Time and the expression level of B7.1 and B7-H1 molecules at their surface. Results from

Time and the expression level of B7.1 and B7-H1 molecules at their surface. Results from in vitro and in vivo experiments suggested that B7.1 and B7-H1 molecules played different roles in Ad5FB4-mediated transduction of murine dormant leukemia cells, with B7.1 involved in cell attachment of Ad5FB4, and B7-H1 in its cellular uptake. Our data also suggested that the interaction between B7.1 and Ad5FB4 was mediated by the penton capsomeres (or penton base-linked fibers) of the vector capsid. In situ BRET analysis showed that B7.1 interacted with B7-H1 to form heterodimers at the cell surface, and that Ad5FB4 penton capsomeres interfered negatively with the formation of these complexes. Our finding that tumor cell surface molecules of the B7 family implicated in immunoevasion mechanisms were recognised by the adenoviral vector Ad5FB4 offered novel opportunities for cancer therapy, using intrinsically B7-targeted Ad5FB4 vectors for therapeutic gene transfer. Alternatively, Ad5FB4 penton capsomeres, via their negative interference with the B7-H1/B7.1 heterodimer formation, might be used as therapeutic agents to decrease the amounts of these complexes at the tumor cell surface, and hence lower their capacity to resist to antitumor T-cell responses.MethodsPlasmidsMouse B7-H1 cDNA was kindly provided by M. Azuma [28], and pSelect-B7.1 was purchased from Invivogen (San Diego, CA). For fusion constructs, the stop codons of the murine coding sequences of B7-H1 and B7.1 were removed from the plasmids. PCR products were cloned in phase with either Rluc8 or YPet into the pcDNA3 vector [29].Cell culture and transfectionDormant leukemia cells (DA1-3b/d35, DA1-3b/d90 and DA1-3b/d365) were established as described previously [12]. DA1-3b and DA1-3b-derived cell lines, Raji andGrellier et al. Molecular Cancer 2011, 10:105 http://www.molecular-cancer.com/content/10/1/Page 3 ofJurkat cells were cultured in RPMI-1640 medium with 10 fetal bovine serum (FBS), 1 non-essential amino acids (NEAA), and 1 mM sodium pyruvate. Epithelial cells HEK-293 and A549 were cultured in DMEM medium supplemented with 10 FBS and 1 NEAA. HeLa cells were grown in DMEM supplemented with 10 FBS, 4.5 g/L glucose, and 1 mM glutamine. Transfections for establishing transient expression were performed using Fugene6 HD (Roche, Meylan, France). Cells were maintained in a humidified incubator at 37 with 5 CO2.Antibodies and immunodetection of cell surface molecules. (i) B7-H1/B7.B7-H1/B7.1 siRNA knockdownDA1-3b/d365 cells were transfected by electroporation with siRNA (Thermo Fisher, Dharmacon technology, Belgium) using 3.3 nmol/cell of siRNA against murine B7-H1 (5′-CACAAUUCgAggAgACgUAUU-3′) or B7.1 (5′-gAAUUACUggCAUCAAUA-3′). Negative control siRNA consisted of scramble sequences. B7-H1 or B7.1 expression was immunodetected as described above, and the silencing effect determined at 24, 48, 96 and 144 h after electroporation.Adenovirus vector amplification and labelingCells (106-aliquots) were first incubated with 5 g/mL of Fc-receptor blocking antibody (rat anti-mouse CD16/CD32; BD Biosciences, Le Pont-De-Claix, France) for 5 min at 4 . Cells were then reacted with monoclonal antibodies against PE-labeled mouse/ human B7-H1, FITC-labeled mouse/human B7.1 (eBioscience, San Diego, CA; http://www.eBioscience. com), and the corresponding control isotype, at 40 g/ mL and 4 for 1 h. Cell surface expression of B7-H1 or B7.1 was quantitated by flow cytometry, using an EPICS PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28300835 XL MLC MonocrotalineMedChemExpress Crotaline Coulter.