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Tumor antigens that might serve as potential targets for adoptive T cell therapy have been defined in different tumor entities. In order to generate conditions to induce primary T cell responses against different HLA-A+0201-restricted melanoma-associated peptides and to allow further expansion of peptide-specific T cells for adoptive transfer, CD8+ purified T cells from healthy donors and melanoma patients were stimulated in vitro with autologous dendritic cells (DC) pulsed either with the natural (EAAGIGILTV) or modified (ELAO1G1LTV) Melan-A (aa26-36) peptide. After 3-4 weekly stimulation cycles, we were able to generate a strong peptide-specific cytotoxic T lymphocyte (CTL) response in vitro. CTL stimulated with the modified Melan-A peptide revealed a frequency of up to 81% CD8+/Melan-A+ T cells measured by MHC tetramer staining. Additional antigenindependent expansion with anti-CD3/anti-CD28 monoclonal antibodies together with IL-2 gave rise up to 3,000-fold expansion of CD8+ CTL that maintained Melan-A specificity and were able to efficiently lyse Melan-A-expressing melanoma cells. In order to enrich antigen-specific T cells in vitro we used the recently developed cell surface affinity matrix technology for analysis and sorting of live cells according to secreted cytokines. Antigen-specific CTL were detected based on stimulation-induced IFN-gamma and IL-4 secretion and further enriched using magnetic beads. The resulting IFN-gammaand IL-4-secreting CTL lines demonstrated a significantly higher cytotoxic activity against Melan-A expressing target cells than unpurified CTL. Direct staining of enriched CTL with HLA-A2-Melan-A tetramers revealed a good correlation between the results obtained from the cell surface affinity matrix technology and those obtained from tetrameric complexes. Altogether, our study demonstrates that we have developed an efficient method for generating large numbers of peptide-specific T cells in vitro that may be used for adoptive T cell transfer in tumor immunotherapy.


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