Ad5 vectors capsid-displaying retro-DAF complement inhibitor significantly reduce Ad-triggered activation of NK cells, including reduced expression of interferon-γ by NK cells. Early activation of NK cells was studied by flow cytometric-based methods: 0.75 × 1011 vp/mouse of Ad5-GFP or Ad5-GFP-IX-dDAF_REO were injected intravenously into C57BL/6 mice. Splenocytes were harvested and processed at 48 hours after injection as described in “Cell staining and flow cytometry.” CD69-phycoerythrin (PE), CD3–allophycocyanin (APC)–cyanine 7 (Cy7), CD19–peridinin chlorophyll protein-cyanine 5.5, NK1.1-PECy7, CD8a-Alexa Fluor 700 antibody cocktail was used for surface staining. Interferon γ (IFNγ)–-APC was used for intracellular staining. Samples were analyzed on BD LSR II instrument and analyzed with the use of FlowJo software. The bars represent mean ± SD. Statistical analysis was completed with the use of 1-way analysis of variance with a Student-Newman-Keuls post hoc test, P < .05 was deemed a statistically significant difference. Values statistically different from those in WT_Mock-injected animals, *P < .05, **P < .001; statistically different values from WT_Ad5-GFP mice, #P < .05. n = 3 for Mock-injected animals, n = 4 for virus-injected mice.
