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Purpose Various tumor antigens can be loaded onto dendritic cells (DCs) to induce a potent cytotoxic T lymphocyte (CTL) response in DC-based immunotherapy against breast cancer. However, in the clinical setting, obtaining a sufficient number of autologous tumor cells as a source of tumor antigens is a laborious process. We therefore investigated the feasibility of immunotherapy using breast-cancer-specific CTLs generated in vitro by use of alpha-type 1 polarized DCs (αDC1s) loaded with ultraviolet B-irradiated cells of the breast cancer cell line MCF-7. Materials and Methods αDC1s were induced by loading allogeneic tumor antigen generated from the MCF-7 UVBirradiated breast cancer cell line. Antigen-pulsed αDC1s were evaluated by morphological and functional assays, and the breast-cancer-specific CTL response was analyzed by cytotoxic assay. Results The αDC1s significantly increased the expression of several molecules related to DC maturation without differences according to whether the αDC1s were loaded with tumor antigens. The αDC1s showed a high production of interleukin-12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumor antigens. Breast-cancer-specific CTLs against autologous breast cancer cells were successfully induced by αDC1s loaded with apoptotic MCF-7 cells. Conclusion Autologous DCs loaded with an allogeneic breast cancer cell line can generate potent breast-cancer-specific CTL responses. This may be a practical method for cellular immunotherapy in patients with breast cancer.