i-iv) or mouse CTLs (v-vii) for 5 minutes at 37°C prior to fixation. Samples were processed for immunofluorescence using anti-CD8 antibody (secondary anti-mouse 488) to identify CTLs, as described in “Microscopy.” Effector–target cell conjugates were identified, and locations of target cell mitochondria were scored. Mitochondrial localization was determined by immunofluorescence using anti–cytochrome c (Jurkat, anti-mouse 555) or Mitotracker Red (L1210). Shown are representative DIC (top) and fluorescent (bottom) images of mitochondria not aligned at conjugate interface (i), or aligned at conjugate interface (ii-vii) of both human (ii-iv) and mouse (v-vii) cells. Only conjugates formed from a 1:1 interaction between target and effector cells were scored, with an average 150 conjugates per experiment scored. The hCD8-Jurkat experiment was performed 2 times, and the mCTL-L1210 experiment was performed 7 times. (B) Target cell mitochondrial polarization to point of contact is not dependent on grB or perforin. The percentage of cells displaying target cell mitochondrial alignment to conjugate interface with WT mCTLs (WT), grB-cluster KO mCTLs (grB KO), and perforin KO mCTLs (Per KO) are shown. Shown are the average values of 4 independent experiments plus or minus SD.