Donor T cells responding to minor histocompatibility (H) antigens on host hematopoietic cells play a role in the graft versus leukemic effect observed in HLA identical stem cell transplantation (SCT). CD8+ cytotoxic T lymphocyte (CTL) clones recognizing minor H antigens eliminate myeloid leukemia in NOD SCID mice, and the adoptive transfer into patients of CTL clones specific for minor H antigens displayed on host leukemic cells to treat leukemic relapse after SCT is under investigation. Currently, strategies for ex vivo isolation and expansion of minor H antigen specific CD8+ CTL clones for immunotherapy rely on isolating donor T cells from the recipient after SCT to allow priming to recipient minor H antigens in vivo. This approach is not uniformly successful in part due to the intense immunosuppression that is administered post transplant. We investigated whether CD8+ CTL specific for minor H antigens can be isolated directly from donor blood by primary in vitro sensitization. We hypothesized that CD8+ T cells capable of recognizing minor H antigens would be contained predominantly in the naïve subset of cells, given the greater diversity of the T cell receptor repertoire in this population. Advances in immunology have provided insights into the requirements for the generation of a primary immune response from naïve T cells, including the critical role of dendritic cells (DC) as antigen presenting cells (APC), and the cytokines that promote differentiation and expansion of CD8 T cells. Mature CD14 derived DCs were generated from peripheral blood mononuclear cells (PBMC) of HLA identical sibling pairs and used as APC. Purified populations of naïve (>95% CD45RO−, CD62L+) or memory (>95% CD45RO+) CD8+ T cells were prepared from PBMC using magnetic bead cell separation, stimulated with DC from the respective sibling and cultured for 14 days in media containing IL12, 7 and 15. CD8+ CTL lines that lysed EBV-LCL derived from the donor used as the source of DC, but not autologous EBV-LCL, were isolated from 8/8 volunteer HLA identical sibling pairs and 2 HLA identical patient donor pairs undergoing SCT. In 7/7 pairs in which cloning by limiting dilution was performed, minor H antigen specific T cell clones were isolated from the T cell lines and could be expanded efficiently in vitro with retention of specific cytotoxicity. Cytotoxic minor H antigen specific CD8 clones were reliably generated from naive CD8 cells, but not from the memory CD8 population. Cytotoxic T cell clones recognizing minor H antigens expressed predominantly on hematopoeitic tissues have been identified amongst the derived clones. The panel of clones from each donor frequently recognized two or more different specificities presented by the same or different HLA class I molecules. Experiments are in progress to determine the recognition of primary leukemic cells and the elimination of leukemic stem cells in NOD SCID mice. Our results show that human CD8+ minor H antigen specific CTL clones can be reproducibly generated by primary in vitro stimulation of naïve T cells with DC from HLA identical siblings. This is a promising approach to generating CTL prior to transplant, that could be used for adoptive transfer into patients for the treatment of overt post transplant leukemic relapse, for preemptive therapy of molecular relapse, and for graft engineering. Furthermore, this technique provides a platform for the discovery of new minor H antigens and, potentially, leukemia specific antigens.