In vivo priming using DC-based vaccination. (A) MART-1-specific multimer staining versus CD8 staining of in vivo-primed human T cells. Differently matured DC of an HLA-A2+ donor, electroporated with MART-1 ivt- RNA, were used for vaccination. Staining was performed one day after spleen isolation. (B) Killing capacity (% specific lysis) of human lymphocyte populations, shown in A, tested individually in a chromium-release assay 24 h after isolation (ex vivo). 2 × 103 mel624.38 target cells were incubated with varying numbers of effector cells. Specific lysis of non-immunized mice is shown as open circles while filled circles represent specific lysis of lymphocytes from immunized mice. THP-1 cells (HLA-A2+, MART-1-) cells were not recognized (data not shown). (C) Killing capacity (% specific lysis) human lymphocyte populations cultured in vitro after isolation from individual mice and tested separately in a chromium-release assay at day 7 after isolation. Responses of non-immunized mice are shown as open circles while filled circles represent responses of immunized mice. Individual mice shown in (A) are indicated with * and the analyzed lymphocyte populations correspond to the populations tested in B. THP-1 (HLA-A2+, MART-1-) and K562 (HLA-A2-, MART-1-) cells were not recognized (data not shown). (D) Amount of secreted IFN-γ by human lymphocyte populations after stimulation with mel624.38 cells following in vitro culture for 7 days. Analysed cell populations correspond to those tested in B and C and were analysed on the same day as C. Values are given as means of four mice with SEM and PMA/I stimulation served as the positive control. (E) Specific lysis of melA375 (HLA-A2+, MART-1-) and mel624.38 (HLA-A2+, MART-1+) melanoma cell lines as target cells. Shown are means (SEM) of four mice vaccinated with MART-1-expressing DC populations using the 4-wk protocol.