Mouse B7-H1 cDNA was kindly provided by M. Azuma , 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 .
Cell culture and transfection
Dormant leukemia cells (DA1-3b/d35, DA1-3b/d90 and DA1-3b/d365) were established as described previously . DA1-3b and DA1-3b-derived cell lines, Raji and Jurkat 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°C with 5% CO2.
Antibodies and immunodetection of cell surface molecules. (i) B7-H1/B7.1
Cells (106-aliquots) were first incubated with 5 μg/mL of Fc-receptor blocking antibody (rat anti-mouse CD16/CD32; BD Biosciences, Le Pont-De-Claix, France) for 5 min at 4°C. 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 μg/mL and 4°C for 1 h. Cell surface expression of B7-H1 or B7.1 was quantitated by flow cytometry, using an EPICS XL MLC Coulter flow cytometer. For detection of cell surface molecules B7.1 and B7-H1 after adenoviral vector cellular uptake, aliquots of DA1-3b/d365 cells in suspension (1.5 × 106) were incubated at 4°C for 90 min with Ad5FB4 vector doses of 5,000 or 10,000 physical particles per cell (vp/cell) in serum-free medium. Cells were then transferred to 37°C for 10 min to allow for vector internalization, and probed for B7-H1 and B7.1 by flow cytometry, as described above. (ii) CAR . CAR was detected by flow cytometry, using polyclonal antibody against mouse CAR (Santa Cruz Biotechnology, Cat.#sc-10313), or the corresponding control isotype (goat IgG; Santa Cruz biotechnology, Cat#sc-3887). (iii) IgFcR. Cell surface expression of immunoglobulin Fc receptors was assessed by flow cytometry, using rat anti-mouse CD16/CD32 (Fc receptor blocking antibody; BD Biosciences, Le Pont-De-Claix, France) or the corresponding control isotype, and Alexa fluor® 488-labeled goat anti-rat IgG (Invitrogen, A11006), and quantitated by flow cytometry, using an EPICS XL MLC Coulter flow cytometer.
DA1-3b cells (107) were suspended in 3 mL of ice-cold PBS containing 1% BSA and 2 mM EDTA, and incubated with monoclonal PE-labeled antibody against B7-H1 or control, irrelevant isotopic antibody, as described above. DA1-3b cells expressing B7-H1 at low and high levels, respectively, were sorted using an Epics Altra Coulter cell sorter.
B7-H1/B7.1 siRNA knockdown
DA1-3b/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 labeling
The genetic constructions of the E1-deleted adenoviral vectors Ad5 and Ad5FB4 containing the ß-gal reporter gene have been described previously [23, 24]. In the chimeric Ad5FB4 fiber, the junction between human serotype 2 fiber (F2) and bovine serotype 4 (BAdV4) fiber was situated in the shaft repeat 7 at the GKL (glycine-lysine-leucine) motif, to generate the chimeric fiber F2/BAdV4 , abbreviated FB4 in the present study. The Ad5FB4 and Ad5 vectors were amplified and purified following conventional protocols. Since Ad5FB4 had a lower tropism for epithelial cells, compared to Ad5, it was not possible to compare their infectious titers by conventional plaque assays on HEK-293 cell monolayers. Stocks of purified vectors were titrated by optical measurement of the viral DNA concentration at 260 nm, and the vector titer expressed as vp/mL. Fluorescent labeling of vector particles with carboxyfluorescein succinimidyl ester (FAM; Invitrogen, Cergy-Pontoise, France) was performed as previously described .
Ad5FB4 and Ad5 infections were carried out as previously described , except for the virus inoculum which was eliminated by low-speed centrifugation of the infected cells (800 × g, 5 min) at 24 h post-infection (pi). Cells were then resuspended in culture medium with 4% FBS and maintained for an extra 48 h for murine cells, or an extra 24 h for human cells. The ß-gal activity was determined using fluorescein-ß-D-galactopyranoside or the colorimetric X-gal staining procedure (Fisher scientific, Belgium), as previously described .
