Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers.
Immunogenic peptides of human tumor Ag have been used to generate antigen-specific CTL. However, the vast majority of these peptide-specific CTL clones are of low avidity and are peptide, but not tumor, reactive. Peptide-MHC tetramers have been shown to bind specific TCRs with sufficient affinity to be useful reagents for flow cytometry. In this paper we demonstrate that peptide-MHC tetramers can also be used to selectively identify high avidity tumor-reactive CTL and enrich, from a heterogeneous population, the subpopulation of peptide-reactive T cells that can lyse tumor targets. The melanoma proteins, MART-1 and gp100, were used to induce potentially tumor-reactive T cells, and the intensity of T cell staining by TCR binding of specific peptide-MHC tetramers was assessed. A range of fluorescence intensity was detected, and the magnitude of tetramer binding was correlated with T cell avidity. The population of peptide-reactive T cells was phenotypically similar with regard to expression of TCR and adhesion molecules, suggesting that this differential avidity for tumor cells reflected differential affinity of the TCR for its peptide-MHC ligand. Sorting, cloning, and expansion of tetramerhigh CTL from a heterogeneous population of peptide-stimulated PBMCs enabled rapid selection of high avidity tumor-reactive CTL clones, which retained their functional and tetramerhigh phenotype on re-expansion. These results demonstrate that the avidity of a T cell for its tumor target is due to the specific affinity of the TCR for its peptide-MHC ligand, that this interaction can be described using peptide-MHC tetramers and used to isolate high avidity tumor-reactive CTL.
Yee
C
C
Clinical Research Division, Fred Hutchinson Cancer Research Center, and Department of Medicine, University of Washington, Seattle 98109, USA. cyee@fhcrc.org
Savage
P A
PA
Lee
P P
PP
Davis
M M
MM
Greenberg
P D
PD
D000938:Antigen-Presenting Cells / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D002460:Cell Line; D002469:Cell Separation; D002999:Clone Cells; D003601:Cytotoxicity Tests, Immunologic; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology; D015789:HLA-A2 Antigen / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology; D009363:Neoplasm Proteins / Q000276:immunology; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology*; D013194:Staining and Labeling; D016176:T-Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry / Q000276:immunology*; D014407:Tumor Cells, Cultured; D058951:gp100 Melanoma Antigen