PROJECT SUMMARY/ABSTRACT The hallmark of systemic lupus erythematosus (SLE) is the production of antibodies (Abs) to nuclear antigens such as ribonucleoproteins and chromatin. High-affinity IgG Abs to double-stranded DNA (dsDNA) are associated with more severe disease and the development of lupus nephritis (LN). DNASE1L3 is a unique secreted DNase that is capable of digesting DNA packaged in chromatin, and null mutations in human DNASE1L3 cause monogenic early-onset SLE with anti-DNA reactivity. Similar to humans, DNASE1L3-deficient mice rapidly develop Abs to chromatin and dsDNA. Our analysis of this model revealed that the anti-DNA Ab response arises from the short-lived extrafollicular plasmablasts supported by the extrafollicular helper T cell (EF-TFH) subset. Confirming the relevance of DNASE1L3 to human patients with sporadic SLE, we demonstrated that many SLE patients with LN have reduced DNASE1L3 activity, which is associated with the presence of blocking Abs to DNASE1L3. These results suggest that by blocking D1L3, anti-D1L3 Abs facilitate autoreactivity to chromatin and thereby contribute to SLE pathogenesis. In this cycle of the award, these advances will be leveraged to dissect the mechanisms and clinical consequences of anti-DNA responses in SLE patients with autoantibody-mediated D1L3 deficiency. The project will continue the team effort that includes experimental immunology (Reizis), human SLE phenotyping and biobanking (Buyon), and immune repertoires analysis (Ippolito). In Aim 1, we will further characterize the dynamics and clinical correlates of anti-DNASE1L3 Abs in SLE patients, and analyze their role in the control of cell-free DNA. In Aim 2, we will clone anti-DNASE1L3 Abs from SLE patients and analyze their reactivity spectrum and pathogenic potential in vivo. In Aim 3, we will use single-cell TCR sequencing to characterize the autoreactive T cell clones in SLE patients with Ab-mediated blockade of DNASE1L3, and in DNASE1L3-deficient mice. Collectively, these studies should provide insights into the origin and mechanisms of the pathogenic anti-DNA responses in SLE, and facilitate more targeted approaches towards their therapeutic blockade.