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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]

Objective: Idiopathic subglottic stenosis (iSGS) is a rare, recurrent, fibroinflammatory disease affecting the larynx and proximal trachea. Given it occurs primarily in adult females, estrogen is speculated to play a central pathophysiological role. This study aimed to evaluate relationships between estrogen exposure, disease progression, and recurrence. Methods: North American Airway Collaborative (NoAAC) data of adults with iSGS obstructive airway lesions, who underwent index endoscopic airway dilation, were used to identify associations between estrogen exposure, disease characteristics, and time to recurrence (TTR), and interventions were analyzed using Kruskal–Wallis test and Pearson coefficient. Cox proportional hazards regression models compared hazard ratios by estrogen exposure. Kaplan–Meier curves were plotted for TTR based on menopausal status. Results: In all, 533 females had complete estrogen data (33% premenopausal, 17% perimenopausal, 50% postmenopausal). Median estrogen exposure was 28 years. Overall, there was no dose–response relationship between estrogen exposure and disease recurrence. Premenopausal patients had significantly shorter time from symptom manifestation to diagnosis (1.17 vs. 1.42 years perimenopausal vs. 2.08 years postmenopausal, p < 0.001), shorter time from diagnosis to index endoscopic airway dilation (1.90 vs. 2.50 vs. 3.76 years, p = 0.005), and higher number of procedures (1.73 vs. 1.20 vs. 1.08 procedures, p < 0.001). Conclusions: We demonstrate premenopausal patients may have a more aggressive disease variant than their peri‐ and postmenopausal counterparts. However, it is unclear as to whether this is related to reduced estrogen in the peri‐ and postmenopausal states or the age‐related physiology of wound healing and inflammation, regardless of estrogen. Level of Evidence: 3 Laryngoscope, 134:825–830, 2024 [ABSTRACT FROM AUTHOR]

Objectives: Idiopathic subglottic stenosis (iSGS) is an unexplained progressive fibrosis of the upper airway. iSGS almost exclusively affects women; as a result, female hormones (estrogen and progesterone) have been proposed to participate in the pathogenesis of iSGS. Our aim was to localize cell‐specific gene expression of estrogen receptors (ESR1 and ESR2) and progesterone receptor (PGR) using an established iSGS single‐cell RNA sequencing (scRNAseq) cell atlas. Study Design: Ex vivo molecular study of airway scar and healthy mucosa from iSGS patients. Methods: An established scRNAseq atlas consisting of 25,974 individually sequenced cells from subglottic scar (n = 7) or matched unaffected mucosa (n = 3) in iSGS patients was interrogated for RNA expression of ESR1, ESR2, and PGR. Results were quantified and compared across cell subsets, then visualized using Uniform Manifold Approximation and Projection (UMAP). Confirmatory protein assessment of endocrine receptors in fibroblasts from iSGS patients (n = 5) was performed via flow cytometry. Results: The proximal airway mucosa in iSGS patients demonstrates differential expression of endocrine receptors (ESR1, ESR2, PGR). Within airway scar, endocrine receptors are primarily expressed by fibroblasts, immune cells, and endothelial cells. Fibroblasts show strong ESR1 and PGR expression, while immune cells possess RNA for both ESR1 and ESR2. Endothelial cells predominantly express ESR2. Epithelial cells in unaffected mucosa express all three receptors, which are all reduced in airway scar. Conclusions: scRNAseq data localized endocrine receptor expression to specific cell subsets. These results provide the foundation for future work interrogating how hormone‐dependent mechanisms promote, sustain, or participate in iSGS disease pathogenesis. Level of Evidence: NA; Basic science Laryngoscope, 133:3506–3511, 2023 [ABSTRACT FROM AUTHOR]

Objective: Laryngotracheal stenosis (LTS) describes fibrotic airway obstruction that is life-threatening without treatment. Targeted therapies are needed as an adjunct to surgical management. We have previously observed the upregulation of immune checkpoint programmed cell death (PD)-1 and its ligand, PD-L1, in patients with LTS. This study aims to determine whether PD-1 and PD-L1 play a role in the pathophysiology of LTS., Study Design: Basic science., Setting: Laboratory., Methods: Fibroblasts derived from the subglottic scar of 5 iSGS patients were cultured ex vivo with transforming growth factor β (TGFβ), PD-L1 agonist (PD-1), and PD-L1 blockade (anti-PD-L1). PD-L1, TGFβ receptor II (TGFβRII), and Collagen-1 expression were quantified by flow cytometry. A validated chemomechanical injury model of subglottic stenosis was applied in PD-1 knockout and wild-type (WT) mice, and subglottic thickening was assessed by histologic analysis., Results: TGFβ significantly increased the expression of PD-L1 and Collagen-1 in human airway scar fibroblasts (P < .05). PD-1 knockout mice demonstrated no significant difference in subglottic airway fibrosis compared to WT mice. Ex vivo PD-L1 modulation had no impact on fibroblast Collagen-1 expression. PD-L1 high-intensity fibroblasts expressed greater Collagen-1 and TGFβRII compared to PD-L1 low-intensity fibroblasts., Conclusion: PD-1 knockout does not protect mice from the development of laryngotracheal fibrosis. However, its ligand, PD-L1 is highly expressed on pathologic fibroblasts unique to scar, characterized by high Collagen-1 and TGFβRII expression. PD-L1 is also upregulated in conjunction with Collagen-1 by TGFβ stimulation. PD-L1 may act independently of PD-1 to sensitize fibroblasts to TGFβ, suggesting direct targeting of PD-L1 may have therapeutic potential in LTS., (© 2024 The Author(s). Otolaryngology–Head and Neck Surgery published by Wiley Periodicals LLC on behalf of American Academy of Otolaryngology–Head and Neck Surgery Foundation.)