BACKGROUND: The need for new therapeutic regimens for cancer has led to the identification of tumor antigens in several tumors. Yet, potent antigen-presenting cells (APC) are being identified and characterized in order to find those most able to induce T-cell-mediated immunity. To facilitate the generation of cytotoxic T-lymphocytes (CTL) specific for known antigens, various sources of antigen-presenting cells (APC) were compared for their efficiency. MATERIALS AND METHODS: Allogeneic dendritic cells (DC), T2 cells and two Epstein Barr virus-transformed B cell lines, JY and TK-6, were used as antigen-presenting cells (APC). T-cells from normal donors were stimulated with solid-phase anti-CD3 antibody and reactivated with peptide-pulsed APC three times at weekly intervals. To minimize any potential alloreactivity, eighteen random combinations of JY, TK6 and T2 were used for three peptide stimulations. The APC were pulsed with the HLA-A2-binding HER2/neu-derived tumor antigen p654-662 (GP2). RESULTS: In addition to either T2 or DC alone, most combinations of APC induced significant recognition of GP-2-pulsed target cells to a varying degree. The stimulation index varied from 0.5 to 8.1 using combinations of APC. The level of peptide recognition correlated with recognition of HLA-A2+, HER2/neu+ pancreatic cancer cells; in contrast, HLA-A2-negative pancreatic cancer cells were not recognized. The recognition of tumor cells was HLA-restricted as evidenced by blocking studies with anti-HLA class I and anti-HLA-A2 antibodies. CONCLUSION: Tumor peptide-specific CTL can be generated using allogeneic B cell lines, which may provide a useful alternative to T2 or dendritic cells.