A clear link has been established between the activation of Toll-like receptors (TLRs) 7 and 9 by self nucleic acids and systemic lupus erythematosus (SLE). The localization of TLR7 and TLR9 is thought to limit activation by self-RNA and DNA, respectively. Anti-nuclear antibodies (ANA), a hallmark of SLE, can bypass this compartmentalization, but this mechanism cannot be the only factor that predicates disease because ANA are typically only produced after an initial break in tolerance. Therefore, two key questions for our understanding of SLE are how and why the initial break in tolerance occurs. In this proposal, I describe experiments that will examine these fundamental issues in light of recent breakthroughs that we’ve made regarding how TLR7 and TLR9 activation are regulated. We have determined how the trafficking chaperone Unc93b1 regulates the localization of these, and the mechanisms may explain how the activation thresholds for TLR7 and TLR9 are set. Moreover, we have discovered that the trafficking and localization of TLR7 and TLR9 are distinct, and we propose that these differences may account for the differing roles these receptors play in SLE. Our ultimate goal is to understand how alterations in TLR regulation lead to the development of SLE.