Cellular mechanisms of self versus non-self discrimination by nucleic acid-sensing Toll-like receptors Olivia Majer*, Bo Liu*, Brian J Woo*, Lieselotte SM Kreuk*, Erik Van Dis*, and Gregory M. Barton* *Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA Recognition of nucleic acids enables the detection of diverse pathogens by a limited number of innate immune receptors, but also exposes the host to potential autoimmunity. At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self-RNA or -DNA, respectively. Despite the structural and functional similarities between these receptors, their contribution to autoimmune diseases such as systemic lupus erythematosus (SLE) can be quite different. For example, TLR7 and TLR9 have opposing effects in mouse models of SLE; disease is exacerbated in TLR9-deficient mice but attenuated in TLR7-deficient mice. However, mechanisms of negative regulation that differentiate between TLR7 and TLR9 have not been described. Here we report a new function for the TLR trafficking chaperone Unc93b1 that specifically limits signaling of TLR7, but not TLR9, and prevents TLR7-dependent autoimmunity in mice. We find that mutations in Unc93b1 leading to enhanced TLR7 signaling also disrupt binding to Syntenin-1, a protein implicated in MVB/exosome biogenesis. Both Unc93b1 and TLR7 are detectable in exosomes, suggesting that Unc93b1-mediated recruitment of Syntenin-1 facilitates sorting of TLR7 into MVBs to terminate signaling. TLR9 is not subject to this negative regulation as it releases from Unc93b1 in endosomes. Our work establishes that Unc93b1 not only enables proper trafficking of nucleic acid-sensing TLRs but also sets the activation threshold of potentially self-reactive TLR7. Furthermore, we provide the first molecular mechanism by which endosomal TLR activation may be distinctly regulated. Understanding the differential regulation of TLRs will be key to explain the opposing roles that TLR7 and TLR9 play in certain autoimmune diseases.