Abstract
Somatic cell hybrids of HLA-A2(+) EBV-transformed B- or dendritic cells (DC) and allogeneic HLA-A2(-) melanoma cell line Me15 were obtained by in vitro electrofusion using an electroporator. Before fusion, melanoma cells were stably transfected with green fluorescent marker protein (GFP) and neomycin resistance gene (neo(+)). Stably growing hybrid antigen-presenting cells (HAPC) expressing HLA-DR and HLA-A2 (or HLA-A30/31), and melanoma-associated antigens (MART-1, gp100) were selected by a double strategy of immunomagnetic MACS and neomycin selection. Fusion efficiency ranged between 3% and 18% (mean: 8.0+/-4.7%) as defined by simultaneous GFP and HLA-A2 detection. Expression of melanoma-associated antigens (MART-1, gp100) in hybrid cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). HLA-restricted antigen-specific presentation of melanoma antigens was demonstrated by killing of semi-allogenic HAPC by HLA-A2-restricted MART-1 or gp100-specific cytotoxic T lymphocyte (CTL) clones. HLA restriction and antigen specificity were confirmed by inhibition of specific cytotoxicity by anti-HLA antibodies and cold target inhibition. During long-term (42-70 days) neomycin selection of HAPC, a drastic loss of antigen-presenting cell (APC)-derived determinants (e.g. HLA-DR, HLA-A2) was observed which, however, could be "reversed" by repeated MACSorting (days 10, 21 and 49). Our method allows the generation of semi-allogenic HAPC that constitutively proliferate in vitro. This opens the possibility of establishing a number of tumor-APC hybrids expressing defined HLA haplotypes and tumor antigens, of investigating their specific properties (e.g. antigen processing), and testing their diagnostic or therapeutic potential.