ORIGINAL ARTICLE Donna O. McCarthy á Nicole Glowacki á Kathleen Schell Carol A. Emler á Mark R. Albertini Antigenicity of human melanoma cells transfected to express the B7-1 co-stimulatory molecule (CD80) varies with the level of B7-1 expression Received: 14 October 1999 / Accepted: 3 December 1999 Abstract The aim of this study was to compare the an- tigenicity of human melanoma cells molecularly modi- ®ed by particle-mediated gene transfer to have transient or stable expression of the B7-1 co-stimulatory molecule (CD80). The unmodi®ed melanoma cells (mel5, m21) had no constitutive expression of B7-1, but 22%±28% of cells had transient B7-1 expression 24 h following transfection with cDNA for B7-1 (mel5-B7, m21-B7). In addition, 85%±90% of cells had stable B7-1 expression following transfection with cDNA for B7-1 and in vitro culture under selection conditions (mel5-B7neo, m21- B7neo). Allogeneic HLA-unmatched normal donor peripheral blood mononuclear cells (PBMC) secreted greater amounts of granulocyte/macrophage-colony- stimulating factor (GM-CSF) when incubated for 3 days with m21-B7neo than did PBMC incubated with m21- B7, which, in turn, secreted greater amount of GM-CSF than PBMC incubated with m21. Similarly, cell-medi- ated cytotoxicity against unmodi®ed melanoma cells by PBMC co-cultured for 5 days with the modi®ed or un- modi®ed melanoma cells was proportional to the level of B7-1 expression on the stimulating cells. This cytolytic activity had both an HLA-class-I-restricted and an HLA-class-I-unrestricted component. Following 5 days of co-culture, PBMC expression of CD28, the ligand for B7-1, was down-regulated in proportion to the level of B7-1 expression on the stimulating melanoma cells. Thus, particle-mediated gene delivery of cDNA for B7-1 into human melanoma cells increased expression of functional B7-1 and enhanced the antigenicity of the gene-modi®ed cells in proportion to their level of B7-1 expression. Key words Human melanoma á B7-1 co-stimulatory molecule á Particle-mediated gene transfer á Cytotoxicity á Flow cytometry Introduction The importance of the T cell response to autologous human melanoma has been suggested for many years [4, 11]. Unfortunately, this response is clearly not eec- tive in patients who develop metastatic disease, but it may be possible to be enhanced by appropriate inter- vention. Treatment approaches that augment expression of MHC antigens on melanoma cells may make these modi®ed melanoma cells better stimulators of T cell immunity [13]. However, T cell activation also requires a second co-stimulatory signal from the antigen-present- ing cell [15]. The best-characterized co-stimulatory molecule is B7-1 (CD80), the T cell ligand of which is the CD28 antigen [14]. The importance of B7-1 in the T cell response to melanoma has been demonstrated in murine melanoma models, in which rejection of murine mela- noma cells was mediated by CD8 + T cells following stimulation by melanoma cells stably transfected with B7-1 cDNA [5, 17]. Systemic immunity against non- transfected melanoma cells was induced, as subsequent tumor challenge with non-transfected melanoma cells did not result in tumor formation [17]. It has been sug- gested that the level of B7-1 expression on the stimu- lating murine melanoma cells may be an important factor in stimulating an anti-melanoma immune re- sponse [6]. There is evidence that human melanoma cells lines stably transfected with B7-1 cDNA are better stimula- tors of a T cell response against melanoma than are the unmodi®ed parental cells [7, 10, 12, 16, 19]. This en- hanced antigenicity has been shown to be partially blocked by addition of anti-B7-1 (CTLA-4) antibody to the cultures of PBMC and tumor cells, indicating the need for the B7-CD28 interaction in activation of the T cell response to the modi®ed tumor cell [7]. In addition, Cancer Immunol Immunother (2000) 49:85±93 Ó Springer-Verlag 2000 D. O. McCarthy á N. Glowacki á K. Schell á M. R. Albertini (&) K4/414 Clinical Science Center, University of Wisconsin-Madison, 600 Highland Avenue, Madison, WI 53792, USA Tel.: +1608-263-0117 C. A. Emler Agracetus Inc., Middleton, Wisconsin 53562, USA