Ex vivo analysis of peptide-specific CD8 T cells before and after vaccination. Peripheral blood mononuclear cells were stained ex vivo with fluorescent HLA-A2/Melan-A tetramers and monoclonal antibodies, and analyzed by flow cytometry. The dot plots show CD8 gated T cells (100% corresponds to the total number of CD8+ lymphocytes). The percentages of tetramer positive cells that are CD28 negative (upper left quadrants) or CD28 positive (upper right quadrants) are indicated. In healthy donors, all tetramer positive (Melan-A-specific) cells are CD28 positive. This was also the case for patient LAU 392 who did not respond to vaccination since Melan-A-specific cells were not more frequent following vaccination. In contrast, patient LAU 567 responded by activation of Melan-A-specific T cells, which were expanded 3.8-fold after vaccination (0.12±0.05% before vaccination, 0.56±0.08% after vaccination).