Vector-cell binding and internalization
Cell aliquots (1.5 × 106) were incubated at 4°C for 1 h in suspension with FAM-labeled Ad5 or FAM-labeled Ad5FB4 at 1011 vp/mL in serum-free medium. Cells were rinsed with PBS containing 1% BSA (PBS-BSA), and cell-bound vector particles were quantitated using flow cytometry (FACS). For internalization assays, cells and vector were incubated at 4°C for 1 h, then transferred to 37°C and further incubated at this temperature for different periods of time, ranging from 5 min to 2 h. Before FACS analysis, cell samples were incubated for 15 min at 37°C with trypsin at 0.25% in 1 mM EDTA to detach vector particles possibly sequestered at the cell surface . Cells were resuspended in PBS-BSA, and the amounts of internalized vector were quantitated by FACS analysis.
Surface plasmon resonance (SPR)
SPR analyses were carried out using a BIAcore 2000. Recombinant mouse B7-H1, B7.1 and PD-1 were covalently immobilized onto separate flow cells of a CM5 biosensor chip by amine coupling according to the manufacturer's instructions. As a reference, another flow-cell surface was activated and deactivated. Protein samples were diluted in HBS (0.01 M Hepes, pH 7.4; 0.15 M NaCl; 0.005% P20), and the binding analyses were performed at 25°C with HBS as running buffer. A flow rate of 10 μL/min was used to inject B7-H1, B7-H2, B7.1 (R&D Systems Europe, Lille, France) and 20 μL/min to inject viral proteins; all samples were run five times. For vector particles analysis, SPR experiments (BIAcore 3000) were run on a CM4 sensorship at 5 μL/min using HBS-N (GE-Healthcare) supplemented with 2 mM CaCl2. Immobilisation of both B7.1 and B7-H1 was performed by interaction of these ligands diluted at 1 μg/mL in 10 mM sodium acetate buffer pH 4.2 on EDC-NHS activated flow-cells for 10 min at room temperature. After ethanolamine deactivation, vector particles were injected (1.1010 vp in 25 μL of running buffer) and the signal from the ligand flow-cells was automatically subtracted from the background of an ethanolamine deactivated EDC-NHS flow-cell.
Human B7.1, B7-H1 and PD-1 proteins and adenoviral capsid proteins
Recombinant B7-1-Fc, B7-H1-Fc, and PD-1-Fc fusion proteins used in SPR analyses were purchased from R&D Systems. Adenovirus penton protein (penton base-linked fiber) was isolated from adenovirus-infected 293 cell lysates, according to a conventional protocol adapted to fast protein liquid chromatography [31–34].
At 24 h before transfection, cells (2 × 105-aliquots) were plated in 6-well plates and transfected with increasing amounts of B7.1-YPet-, B7-H1-YPet- or IR-YPet-expressing plasmids (10 to 500 ng/well), and constant amounts (10 ng-aliquots) of plasmid expressing B7-H1-Rluc8 or B7.1-Rluc8 fusion protein. 48 h later, cells were collected and washed twice with PBS, and aliquots were placed in 384-well plates. Coelenterazine H substrate was added at a final molarity of 5 μM, and BRET was measured immediately. To analyze the effect of the penton, cells were incubated for 5 min without or with penton protein solution at 0.33, 0.66 or 132 ng/μL, followed by Coelenterazine H addition and BRET measurement. BRET was monitored using a lumino/fluorometer (Mithras; Berthold Technologies, France), allowing for the sequential integration of luminescence with two filter settings (Rluc filter, 485 ± 10 nm; YFP filter, 530 ± 12.5 nm). The emission signal values obtained at 530 nm were divided by the emission signal values obtained at 485 nm. The BRET ratio was calculated by dividing the emission signal value obtained with coexpressed donor and acceptor by that obtained with the donor protein expressed alone. Data from at least three independent experiments were averaged, and results expressed as milliBRET (mBRET), corresponding to the BRET ratio multiplied by 1,000. Donor saturation curves were determined as previously described [35, 36].
Data were presented as the mean of triplicate experiments (m ± SEM), and were representative of the results obtained from three independent experiments that produced similar results. Statistical analyses were performed using the Mann-Whitney test.