Antigen-specific T cells provide a therapy for cancer that is highly specific, self-replicating, and potentially devoid of toxicity. Ideally, tumor-specific T cells should recognize multiple epitopes on multiple antigens to prevent tumor immune escape. However the large-scale expansion of such broad-spectrum T cells has been limited by the availability of potent autologous antigen-presenting cells that can present antigens on the polymorphic array of each patient's HLA allotype. We evaluated a novel antigen-presenting complex (KATpx) in which antigens in the form of peptide libraries can be presented by autologous activated T cells, whereas costimulation is complemented in trans by an HLA-negative K562 cell line genetically modified to express CD80, CD83, CD86, and 4-1BBL (K562cs). The additional costimulation provided by K562cs significantly enhanced T-cell expansion in culture over autologous activated T cells alone while maintaining antigen specificity. We validated this antigen-presenting system by generating Epstein-Barr virus (EBV) antigen-specific T cells from healthy donors and from patients with EBV-positive malignancies including nasopharyngeal carcinoma and multiply relapsed EBV-positive lymphoma. These T cells were specific for EBNA1, LMP1, and LMP2, the viral antigens expressed in these type 2 latency EBV-associated malignancies. The KATpx system consistently activated and expanded antigen-specific T cells both from healthy donors and from 5 of 6 patients with lymphoma and 6 of 6 with nasopharyngeal carcinoma, while simplifying the process for generating APCs by eliminating the need for live virus (EBV) or viral vectors to force expression of transgenic EBV antigens. Hence, KATpx provides a robust, reliable, and scalable process to expand tumor-directed T cells for the treatment of virus-associated cancers.