papillomavirus (BPV 69%) were excised by digesting BCMG-neo with Hind III and 
Not I. The large DNA fragment resulting from this digestion was gel-purified 
subcloned into the vector Bluescript SK-. This plasmid (CMV-B7, appendix II) 
contains the CMV promoter driving expression of the human B7 gene and the SV40 
promoter upstream of the neomycin phosphotransferase gene (allowing for 
selection of transformants by growth in G418). No other eukaryotic genes are 
expressed, and this plasmid is not capable of episomal replication. The entire 
970 bp of the B7 gene contained in the final construct were sequenced and 
found to match exactly the published sequence for the human B7 gene. 
Parental melanoma cell lines DM-150, DM-93, and DM- 13 were transfected with 
this plasmid using the Lipofectin reagent (Gibco/BRL) , and G4 18-resistant 
colonies were selected utilizing cloning rings to ensure clonality. Twelve 
clones derived from each parental melanoma line were expanded and analyzed by 
FACS using the monoclonal antibody BB1 which is specific for B7 . One BB1+ 
clone from each parental cell line was selected for use in this trial; the 
clones are referred to as DM150/B7-8, DM13/B7-7, and DM93/B7-3. The cell 
surface phenotype of the clones was assayed by FACS and was identical to the 
parental cell phenotype with the exception of the expression of B7 . 
DM150/B7-8, DM13/B7-7, and DM93/B7-3 cell lines have been analyzed for the 
expression of MAGE and tyrosinase genes. MAGE expression was examined by RT- 
PCR analyses of RNA extracted from the B7- trans fectants . Tyrosinase gene 
expression was determined by Northern blot analysis. DM-150 trans fectants 
express MAGE-1, 2 and 3 RNA, while DM13 and DM93 express MAGE-2 and 3 but only 
low levels of MAGE-1. Although tyrosinase was detected in all three cell 
lines, DM150 expressed only low levels. HLA-A2 TIL cell lines obtained from 
Dr. Kawakami and Dr. Rosenberg (Surgery Branch, NCI) have demonstrated lytic 
activity at low eff ector : target ratios against both parental and B7- 
transfected melanoma cell lines. 
We examined the capacity of the B7-transfectants versus the parental cell 
lines to stimulate allogeneic peripheral blood lymphocytes (PBL) of a normal 
donor in a 7-day in vitro culture. PBL were depleted of B cells and 
macrophages. In 5 separate experiments, parental tumor lines failed to 
stimulate allogeneic T cells as determined by the lack of increased expression 
of HLA-DR or CD25 on the T cells. In contrast, B7-trans f ected lines induced 
increased expression of CD25 and HLA-DR on both CD4 + and CD8+ allogeneic T 
cells. The B7-trans fectants also induced a 5-10 fold increase in T cell number 
in comparison to the parental tumor cell lines. Exogenous IL-2 could not 
rescue T cells cultured with the parental cell line, and had variable 
stimulatory effects on T cells cultured with the B7-transfectants . Only T 
Recombinant DNA Research, Volume 18 
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