Fig. 1.

In vitro characterization of aAPC generated Mart-1 specific CTL. a Human peripheral blood CD8⁺ T cells were cultured with Mart-1 peptide loaded aAPC for 4 weeks to induce and expand antigen specific CTL as detailed in “Materials and methods”. After 4 weeks, the CTL were stained with anti-human CD8 antibody and with either Mart-1 tetramer (upper panel) or control CMV-pp65 tetramer (lower panel) and analyzed by flow cytometry. The percentage of peptide specific CTL within total CD8⁺ T lymphocytes is shown in the upper right corner. b Cytotoxic activity of Mart-1-specific CTL was tested against melanoma cells expressing endogenous antigen as targets using ⁵¹Cr-release assay. % specific lysis by Mart-1 specific CTL is shown for HLA-A2⁺ and HLA-A2⁻ melanoma targets. Values represent triplicates at varying effector-target ratios. The effector cell numbers represent total CD8⁺ T cells. c Mart-1 specific CTL were incubated alone or stimulated with either HLA-A2⁺ or HLA-A2⁻ melanoma cells for 6 h and the supernatants were analyzed for IL-2, IL-4, IL-5, IL-10, TNF-α and IFN-γ using cytometric bead array. Values are shown as mean ± SD of duplicate wells in the assay. The Mart-1 specific CTL for both assays were obtained after four stimulations with peptide loaded aAPC. Data is representative of three independent experiments