Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder. Although allogeneic stem cell transplantation can induce long-term remissions, relapse rates remain high and innovative approaches are needed. Since donor lymphocyte infusions have clinical activity in JMML, T-cell-mediated immunotherapy could provide a nonredundant treatment approach to compliment current therapies. Gamma-globin, an oncofetal protein overexpressed by clonogenic JMML cells, may serve as a target of an antitumor immune response. We predicted 5 gamma-globin-derived peptides as potential human leukocyte antigen (HLA)-A2 restricted cytotoxic T lymphocyte (CTL) epitopes and showed that 4 (g031, g071, g105, and g106) bind A2 molecules in vitro. Using an artificial antigen-presenting cell (aAPC) that can process both the N- and C-termini of endogenously expressed proteins, we biochemically confirmed that g105 is naturally processed and presented by cell surface A2. Furthermore, g105-specific CD8(+) CTLs generated from A2-positive healthy donors were able to specifically cytolyze gamma-globin(+), but not gamma-globin(-) JMML cells in an A2-restricted manner. These results suggest that this aAPC-based approach enables the biochemical identification of CD8(+) T-cell epitopes that are processed and presented by intact cells, and that CTL immunotherapy of JMML could be directed against the gamma-globin-derived epitope g105.