Spleens were isolated 2 weeks after the boost immunization, and the number of OVA-specific interferon- spot-forming CD8+ and CD4+ T cells (SFC) was determined by enzyme-linked immunosorbent spot (ELISPOT). Figure S1). We evaluated two N/P ratios that give yield to mRNA lipoplexes of similar size ( 300C400?nm) but opposite charge, namely lipoplexes at N/P1 had a negative zeta-potential of ?18 mV and N/P10 lipoplexes displayed a positive charge of +32 HBGF-4 mV (see Supplementary Figure S1a,b). Further, we addressed mRNA lipoplexes of ratio N/P1 as most suited to yield high expression levels of the delivered mRNA (see Supplementary Figure S1c) and to induce proper induction of IFN-? producing CD8+ and CD4+ T cells upon subcutaneous injection (see Supplementary Figure S1d). As a consequence, N/P1 was selected in all further experiments aimed at addressing the impact of type I IFNs on the efficacy of mRNA lipoplexes to yield T cell immunity. Previously, we have demonstrated that DOTAP-based mRNA lipoplexes elicit strong type I IFN secretion upon incubation with bone marrow derived DCs upon subcutaneous injection, we used an IFN- reporter mouse in which a firefly luciferase encoding sequence has been placed under the control of the IFN- promoter (Figure 1a).27 As type I IFN production is regulated by self-enforcing feedforward loops, heterozygous reporter mice (IFN-+/-luc) were used to allow signal amplification by early induced IFN-. Mice were injected subcutaneously with respectively DOTAP liposomes (no mRNA), unformulated mRNA or mRNA lipoplexes. bioluminescence imaging revealed a strong induction of the IFN- promoter to injection d-Atabrine dihydrochloride of naked mRNA and of mRNA lipoplexes, but not to liposomes without mRNA (Figure 1b,?cc). Strikingly, naked ovalbumin (OVA) mRNA elicited the most prominent induction of type I IFNs, clearly indicating that type I IFN induction to mRNA is inherent to the mRNA itself rather than to unique features of the d-Atabrine dihydrochloride mRNA lipoplexes. Open in a separate window Figure 1 mRNA lipoplexes induce a potent type I IFN response (a) Graphical scheme of the IFN- reporter construct. The myc-tagged luciferase gene is brought under the control of the IFN- promoter by the Cre-Lox system. (b,c) IFN-+/-luc mice were subcutaneous (s.c.) injected with 10 g of OVA mRNA, mRNA lipoplexes and liposomes. Luminescence was measured 6 hours postinjection. Data are shown as mean SD of four mice. **< 0.001. *< 0.05 (MannCWhitney test). Control = 5% glucose water; liposomes = DOTAP/DOPE lipids; mRNA lipoplexes = messenger RNA complexed to liposomes. mRNA, messenger RNA; IFN, interferon, OVA, ovalbumin; SD, standard deviation. Type I IFNs impact the magnitude and functional characteristics of the vaccine elicited CD8+ d-Atabrine dihydrochloride T cell response Depending on the context, type I IFNs have been reported to either promote or interfere with the generation of T cell responses. As a consequence, we thoroughly addressed the impact of type I IFN signaling on the magnitude and functionality of the T cell response generated by mRNA lipoplex vaccination through comparative immunization studies in wild type mice and in mice lacking he common IFN-/ receptor IFNAR1 (Ifnar?/?). First, we addressed the effects of type I IFNs on the initial priming of antigen-specific T cells. To this end, carboxyfluorescein diacetate succinimedyl ester (CFSE) labeled transgenic OVA-specific CD8+ T cells (OT-I T cells) were transferred to respectively wild type and Ifnar?/? mice, which were subsequently immunized with OVA mRNA lipoplexes. Four days postimmunization, the draining popliteal lymph nodes were dissected and OT-I T cell proliferation was analyzed by flow cytometry (Figure 2a). As shown in Figure 2b, Ifnar?/? mice showed strongly elevated OT-I proliferation when compared with wild type mice. This negative impact of type I IFNs on the magnitude of the vaccine evoked CD8+ T cell response was confirmed by quantification of vaccine elicited OVA-specific CD8+ T cells in the blood of wild type d-Atabrine dihydrochloride versus Ifnar?/? mice (Figure 2c). Five days after immunization, OVA-specific CD8+ T cells were hardly detectable in the blood of wild type mice but reached up to 3% of all CD8+ T cells in the blood of Ifnar?/? mice. No significant numbers of OVA-specific T cells were detected in response to unformulated OVA mRNA. Next, we analyzed the impact of IFNAR.