CTL targeting induces Src Kinase signaling leading to cytotoxicity and Erk phosphorylation. (A) Src mediates the cytotoxic response of targeted CTLs. H-2^d CTLs preincubated with PP2/PP3 for 1 hour were targeted with calcein-loaded 2C CD8⁺ cells (1:1 ratio). After 2 and 5 hours, 2C cell depletion was evaluated and compared with untreated CTLs. H-2^s CTLs served as the control for nonspecific activity. (B) PP2 does not affect conjugate formation. Cells were fixed, differentially stained, and imaged 1 hour into culture. Conjugation was assessed counting conjugated CTLs per total CTLs in several microscopic fields. Error bars represent SD from triplicate datasets, n > 40 in each. (C) Lck is responsible for mediating the CTL response. H-2^d CTLs were transfected with Lck siRNA. After 60 hours, CTLs were targeted with 2C CD8⁺ cells, and 2C cell depletion was compared with depletion induced by untreated CTLs or CTLs transfected with non-target siRNA. (D) Src activation-loop phosphorylation. H-2^d CTLs were targeted with 2C CD8⁺ cells for 90 minutes and evaluated by FACS for activating Src kinase phosphorylation (Tyr416). H-2^s CTLs controlled nonspecific phosphorylation. The effect of PP2 is shown. (E) Erk phosphorylation in targeted CTLs. CTLs were targeted as in panel D and evaluated for ERK phosphorylation. The effects of Erk inhibitor U0126 and Src inhibitor PP2 are shown. (F) Overlay of FACS plots representative of the Erk phosphorylation shown in panel E. Solid dark gray, targeted CTLs; black line, untargeted CTLs. Panels A-F: Data from single experiment representative of 3 experiments. Error bars represent SD from triplicate wells. **P < .01, ***P < .001.