Description: Purpose: Although modern cure rates for childhood acute lymphoblastic leukemia (ALL) exceed 80%, the outlook remains poor in patients with high-risk disease and those who relapse, especially when allogeneic hematopoietic stem cell transplantation is not feasible. Strategies to improve outcome and prevent relapse are therefore required. Immunotherapy with antigen-specific T cells can have antileukemic activity without the toxicities seen with intensive chemotherapy, and therefore represents an attractive strategy to improve the outcome of high-risk patients with ALL. We explored the feasibility of generating tumor antigen-specific T cells ex vivo from the peripheral blood of 50 patients with ALL [26 National Cancer Institute (NCI) high-risk and 24 standard-risk] receiving maintenance therapy. Experimental Design: Peripheral blood mononuclear cells were stimulated with autologous dendritic cells pulsed with complete peptide libraries of WT1, Survivin, MAGE-A3, and PRAME, antigens frequently expressed on ALL blasts. Results: T-cell lines were successfully expanded from all patients, despite low lymphocyte counts and irrespective of NCI risk group. Antigen-specificity was observed in more than 50% of patients after the initial stimulation and increased to more than 90% after three stimulations as assessed in IFN--enzyme-linked immunospot (ELISpot) and 51 Cr-release assays. Moreover, tumor-specific responses were observed by reduction of autologous leukemia blasts in short- and long-term coculture experiments. Conclusion: This study supports the use of immunotherapy with adoptively transferred autologous tumor antigen-specific T cells to prevent relapse and improve the prognosis of patients with high-risk ALL. Clin Cancer Res; 19(18); 5079–91. ©2013 AACR .