Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones

The induction of an in vitro T cell response against tumour-associated anti gens with subsequent expansion of the individual cytotoxic T lymphocyte (CT L) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART -1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The huma n melanoma cell subline FM3.P was cloned and the immunological properties o f individual clones were compared. Melanoma cell clone FM3.29, having a hig h level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, wa s used for the establishment of a CTL line that was subsequently cloned. Fo r optimization of the conditions of growth of established CTL clones, a par ticular melanoma subline FM3.D/40 was selected for supporting the prolifera tion of CTL clones. The majority of the established CTL clones recognized t he melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. E pitope analysis indicated that two different epitopes derived from gp100 (1 54-162 and 280-288) and a single epitope from MART-1/Melan-A (27-35) were r ecognized by these CTL clones. The gp 100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/ Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstra te that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL c lones even against such an inherently "difficult" differentiation antigen a s gp100.