Ex vivo assays for T cells

Mice were immunized twice at 2-week intervals with DCs pulsed with chaperone vaccines s.c. or with the chitosan-siRNA complex plus hsp110-gp100 vaccine. For intracellular IFN-γ staining, cells were treated with brefeldin A (5 μg/mL, BD GolgiPlug; BD Biosciences) for 3 h at 37 °C. In some cases, cells were stimulated with phorbol-12-myristate-13-acetate (PMA, 50 ng/mL) plus ionomycin (1 μg/mL). Cells were stained with anti-CD8 or CD90.1 antibodies, followed by fixation, permeabilization and staining with anti-IFN-γ antibodies (BD Biosciences). Cells were analyzed by gating on CD8⁺ or CD8⁺CD90.1⁺ T cells. To examine the frequency of IFN-γ-producing T cells, cells were stimulated with gp100_25–33 peptide and subjected to ELISPOT analysis (25). An in vivo CTL assay was performed to determine the cytolytic activity of gp100-specific CTLs as we previously described (17).