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Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

Original publication




Journal article


J Exp Med

Publication Date





1641 - 1650


Adult, Aged, Amino Acid Sequence, Antigens, Neoplasm, Female, Histocompatibility Antigens Class I, Humans, Immunotherapy, Adoptive, Indicators and Reagents, Lymphatic Metastasis, Lymphocytes, Tumor-Infiltrating, MART-1 Antigen, Male, Melanoma, Middle Aged, Monophenol Monooxygenase, Neoplasm Proteins, Peptide Fragments, Protein Conformation, Staining and Labeling, T-Lymphocytes, Cytotoxic