Research paper In vitro methods for generating CD8 + T-cell clones for immunotherapy from the naïve repertoire William Y. Ho, Hieu N. Nguyen, Matthias Wolfl, Juergen Kuball, Philip D. Greenberg ⁎ Program in Immunology, Fred Hutchinson Cancer Research Center and the Departments of Medicine and Immunology, University of Washington, P.O. Box 19024, D3-100, 1100 Fairview Ave N, Seattle, WA 98109, United States Received 2 September 2005; received in revised form 28 November 2005; accepted 30 November 2005 Available online 26 January 2006 Abstract Innovations in gene discovery and the analysis of gene expression are facilitating the identification of a growing number of antigens that could potentially be targeted for immunotherapy of tumors. Methods to reliably generate antigen-specific T-cell responses in vitro would be useful not only to screen candidate antigens for immunogenicity prior to embarking on in vivo vaccination trials, but also to generate T-cell lines or clones that could be used directly for adoptive immunotherapy approaches. Although many techniques have proven successful for expanding ex vivo effector cells from antigen-specific memory CD8 + cells that have been primed in vivo, methods to reliably generate high-avidity CTL clones from the naïve repertoire have not been well described. Various methods for the induction and expansion of antigen-specific CD8 + CTL clones from healthy A2 + donors were compared, using WT1 as a model tumor-associated antigen for which there is a low frequency of precursor T cells in naïve individuals. In contrast to the well-studied Melan-A/MART-1 (Melan-A) A2-restricted response, for which the CD8 + T-cell precursor frequency in the naïve repertoire is unusually high, successful expansion of WT1-specific CD8 + T cells appeared to be more dependent upon cell culture conditions. In particular, primary stimulation with autologous peptide-loaded monocyte-derived DC generated in 48 h (DC2d) was more effective in expanding WT1-reactive populations of CTL than stimulation with DC generated using the more standard week-long protocol (DC7d). Adding supplemental IL-7 2 to 3 days after initiation of a stimulation cycle expanded antigen-specific cells within CTL lines more efficiently than including the cytokine from the beginning of the cycle. Following primary stimulation with peptide-loaded mature DC, subsequent restimulation with peptide-loaded PBMC as the stimulators was more effective at expanding antigen-specific cells than repeated stimulation with mature DC. Using these techniques, high-avidity CTL clones specific for an A ⁎ 0201-restricted epitope of WT1 have been generated from nearly all normal A2 + donors tested. Such clones have been demonstrated to be capable of recognizing and lysing leukemic cells, and will soon be tested for therapeutic activity in clinical trials of adoptive immunotherapy in patients with relapsed leukemia after transplantation. © 2006 Elsevier B.V. All rights reserved. Keywords: WT1; CTL priming; Tumor immunity; Adoptive immunotherapy; In vitro 1. Introduction Enhancing or supplementing the body's own im- mune responses against cancer is a goal of the field of immunotherapy. Although non-specific approaches such as the administration of cytokines can generally Journal of Immunological Methods 310 (2006) 40 – 52 www.elsevier.com/locate/jim Abbreviations: HSCT, hematopoietic stem cell transplant; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cells; CTL, cytotoxic T lymphocytes; DC2d, 2-day monocyte-derived dendritic cells; DC7d, 7-day monocyte-derived dendritic cells; CRA, chromium-release assay; AICD, activation-induced cell death. ⁎ Corresponding author. Tel.: +1 206 667 4462; fax: +1 206 667 7983. E-mail address: pgreen@u.washington.edu (P.D. Greenberg). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2005.11.023