Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Diverse expansion potential and heterogeneous avidity in tumor associated antigen-specific T lymphocytes from primary melanoma patients

B Palermo PhD 1, R Campanelli DSc 1, S Garbelli DSc 1, S Mantovani DSc 1, AM Manganoni MD 2, G Carella PhD 2, GA Da Prada MD 1, G Robustelli-della-Cuna MD 1, A Necker PhD 3, E Lantelme PhD 4, C Giachino PhD 1,4

1 IRCCS Maugeri Foundation Pavia, I, 2 Spedali Civili Brescia, I, 3 Immunotech Marseille, F, 4 Department of Clinical and Biological Sciences, University of Turin Orbassano, I

AIMS While tumor-associated-antigen (TAA)-specific CTLs have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We sought to enumerate and functionally characterize the circulating TAA-specific T lymphocytes from primary melanoma patients. METHODS Fluorescent HLA-A2 tetramers were synthesized and complexed with peptides derived from four melanocyte differentiation antigens: Melan-A/MART1, tyrosinase, gp100 and MAGE-3. Six patients with a diagnosis of primary cutaneous melanoma were included in this study. RESULTS In five out of six cases high numbers of CD8+/tetramer+ cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan-A/MART1 and tyrosinase) were contemporarily present. The TAA-specific cells could represent as much as 1/220 T lymphocytes in the circulating CD8+ population. When tetramers were used to monitor the in vitro expansion of TAA-specific CTL precursors upon antigen-specific stimulation, a diverse expansion potential was evidenced in CTLs from the different donors and, more strikingly, in CTLs specific for the different TAAs. Melan-A/MART1-specific CTL clones derived from two patients exhibited a broad range of avidity in their peptide/MHC recognition. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall avidity. CONCLUSIONS We identified frequent circulating CTLs directed against four different TAAs in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA-A2 tetramers. These CTLs were characterized by diverse expansion potential and heterogeneous avidity in antigen recognition.

KEY WORDS: HLA tetramer, tumor antigen.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on prognostic markers.