Binding, uptake, and Ag presentation of NPs by mouse DCs. (A) Mouse BMDCs were incubated with NPs harboring OVA and TLRLs for 1 hour on ice. Particles carried no (NP-OVA-TLRL), rat anti–mouse DEC205 (NP-DEC205-OVA-TLRL), or control (NP-Isotype-OVA-TLRL) F(ab′)2 fragments. NP binding was analyzed by staining with fluorescently labeled secondary Abs recognizing rat IgG and FACS analyses. (B-C) CD8⁺ T-cell proliferation and activation was determined after incubation of mouse BMDCs and OT-1 T cells with various concentrations of OVA, either in soluble form targeted or nontargeted within NPs harboring both OVA and TLRLs (NP-DEC205-OVA-TLRL and NP-Isotype-OVA-TLRL, respectively) or targeted or nontargeted within NPs harboring only OVA (NP-DEC205-OVA and NP-Isotype-OVA, respectively). Note that NPs carrying TLRLs contain 0.06 ng of R848 and 0.2 ng of poly I:C per nanogram of OVA. After 3 days, CD8⁺ T-cell proliferation was measured by tritium thymidine incorporation assay (B) and CD8⁺ T-cell activation was determined by measuring IFN-γ levels in the supernatant (C). Two independent experiments were performed, yielding similar results. Data represent mean values of 1 experiment performed in duplicate ± SEM.