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. 2012 Sep 25;21(1):251–259. doi: 10.1038/mt.2012.202

Figure 3.

Figure 3

DOTAP/DOPE complexed mRNA displays intrinsic adjuvant activity. (a) Expression pattern of maturation markers of bone marrow-derived DCs (BMDCs) transfected with DOTAP gag. BMDCs were transfected with 2.5 µg DOTAP gag and the expression of major histocompatibility complex (MHC)-II, CD80, CD86, and CD40 on Gag+ and Gag-CD11c+ cells was analyzed 24 hours later by flow cytometry. (b) Transcript levels of interleukin (IL)-6, IL-1β, IFN-α and interferon (IFN)-β mRNA were determined by RT-qPCR 24 hours (IL-6 and IL-1β) or 6 hours (IFN-α and IFN- β) after transfection of BMDCs with 2.5 µg DOTAP gag. Results represent mean n-fold induction levels compared to unstimulated control cells ± SD from triplicate PCR reactions. (c and d) Nuclear translocation of IFN regulatory factors (IRF)-3 and IRF-7 was visualized by confocal microscopy at different time points after stimulation of BMDCs with 2.5 µg DOTAP gag. (c) Confocal images taken 5 minutes after incubation with DOTAP or DOTAP gag and (d) ratios of protein present in the nucleus to total protein. Results represent mean ± SD of 100 cells. (e) Transcript levels of IFN-α and IFN-β mRNA were determined by RT-qPCR 6 hours after transfection of WT, MyD88−/− or TRIF−/− DCs with 2.5 µg DOTAP gag. Results represent mean n-fold induction levels compared to unstimulated control cells ± SD from triplicate PCR reactions. TRIF, TIR-domain-containing adapter-inducing interferon-β. (f) Mice were injected i.v. with 20 µg naked gag or DOTAP gag. Serum levels of IFN-α were determined 1, 3, 6, and 24 hours later by enzyme-linked immunosorbent assay. Mean ± SEM of 4 mice per group is shown. (g) Flowcytometric analysis of DC subsets and inflammatory monocytes present in the draining lymph nodes 1 and 3 days after footpad injection of 4µg of DOTAP gag. Conventional DCs were defined as CD11chi MHCIIint, Tissue-derived DCs were CD11cint MHCIIhi and inflammatory monocytes were MHC-II+ Ly6chi CD11bhi. Mean ± SEM 6 mice per group is shown. ***P < 0.001 as compared with the corresponding inflammatory monocyte numbers in DOTAP injected control mice and mice injected with naked gag.