Allogeneic human histocompatibility leukocyte antigen (HLA)-matched tumor cell lines that have been made immunogenic by the transfer of genes encoding for costimulatory molecules such as CD80 are considered to be potential vaccines for the induction of systemic immune reactions in cancer patients. We used a human HLA-A2.1+ CD80-transfected breast carcinoma cell line (KS-CD80) and investigated in vitro the efficiency at which antigen (Ag)-specific responses were induced following the stimulation of allogeneic HLA-A2.1-matched T lymphocytes. The influenza matrix protein M1 was used as a model Ag. It was either endogenously expressed or exogenously loaded as a peptide (matrix protein), and the frequency of the generated specific T cells was determined. The expression of CD80 in KS cells was required for an effective activation and expansion of Ag-specific T cells. This response was augmented following the pretreatment of KS-CD80 cells with interferon-gamma and tumor necrosis factor-alpha. Interleukin-4 (IL-4), IL-7, and IL-12 further increased T-cell expansion. IL-7 was best at supporting the generation of T cells with Ag specificity. This investigation demonstrates that allogeneic CD80+ tumor cells can induce Ag-specific, HLA-restricted T lymphocytes at a high frequency. Our study supports the use of allogeneic cell lines for the induction of specific T-cell responses in tumor patients.