Figure 1.

Lentivector display of “Marker of Self” CD47. (a) In HEK-CD47-GFP Producer cells the CD47-GFP (green) and anti-CD47 (red) signals are localized to the cell periphery, distinct from the nucleus (Hoechst, blue). CD47-GFP signal colocalizes with anti-CD47 immunostaining of the HEK-CD47-GFP cell membrane. Bivalent anti-CD47 localizes to cell-cell junctions. (a, schematic) Lentiviruses are enveloped viruses, where the viral capsid and genome self-assemble beneath the cell membrane and the assembled lentivirus then buds taking a piece of the cell membrane as the viral envelope (a, schematic). (b) Western blotting of HEK-CD47-GFP cells with an anti-GFP antibody confirms the presence of a 60 kDa protein, consistent in weight with a CD47-GFP fusion that is absent in control HEK cells. (c) HEK-CD47 lentiviral transfection supernatants were incubated with anti-CD47 coated coverslips, and adherent material was stained for acridine orange, which colocalized with the CD47-GFP fusion protein. The frequency of fluorescent and double positive particles was quantified (inset c). (d) CD47-Lenti binds IgG coated glass in an anti-CD47-dependent manner (n ≥ 3, P ≤ 0.05), dependence of binding kinetics on anti-CD47 is described by rate = 1/τ = a (anti-CD47)^m, m=1.8. (e) Lentiviral vectors were exposed to the TIRFM blue laser (~480 nm) for at least 2 minutes, and the CD47-GFP intensity of GFP+ particles over time was quantified, showing evidence of stepwise photobleaching (representative of n = 3). Red steps indicate quantal loss of 1–2 GFP fluorophores every ~10 seconds. (f) Flow cytometry was used to assess the fusion and binding of CD47-GFP+ lentivectors to human cells, which confirmed both that these vectors bind to the cell surface within 15 minutes of coincubation at 4*C and display the CD47-GFP fusion protein as detected by both GFP and anti-CD47 fluorescence.