High avidity melanoma-reactive cytotoxic T lymphocytes are efficiently induced from peripheral blood lymphocytes on stimulation by peptide-pulsed melanoma cells

To design an efficient procedure to expand high avidity melanoma reactive T cells and to perform immunotherapies, we compared conditions of peripheral blood lymphocyte (PEL) stimulation by Melan-A/MART-1 peptides, Avidity of induced CTLs was evaluated by measuring their lysis and cytokine secretion to peptide-pulsed transporter-associated protein-deficient cells and to mel anoma cells. In side-by-side experiments, we show that melanoma cells, eith er allogeneic or autologous, induced the growth of high avidity Melan-A-rea ctive CTLs from all donors, whereas essentially low avidity T cells were in duced by peptide-pulsed PBLs, We also show that at least two cytokines, int erleukin-6 and interleukin-2, were required to promote the growth of high a vidity CTLs, Once sorted by tetramer labeling or cloning, the specificity a nd reactivity to tumor cells of peptide-specific T cells induced by allogen eic melanoma cells were confirmed. We then describe a relatively simple and efficient procedure that allowed u s to obtain systematically high amounts (in the range of billion) of high a vidity Melan-A/MART-1-specific T cells from the PBLs of HLA-A2 melanoma pat ients and healthy donors in 3 months. Because this antigen is expressed by most melanoma tumors, this procedure should be useful for checking the effi ciency of adoptive immunotherapy of melanoma tumors and using functionally well-defined Melan-A/MART-1-specific CTLs in a large group of patients.