title: Protein transfer of glycosyl-phosphatidylinositol (GPI)-modified murine B7-1 and B7-2 costimulators.
abstract: The feasibility of using protein transfer as a means for enhancing the immunogenicity of murine tumor cells was evaluated. Glycosyl-phosphatidylinositol (GPI)-modified variants of the murine costimulators B7-1 (CD80) and B7-2 (CD86), designated B7-1.GPI and B7-2.GPI, respectively, were immunoaffinity-purified from CHO-K1 cells transfected with glutamine synthetase amplification/expression constructs encoding each of these chimeric proteins. The proteins, once purified in detergent-depleted pseudomicelles, were exogenously incorporated into the membranes of several different murine tumor lines (EL-4, SMUCC-1, BW5147.3, P815, Ag104A, and EMT6). Successful membrane painting with the B7.GPI proteins was documented by immunofluorescence and flow cytometry, and membrane integration was verified by demonstrating that the reincorporated proteins were phosphatidylinositol-phospholipase C-sensitive, glycosyl-phosphatidylinositol-phospholipase D-resistant, and refractory to removal with dimyristylphosphatidylcholine vesicles. Significantly, B7-1.GPI and B7-2.GPI could be together copainted onto EL-4 cell surfaces with no interference observed between the two. A standard in vitro proliferation assay was used to show that both of the B7.GPI proteins retained costimulator function after membrane reincorporation. These findings further validate the therapeutic potential of protein-transferred costimulator.GPIs and pave the way for their combinatorial use in animal tumor models.
authors.affiliation: Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: E B
authors.initials: EB
authors.lastname: Brunschwig
authors.forename: J D
authors.initials: JD
authors.lastname: Fayen
authors.forename: M E
authors.initials: ME
authors.lastname: Medof
authors.forename: M L
authors.initials: ML
authors.lastname: Tykocinski
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D018122:B7-1 Antigen / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D051940:B7-2 Antigen; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000138:chemical synthesis; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; D018122:B7-1 Antigen; D051940:B7-2 Antigen; C497964:CD86 protein, human; C497965:Cd86 protein, mouse; C497966:Cd86 protein, rat; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins