Antigens implicated in the graft-versus-leukemia (GVL) effect in chronic myeloid leukemia (CML) include WT1, PR1 and BCR-ABL. To detect very low frequencies of these antigen-specific CD8 + T cells, we used quantitative PCR (qPCR) to measure interferon-gamma (IFN-) mRNA production by peptide-pulsed CD8 + T cells from HLAA* 0201 + healthy volunteers, and CML patients before and after allogeneic stem cell transplantation (SCT). Parallel assays using CMV pp65 tetramers demonstrated the IFN-copy number to be linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8 + T cell/100,000 CD8 + T cells. Responses to WT1 and + T cells comprised central memory +CD27+CD57-) and effector memory (CD45RO - CD27 - CD57 + ) T cells. In + T cells derive from memory T cells and occur at low PR1 but not BCR-ABL were detected in 10/18 healthy donors. Responses to WT1, PR or BCR-ABL were observed in 9/14 CML patients pre-SCT and 5/6 post SCT, often to multiple epitopes. Responses were higher in CML patients compared to healthy donors and highest after SCT. These antigen specific CD8 (CD45RO conclusion, leukemia-reactive CD8 frequencies in healthy individuals and at higher frequencies in CML patients. The increased response in patients post-SCT suggests a quantitative explanation for the greater effect of allogeneic SCT.