Dendritic cells are professional antigen-presenting cells (APC) with the unique capacity to initiate primary immune responses. Recently, it was demonstrated that transfection of DC with RNA coding for specific antigens can elicit an effective T cell response in vitro and in vivo.4-1BB (CD137) is a member of a family of receptors found on the surface of cells of the immune system. It plays an important role in the costimulation of T-cells: it enhances T-cell proliferation, cytokine production and cytotoxic effector functions and suppresses the induction of apoptosis. It is predominantly expressed on activated T-cells, and its ligand 4-1BBL is expressed on activated APC including DC.In the present study we analyzed the effects of 4-1BBL on the stimulatory capacity of DC. Monocyte derived DC were electroporated with in vitro transcribed RNA encoding the tumor-associated antigen Her-2/neu. Additionally these cells were cotransfected with in vitro transcribed RNA coding for the human 4-1BBL with the aim to enhance the costimulatory functions of the cells and therefore the induction of Her-2/neu specific cytotoxic T-cells (CTL).Standard 51Cr-release assays performed after two restimulations showed specific cytotoxic activity of the induced CTL against autologous DC transfected with Her-2/neu RNA and against the HLA-matched allogeneic Her-2/neu+/HLA-A2+ renal cell carcinoma cell line A498 whereas DC transfected with irrelevant RNA as well as Her-2/neu-/HLA-A2+ CROFT cells and the Her-2/neu+/HLA-A2- cell line SK-OV-3 were spared. There was no lysis of K562 cells, thus excluding a NK-cell mediated killing. In this experimental setting the specific lysis rate of target cells could nearly be doubled by the coadministration of Her-2/neu RNA and an equal amount of 4-1BBL RNA (50% vs 90% specific lysis at effector to target ratio of 30:1). Interestingly, overexpression of 4-1BBL had no effect on the stimulation of allogeneic T cells by electroporated DC in a mixed lymphocyte reaction, indicating that 4-1BBL increases the induction of antigen specific T cell responses rather than the unspecific stimulation of bulk T lymphocytes.Furthermore, FACS analysis of DC transfected with 4-1BBL plasmid DNA or in vitro transcribed 4-1BBL RNA showed higher expression of the costimulatory molecules CD80, CD86, CD40 and the T-cell adhesion molecule CD54 as compared to the controls indicating that 4-1BBL signaling can mediate activation of DC and thus increase their stimulatory capacity. In the context of immunotherapies for cancer patients the findings of our study could contribute to optimise the in vitro manipulation of DC for the induction of antigen-specific CTL responses.