A PR1-human leukocyte antigen-A2 tetramer can be used to isolate low-frequency cytotoxic T lymphocytes from healthy donors that selectively lyse chronic myelogenous leukemia

We previously showed (E, Clave et at, J, Immunother,, 22: 1-6, 1999; J. Mol ldrem st at, Blood, 88: 2450-2457, 1996) that PR1, a human-lymphocyte-antig en (HLA)-A2.1-restricted peptide from proteinase 3, could be used to elicit CTLs from normal individuals. These CTLs showed HLA-restricted cytotoxicit y and colony inhibition of myeloid leukemia cells that overexpress proteina se 3, In this study, we constructed a phycoerythrin-labeled PR1-HRLA-A2 tet ramer to identify PR1-specific CTLs by flow cytometry, No peripheral blood lymphocytes from three HLA-2.1(+) donors stained with the tetramer, but, af ter 20 days in culture with weekly PR1 stimulation, 2-8% became tetramer(+) , Tetramer staining identified up to 40-fold more PR1-specific CTLs than we re identified by limiting dilution analysis and correlated better with lysi s of PR1-coated T2 cells (R-2 = 0.95 versus R-2 = 0.76), Tetramer(+) CTLs w ere memory phenotype (91% CD45RO(+)), and mast (58% CD95(+)) were activated . Tetramer-sorted allogeneic CTLs produced 83% lysis of HLA-A2.1(+) chronic myelogenous leukemia (CML) blasts at an E:T ratio of 2.5:1, compared with 23% lysis by nonsorted CTLs, with no background lysis of HLA-A2.1(+) normal cells, Cytoplasmic proteinase-3 expression was one log greater in CML blas ts than in normal granulocytes, These results show that a PR1-HLA-A2 tetram er can be used to identify and select CTLs from normal donors that preferen tially lyse CML cells, which could be used for leukemia-specific adoptive i mmunotherapy.