Targeted CTL activation is CD8 dependent. (A) CD8 mediates Src activation loop phosphorylation. H-2^d CTLs were targeted with 2C CD8⁺ cells for 90 minutes, and Src phosphorylation was evaluated by FACS. The role of the CTL CD8 molecule in phosphorylation was determined by applying a blocking antibody specific for the 2C MHC-I α3 domain (H-2^b). (B) Contribution of the CTL CD8 molecule to killing. Depletion of targeting 2C CD8⁺ cells was determined in the presence or absence of the blocking antibody from A. In A and B nonspecific CTL activity was controlled by H-2^s CTLs. (C) Relevance of CD8 in adhesion. CTLs were targeted in the presence or absence of the blocking antibody from panel A, and adhesion was assessed by counting CTL-2C conjugates per total CTLs in several microscopic fields. (D) Polarization dependence on CD8. The effect of blocking CD8 ligation on CTL granule polarization toward conjugated 2C cells was evaluated by confocal microscopy. (E) CD8 contributes to CTL survival and proliferation. CD8⁺ and CD8⁻ 2C CTLs (H-2^b) were loaded with SIINFEKL peptide and targeted with OT-1 CD8⁺ cells. After 72 hours, survival and proliferation of the two CTL populations were evaluated by viable cell count and CFSE dilution. (F) ERK phosphorylation depends on CTL CD8 level. H-2^d CTLs were targeted with 2C CD8⁺ cells for 90 minutes. The relationship between CD8 levels and targeting-induced ERK phosphorylation was determined by FACS analysis. In panels A-F, single experiments representative of 3 experiments are shown. SD is from triplicate wells/microscopic fields. *P < .05, **P < .01, ***P < .001; n > 50 (C); n > 100 (D).