SUMMARY Membranous nephropathy (MN) is a glomerular disease due to the deposition of anti-podocyte antibodies in the subepithelial space of the glomerular basement membrane. This leadis to complement-mediated podocyte injury and, in ~30% of patients, to the development of end stage kidney disease within 10 years from diagnosis. Most of the research in MN has focused on the autoantibody specificities and has led to the identification of phospholipase 2A receptor (PLA2R) as the main target antigen in 70%-80% of the patients. Conversely, little is known on autoreactive T cells, despite the evidence from other antibody-mediated autoimmune diseases clearly showing a critical pathogenic role of autoreactive T cells, efficacy of T cell targeting treatments, and our preliminary data documenting that autoreactive T cells are present in the circulation of MN patients. Aim 1 of the present project will develop a new strategy to capture the T cell receptor (TCR) repertoire of PLA2R-reactive T cells in MN patients. We will apply high-throughput paired T cell receptor alpha and beta chain DNA sequence capture for renewable TCR gene library generation and functional screening from patient T cells. We will physically link TCR gene sequences for cloning into cellular display libraries, enabling repeated in vitro functional analyses of TCRs in ways that are impossible with alternative available techniques. Aim 2 will apply these technologies for the study of the single-cell transcriptional profile of CD4+ T cells. These data will reveal the characteristics of autoreactive TCRs in a previously collected samples from MN patients with remission versus active disease, setting the stage for expanded clinical studies. Overall, this project will collect and analyze immune response data from MN patients with unprecedented molecular scope and scale. Our approach is innovative because it couples carefully designed clinical sample sets with new high-throughput approaches in TCR analysis to comprehensively interrogate the molecular mechanisms of T cell responses in MN patients. The proposed research is significant because it will provide important tools for mechanistic studies aimed at identifying new biomarkers and therapeutic targets for MN patients.