Activation of T cells after stimulation with DC-tumor cell hybrids. (A) T cells were stimulated with cells prepared from autologous DCs and allogeneic (Mel 108) melanoma cells. Electrofusion was performed at 50 V/cm combined with a 25 µF - 1000 V/cm pulse. Cocultures of DCs and tumor cells, as well as of DCs and melanoma cells (Mel 108) were used as control samples. After two weekly cycles of stimulation, T cells were tested for their cytotoxic activity in a 4-h ⁵¹Cr-release assay against Mel 108 cells. Values represent triplicates at E:T ratios of 1:1, 5:1 and 25:1 (left). For specificity controls, T cells were tested for their cytotoxic activity against irrelevant ROT129 tumor cells (right). (B) The frequency of IFN-gamma secreting T cells in response to fused cells was determined in an ELISPOT assay. T cells were prestimulated for two weeks in vitro with either electrofused cells, a coculture of DCs and melanoma cells, DCs alone, or melanoma (Mel 108) cells alone as described in 5A. Restimulation during the assay was performed with Mel 108 cells. Results are shown as the mean number of spots per well (left). For specificity controls, restimulation was performed with irrelevant ROT129 tumor cells (right).