HLA A*0201⁺ DCs were infected with the KBMA ΔactAΔinlBΔuvrABL. monocytogenes strain expressing the HLA A*0201–restricted CD8⁺ T cell epitopes of MP and MelanA/Mart–1. (A) The naive T cell population lacks the MP-tetramer–positive cells (left panels). After stimulation, MP-specific CD8⁺ T cells were generated at various frequencies (each middle panel representing a single well). Lack of staining with HIV Gag tetramers assured specificity (right panels). The numbers indicate the percentage of tetramer-positive cells. (B) Both live and KBMA recombinant L. monocytogenes primed naive Mart-1_26–35–specific T cells at similar frequencies. Percentage of CD3-gated, CD8, and tetramer double-positive cells after priming (left panels) or after the enrichment with limiting dilution assay is shown (right panels). HIV Gag tetramers were used as a negative control. The population of cells enriched for Mart-1–tetramer-positive cells was used for the functional assays shown in C–E. (C and D) T cells were stimulated with T2 cells pulsed with Mart-1_26–35 peptide, and Mart-1–tetramer-positive cells were analyzed for secreted cytokines by intracellular staining. HIV Gag-tetramers were used as specificity controls. In C, the dot plots of 1, and in D, pie charts representing the spectrum of secreted cytokines by 2 tested donors are shown. In C, the numbers indicate the percentage of cells in each dot plot quadrant. (E) The same T cell populations were tested for cytotoxicity of melanoma cell lines. Gmel and 888 melanoma cell lines express MelanA/Mart-1 protein (left panel), but only Gmel is HLA A*0201–restricted (middle panels), which correlates with the latter’s ability to be lysed by MelanA/Mart-1–specific T cells (right panel). Original magnification, ×100. Preincubation of Gmel with anti-HLA A*0201 antibodies inhibited lysis. A representative result for 1 of 2 donors is shown in A–C and E. In E, error bars represent SD of triplicate culture wells.