PROJECT SUMMARY/ABSTRACT Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by sterile inflammation and high titer antibodies against self-antigens. Although the etiology of SLE is still unknown, cumulative evidence suggest that interferons (IFNs) might play a critical role in disease pathogenesis, likely with type I IFN (IFN-I) as the dominant mediator. While different mechanisms may dysregulate the production of IFN-I in SLE, studies for the last 20 years have been focused on immune complexes (ICs) containing genomic DNA and RNA–protein (RNP) complexes as critical players in TLR activation and the induction of IFN-I by plasmacytoid dendritic cells (pDCs). Recent paradigm shifting data, however, suggest that the mitochondria is an important source of interferogenic signals in SLE. In particular, upon activation with TLR7-agonist autoantibodies, SLE neutrophils release oxidized (Ox) mitochondrial DNA (mtDNA) in complex with transcription factor A mitochondria (TFAM), which together have an exceptional capacity to activate pDCs to produce IFN-I via TLR9 activation. Yet, mechanisms that modulate the delivery of Ox-mtDNA/TFAM complexes into TLR9 endosomes in SLE are not fully understood. Interestingly, our preliminary studies demonstrate for the first time that patients with SLE have autoantibodies to TFAM. Based on this premise, we hypothesize that anti-TFAM antibodies may facilitate the internalization and interferogenic response to Ox-mtDNA/TFAM complexes by pDCs, which may influence disease activity in SLE. The major goal of this proposal is to gain further insights into the potential significance of these novel hypotheses and preliminary findings in the context of SLE pathogenesis. In Aim 1, we will determine the prevalence and clinical associations of antibodies to TFAM and their relationship to the IFN-signature in a prospective observational cohort of patients with SLE, for which extensive clinical and serologic data is available, as well as IFN-induced gene expression analysis. In Aim 2, we will generate anti-TFAM monoclonal antibodies from single SLE anti-TFAM memory B cells to define their autoreactive origin [e.g. V(D)J usage, somatic hypermutations and determinants of antigen recognition], as well as their capacity and mechanism to activate pDCs in complex with Ox-mtDNA/TFAM. Together, these studies seek to enhance our understanding of self-immunogenic pathways underlying sterile inflammation and IFN production in SLE. The final goal of this work is to gain new insights into disease mechanisms, thus laying the foundation to explore novel therapies.