Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers.

Publication Type:

Journal Article


Journal of immunology (Baltimore, Md. : 1950), Volume 162, Issue 4, p.2227-34 (1999)


Antigen-Presenting Cells, Antigens, Neoplasm, Cell Line, Cell Separation, Clone Cells, Cytotoxicity Tests, Immunologic, Epitopes, T-Lymphocyte, gp100 Melanoma Antigen, HLA-A2 Antigen, Humans, MART-1 Antigen, MELANOMA, Membrane Glycoproteins, Neoplasm Proteins, Oligopeptides, Staining and Labeling, T-Lymphocyte Subsets, T-Lymphocytes, Cytotoxic, Tumor Cells, Cultured


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.