In vivo cytotoxicity assays were performed by injecting 50:50 mixtures of CFSE^hi SIINFEKL-pulsed B6CBAF1 splenocytes (target cells) and CFSE^lo unpulsed splenocytes (control cells). The ratio (r) was calculated as the number of CFSE^hi to CFSE^lo cells remaining 16 hours later; low ratios imply high levels of SIINFEKL-specific killing. (A) Low levels of SIINFEKL-specific cytotoxicity in Act-mOVA–mated mice receiving OT-I cells. E10.5–E11.5 pregnant or virgin females injected with OT-I cells were treated as indicated, then assayed 6 days later. Data are representative of 2 independent experiments, encompassing n = 4 Act-mOVA–mated mice. (B) Absent SIINFEKL-specific cytotoxicity in Act-mOVA–mated mice with unmanipulated T cell repertoires, unless treated with anti-CD40 antibodies and poly(I:C). E10.5–E13.5 pregnant or virgin females were treated as indicated, then assayed 6 days later. Data are from of 1 of 6 independent experiments. Each histogram shows representative data for a treatment group, except in the case of Act-mOVA–mated females treated with anti-CD40 antibodies and poly(I:C), for which results for the mouse with the highest cytotoxicity are shown. The total number of mice in each group over all experiments is indicated. (C) CTL responses in Act-mOVA–mated females treated with CD40 antibodies and poly(I:C). The percentage of induced SIINFEKL-specific killing was calculated by normalizing the CFSE^hi to CFSE^lo ratio for individual anti-CD40/poly(I:C)-treated mice to the mean value of this ratio for their respective nonadjuvant-treated group. Act-mOVA–mated mice were assayed in 3 independent experiments; the number of Act-mOVA concepti/total concepti for each of these mice is shown.