Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dendritic cell/melanoma cell hybridomas

Due to their central role in controlling immunity, dendritic cells are logi cal targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma ce lls, both of which were derived from patients with metastatic malignant mel anoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interle ukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activ ity and released Th-1-associated, but not Th-2-associated, cytokines. Furth ermore they showed effective anti-melanoma activity, resulting in death of 70 +/- 9% of autologous melanoma cells. After depletion of CD4(+) cells fro m the mixed population of primed T lymphocytes, the remaining CD8(+) cells were able to kill 63 +/- 8% of autologous melanoma cells. Following depleti on of CD8+ cells, however, the cytotoxic capacity of the remaining T lympho cytes caused death in only 32 +/- 6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytot oxic activity of primed T cells. These findings strongly suggest that hybri domas deliver melanoma-associated antigens via MHC class I molecules to T l ymphocytes, resulting in the generation of CD8(+) cytotoxic T lymphocytes w ith effective anti-melanoma activity in vitro. The data may serve as a basi s for the use of hybridomas in the immunotherapy of malignant melanoma in v ivo.