Endosomal escape triggers the Ad-induced production of IFN-ab and IL-6, but prevents TLR9-dependent innate recognition During the course of our adenovirus preparations, we regularly found ''empty capsids'' which we separated and purified in addition to the mature virions. We tested the IFN-ab stimulating activity of these preparations in vivo and observed that they were not active (Fig. S12A ). Since empty capsids lack viral DNA and exhibit an altered protein composition [69] , the absence of an inducing viral constituent(s) from these capsids could explain their inability to provoke an IFN-ab response. Another possible explanation could be that endosomal escape is required for IFNab induction, since empty capsids cannot escape from the endosome [69] . To test the latter possibility, we infected mice with 3.6610 10 viral particles of wt Ad2 and Ad2Ts1, a viral mutant deficient in endosomal escape [5] . As shown in Fig. 10A , in contrast to the wt virus, Ad2Ts1 did not induce detectable levels of type I IFN at 4 and 8 h after infection. Furthermore, the IL-6 response was also severely reduced in these animals (Fig. S12B) . In a control experiment, mice infected with Ad2Ts1 grown at permissive temperature (32uC) and thus capable of endosomal escape, exhibited normal IFN-ab responses (Fig. 10B) . The inability of Ad2Ts1 to escape from endosomes of mDCs was confirmed by electron microscopy (Fig. 10E-H) and in vivo by the lack of Ad early E1A gene expression in the spleen of mice infected with Ad2Ts1 (Fig. 10I) . Thus, escape from the endosome is critical for the induction of IFN-ab and IL-6 by adenoviruses.