Identifying Pathogenic Epitopes and Autoreactive Cells in ANCA Vasculitis

This dissertation is focused on the interaction between the humoral (B cell) and cellular (T cell) arms of the adaptive immune system, and known autoantigens in patients with anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. ANCA vasculitis is characterized by vascular inflammation that leads to organ damage. Current treatment involves medications designed to induce disease remission, but many patients suffer from side effects including severe infection. We hypothesized that elucidating specific interactions between autoreactive lymphocytes and known autoantigens in patients would reveal targets for novel therapies that could halt the autoimmune response without side effects. In Chapter 1, I use high-resolution human leukocyte antigen (HLA) typing to identify alleles prevalent in our patients and demonstrate using Kaplan Meier estimates that carriage of HLA-DPB1*04:01 does not increase risk of relapse, contrary to previous results (1). Based on our independent serotype analysis, we hypothesize that carriage of HLA-DPB1*04:01 is a risk factor for patients with PR3-ANCA vasculitis (not MPO-ANCA vasculitis). Also addressed are significant differences between patients with MPO- and PR3-ANCA vasculitis, to clarify the rationale for focusing exclusively on MPO-ANCA vasculitis for the remainder of this dissertation. Chapter 2 focuses on potentially immunopathogenic regions of MPO. Patient autoantibody reactivity was assessed by enzyme-linked immunosorbent assay (ELISA) and anti-MPO447-461 antibodies were detected in 53% of patients. Epitopes were characterized using alanine scanning, circular dichroism, and solvent exposure experiments. Finally, in silico and in vitro binding studies confirmed the ability of MPO epitopes to bind patient HLA. In Chapter 3, I assess patient CD4+ T cell recognition of MPO epitopes. MHC II tetramers were created with patient HLA alleles and MPO epitopes, and used to stain patient cells. Patients demonstrated specific CD4+CD25intermediate T cell binding to tetramers and these cells secreted the pro-inflammatory cytokine IL-17A. TCR sequencing studies revealed clonally expanded autoreactive cells compared to controls, and some public TCR clones. Elucidation of these specific immune interactions will allow for new antigen-specific treatments using a variety of emerging techniques including chimeric antigen receptor (CAR) T cells, tolerogenic dendritic cells (DC), nanoparticle presentation of antigens, gene therapy, or removal of pathogenic T cells by apheresis.Doctor of Philosoph