Enhanced CTL induction by co-targeting of Ags and TLRLs to DCs in mice. Mice were vaccinated intravenously with various amounts OVA Ag encapsulated in targeted NPs containing TLRLs (NP-DEC205-OVA-TLRL), nontargeted NPs containing TLRLs (NP-Isotype-OVA-TLRL), targeted NPs combined with soluble TLRLs (NP-DEC205-OVA + soluble TLRL), or PBS. Note that per microgram of OVA, 0.06 μg of R848 and 0.20 μg of poly I:C were injected, either co-encapsulated with the OVA in NPs or in soluble form. (A) To determine CD8⁺ T-cell proliferation, CFSE-labeled OVA-specific OT-1 CD8⁺ T cells were injected and 3 days later lymph nodes were harvested. OT-1 T-cell proliferation was assessed by FACS analysis. Histograms show proliferation as measured by CFSE dye dilution in OT-I T cells for mice receiving NPs carrying 3 μg of OVA. Data in graph represent means ± SEM of 3 independent experiments with 2 mice per group. ***P < .001 for the significant difference from NP-DEC205-OVA-TLRL. (B) Ag-dependent cytotoxic T-cell activity of the endogenous T-cell pool was assessed by an in vivo cytotoxicity assay, determining killing of differentially labeled target and control cells pulsed with OVA peptide and a high CFSE concentration or irrelevant peptide and a low CFSE concentration, respectively. Seven days after vaccination, the CFSE^high target and CFSE^low control cells were injected and the next day, spleens were harvested and specific killing was determined by flow cytometry. Representative histograms of CFSE^high target and CFSE^low control cells from mice receiving the highest Ag dose (3 μg of OVA) or PBS are shown. Data in graph represent means ± SEM of 3 independent experiments with 2 mice per group. *P < .05 for the significant difference from NP-Isotype-OVA-TLRL and NP-DEC205-OVA + soluble TLRL