Figure 4
Enhanced antigen presentation and CTL induction by NP-loaded DC. DC loaded with nanoparticles containing the peptides: (A) MART-127-35, or (B) gp100209-217. DC were incubated with: soluble peptide (DC+peptide); empty nanoparticles (CNP); or with nanoparticles formulated with the same peptides using 300 μg (DC+NP300) or 600 μg (DC+NP600) peptide per batch. Four days later, DC were co-cultured for 20 hours with TIL1235 (recognizing MART-127-35) or TIL1520 (recognizing gp100209-217) cells, and the antigen presentation was evaluated in an IFN-γ ELISPOT assay. (C) Cytotoxic activity of CTL induced in vitro with peptide-pulsed or NP-loaded dendritic cells: Dendritic cells were pulsed with the peptide MART-127-35 or with MART-127-35-containing NP and used as APC to induce MART-127-35-specific CTL. The experimental groups include: T2 target cells incubated with peptide-DC induced CTL (□), or with NP-DC induced CTL (■); peptide-pulsed T2 cells incubated with peptide-DC induced CTL (∓), or with NP-DC induced CTL (ℓ); HLA-A2+ melanoma cells 624 incubated with peptide-DC induced CTL (△), or with NP-DC induced CTL (▲); and HLA-A2- melanoma cells 1351 incubated with peptide-DC induced CTL (▽), or with NP-DC induced CTL (▼). The CTL lines were incubated with the target cells for 4 hours and the cytotoxicity was determined with a standard LDH-release assay (Promega). Data is representative of 3 independent experiments; bars, SD. *, significant differences (P < 0.05) between experimental and control cultures (non-pulsed DC or CNP-loaded DC).