(182t) High-Throughput Discovery of Autoreactive T-Cell Receptors in Membranous Nephropathy

Membranous nephropathy (MN) is an autoimmune disease in which immune complexes inhibit kidney function, which can lead to severe kidney failure. MN affects approximately 8-10 people per 1 million globally and is a leading cause of nephrotic syndrome. Current studies have focused on the B-cell response in MN, leading to the discovery of phospholipase A2 receptor (PLA2R) as the most common antigen in patients. However, the current treatment model of B-cell depletion therapy faces limitations of toxicity and potency. For example, depletion via rituximab is safely tolerated but often results in the return of resistant autoreactive B-cells. Some studies have noted the involvement of CD4 helper T-cells in the MN autoimmune response, but their exact role is still unclear. Understanding the role of the autoreactive CD4 T-cell repertoire in MN patients can lead to improved treatments that are safe, efficacious, and durable. In addition, exploring the binding of T-cell receptors (TCRs) to PLA2R will enhance knowledge of the protein-protein interactions that govern the adaptive immune system.

To investigate the CD4 T-cell immune response in MN and mine immune system protein-protein interactions, a high-throughput single-cell emulsion-based platform is used to capture natively paired alpha and beta TCR genes from patient samples. These natively paired TCR gene libraries are cloned into immortalized cell lines, which are then functionally profiled for autoreactive responses. The patient-derived TCR libraries are co-cultured with antigen-presenting cells (APCs) displaying PLA2R peptides, and successful TCR-PLA2R interactions can be identified via fluorescently-stained activation markers. CD4 TCRs published in literature are utilized to validate the activation-based functional screening and ensure activated TCRs are sufficiently captured. This high-throughput technique allows extensive examination of TCR-peptide recognition and the CD4 T-cell immune response in MN patients with potential for immunotherapy applications. By comparing the change in T-cell repertoire in patients during active disease and remission following successful therapy, the pro-inflammatory and immunosuppressive T-cells can be identified to inform disease prognosis. Overall, this study provides a basis for developing better immunotherapeutics in the autoimmune disease sphere and advances current understanding of TCR protein immune interactions in MN.