Targeted TLRLs are effective at low doses, preventing high serum IFN-α and IFN-β levels and hypothermia. (A) Mice were vaccinated intravenously with targeted NPs carrying 3 μg of OVA, 0.18 μg of R848, and 0.61 μg of poly I:C (NP-DEC205-OVA-TLRL) or soluble R848 and poly I:C at 1, 10, or 100 times the dose in combination with targeted NPs carrying 3 μg of OVA (1×, 10×, and 100× TLRL + NP-DEC205-OVA). Control mice received IV injections with PBS or the highest dose of soluble TLRLs in the absence of Ag (100× TLRL without Ag). Seven days after vaccination, Ag-dependent CTL activity was assessed by in vivo cytotoxicity assay. Representative histograms of CFSE^high target and CFSE^low control cells isolated from mice injected with NPs, 100× TLRLs without Ag, or PBS are shown. Data in bar graph represent means ± SEM for mice receiving NP-DEC205-OVA-TLRL and NP-DEC205-OVA combined with soluble TLRLs. *P < .05 for the significant difference from NP-DEC205-OVA-TLRL. (B) Mice were vaccinated as in panel A with NP-DEC205-OVA-TLRL, 100× TLRL + NP-DEC205-OVA, or PBS. Body temperature was measured 3 hours after vaccination. **P < .01 for the significant difference from PBS control. (C) Mice were vaccinated with NP-DEC205-OVA-TLRL or 100× TLRL + NP-DEC205-OVA as in panel A. Blood was drawn 3 hours after vaccination and serum cytokine levels determined. *P < .05 and **P < .01 for the significant difference from NP-DEC205-OVA-TLRL. Data in graphs represent means ± SEM from pooled data of 2 independent experiments with 2 mice per group.