PI3K-Dock2 signaling modulates the efficiency of target cell killing. (A–C) RMA-s target cells loaded with 100 nM OVA peptide were mixed with OT-1 CTLs expressing either nontargeting shRNA or shRNA specific for Dock2 or PTEN. (A and B) Specific lysis of target cells at the indicated effector to target (E:T) ratios after 6 h (A), or at the indicated times at an E:T ratio of 2:1 (B). Killing assays were performed in triplicate. (C) Cell surface exposure of CD107a was determined by flow cytometry. (D) OT-1 CTLs expressing the indicated shRNA constructs were stained using antibodies against granzyme B and analyzed by flow cytometry. (E and F) RMA-s target cells loaded with 100 nM OVA peptide were mixed with shRNA-expressing (E) or wortmannin-treated (F) OT-1 CTLs as indicated. CTL–target cell conjugate formation was assessed by gating on the GFP⁺ (CTLs), PKH26⁺ (RMA-s targets) population (see Materials and methods). Degranulation and conjugate assays were performed in duplicate at an E:T ratio of 1:1. Error bars indicate SEM. (G and H) OT-1 CTLs and RMA-s targets were imaged in PDMS wells in the presence of PI to detect killing events. (G) Time-lapse montage showing the killing of two RMA-s cells by a CTL expressing shRNA against PTEN. Time is shown (hours:minutes:seconds) at the top of each image. White arrows indicate conjugate formation and killing events. (H) Top, quantification of killing time, with red lines and bars indicating mean and SEM, respectively. Bottom, killing efficiency of monogamous CTL–target cell contacts is shown in blue. In scatter plots, red lines and error bars denote the mean and SEM, respectively. P-values were calculated using the Mann-Whitney test. **, P < 0.01. Data in A–F are representative of at least two independent experiments. Data in G and H were pooled from two independent experiments.