Project Summary/Abstract Idiopathic subglottic stenosis (iSGS) is an unexplained mucosal fibroinflammatory disease of the upper airway that occurs almost exclusively in adult, Caucasian women. Patients require urgent surgery to prevent death from airway obstruction. Novel therapies aimed at halting the progressive airway fibrosis are critical to reduce the burden of this disease. Given its rarity, the geographical dispersal of affected patients has previously limited investigations to define the basic biology of the disease. Our recent publications reveal microbial dysbiosis in iSGS airway scar, along with inflammatory pathway activation. Leveraging a 1000-patient international iSGS cohort led by this proposal’s PI (iSGS1000), this project is designed to understand the pathogenesis of iSGS by answering the question: Is iSGS related to an active infection, or does bacteria trigger a self-reactive immune response. Our proposal utilizes independent but interrelated approaches to address this question. In Aim 1 we will apply cutting edge molecular immunology and bioinformatic techniques to sort infiltrating CD8+ T cells from airway scar, directly sequence individual T cell receptors (TCR) via RNAseq, then clone high frequency TCRs into an in vitro model system to map antigen specificity. This will allow us to investigate the hypothesis that CD8+ T cells in the mucosa of iSGS airway scar demonstrate a clonal response directed at a bacterial antigen. Then in Aim 2 we will utilize whole genome sequencing (WGS) of the bacteria in iSGS airway scar to confirm a unique bacterial association with iSGS. Precise molecular characterization of the bacteria with metatranscriptomic analysis will provide new information on genetic features impacting pathogenicity. In Aim 3 we will investigate how current treatments for iSGS impact both the local microbiome as well as host immunity. Taken together our independent but interrelated approaches will help define how host and pathogen collide to produce pathogenic tissue fibrosis in iSGS.