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Objectives: Recent immunologic study of the adaptive immune repertoire in the subglottic airway demonstrated high‐frequency T cell clones that do not overlap between individuals. However, the anatomic distribution and antigenic target of the T cell repertoire in the proximal airway mucosa remain unresolved. Methods: Single‐cell RNA sequencing of matched scar and unaffected mucosa from idiopathic subglottic stenosis patients (iSGS, n = 32) was performed and compared with airway mucosa from healthy controls (n = 10). T cell receptor (TCR) sequences were interrogated via similarity network analysis to explore antigenic targets using the published algorithm: Grouping of Lymphocyte Interactions by Paratope Hotspots (GLIPH2). Results: The mucosal T cell repertoire in healthy control airways consisted of highly expressed T cell clones conserved across anatomic subsites (trachea, bronchi, bronchioles, and lung). In iSGS, high‐frequency clones were equally represented in both scar and adjacent non‐scar tissue. Significant differences in repertoire structure between iSGS scar and unaffected mucosa was observed, driven by unique low‐frequency clones. GLIPH2 results suggest low‐frequency clones share targets between multiple iSGS patients. Conclusion: Healthy airway mucosa has a highly conserved T cell repertoire across multiple anatomic subsites. Similarly, iSGS patients have highly expressed T cell clones present in both scar and unaffected mucosa. iSGS airway scar possesses an abundance of less highly expanded clones with predicted antigen targets shared between patients. Interrogation of these shared motifs suggests abundant adaptive immunity to viral targets in iSGS airway scar. These results provide insight into disease pathogenesis and illuminate new treatment strategies in iSGS. Level of Evidence: NA Laryngoscope, 134:3245–3252, 2024 [ABSTRACT FROM AUTHOR]

Objectives: Recent translational scientific efforts in subglottic stenosis (SGS) support a disease model where epithelial alterations facilitate microbiome displacement, dysregulated immune activation, and localized fibrosis. Given the observed immune cell infiltrate in SGS, we sought to test the hypothesis that SGS cases possessed a low diversity (highly clonal) adaptive immune response when compared with healthy controls. Methods: Single cell RNA sequencing (scRNA‐seq) of subglottic mucosal scar in iSGS (n = 24), iLTS (n = 8), and healthy controls (n = 7) was performed. T cell receptor (TCR) sequences were extracted, analyzed, and used to construct repertoire structure, compare diversity, interrogate overlap, and define antigenic targets using the Immunarch bioinformatics pipeline. Results: The proximal airway mucosa in health and disease are equally diverse via Hill framework quantitation (iSGS vs. iLTS vs. Control, p > 0.05). Repertoires do not significantly overlap between individuals (Morisita <0.02). Among iSGS patients, clonality of the TCR repertoire is driven by CD8+ T cells, and iSGS patients possess numerous TCRs targeting viral and intercellular pathogens. High frequency clonotypes do not map to known targets in public datasets. Conclusion: SGS cases do not possess a lower diversity adaptive immune infiltrate when compared with healthy controls. Interestingly, the TCR repertoire in both health and disease contains a restricted number of high frequency clonotypes that do not significantly overlap between individuals. The target of the high frequency clonotypes in health and disease remain unresolved. Level of Evidence: NA Laryngoscope, 134:1757–1764, 2024 [ABSTRACT FROM AUTHOR]