Phenotypic characterization of tetramer-positive CD3⁺CD8⁺ T cells. Analysis of PBMCs was performed by 6-color flow cytometry in patients with CML patients before and after SCT and 2 healthy donors whose CD8⁺ T-cell frequencies to PR1 and WT1 were high enough to be visualized by tetramer staining. CD45RO, CD27, and CD57 phenotype of CD3/CD8-gated tetramer-positive lymphocytes was analyzed. Cells were gated on CD3⁺ lymphocytes, and the number in the top subpanel represents the percentage of CD3⁺ T cells that express CD8. Most of the CMV tetramer CD8⁺ T cells were CD45RO^–CD27^–CD57⁺ (blue), consistent with an effector memory phenotype (A). PR1 and WT1 tetramer-positive CD3⁺CD8⁺ T cells in 2 patients with CML before SCT and 2 patients after allogeneic SCT showed a mixture of CD45RO⁺CD27⁺CD57^– (red) and CD45RO^–CD27^–CD57⁺ (blue), with most of the tetramer-gated cells showing the former phenotype (B-C). The same held true when PR1 and WT1 tetramer-positive T cells in 2 healthy donors were studied. Representative data are presented here. (A) CMV-positive control (UPN 283). (B) Patient with CML before SCT PR1 (UPN 210). (C) Patient with CML after SCT PR1 (UPN 319). (D) Patient with CML after SCT WT1 (UPN 319). (E) Healthy donor (UPN 12) WT1. (F) Healthy donor (UPN 2) PR1.