(A1220) Single Cell Transcriptomics and Spatial Characterization of Tonsils from Pediatric Obstructive Sleep Apnea Patients

Introduction: Obstructive sleep apnea (OSA) in children is characterized by upper airway blockage during sleep and is associated with tonsillar hypertrophy. Previous studies identified increased IL-17A–producing T-cells in OSA tonsils compared to recurrent tonsillitis, with IL-17A correlating with disease severity (Apnea-Hypopnea Index, AHI). IL-17A influences tissue pathology by recruiting immune and structural cells, including endothelial cells, fibroblasts, and B- and T-cells. This study aims to identify IL-17A-responsive cell types and their spatial co-localization.

Methods: From 2023–2024, tonsils were collected from 16 pediatric OSA patients (AHI ≥ 5). Five tonsils underwent single-cell RNA sequencing (10X Genomics) to profile B-cells, T-cells, smooth muscle cells, macrophages, NK cells, dendritic cells, fibroblasts, and neutrophils. The remaining 11 tonsils were sectioned (100–200 µm) for histological and immunohistochemical (IHC) analysis, including H&E, Masson’s Trichrome, Ki-67 (proliferation), CD3 (T-cells), CD20 (B-cells), and aSMA (vasculature). ImageJ Fiji Software was used for analysis.

Results: Single-cell RNA sequencing showed expansion of effector T-cells and germinal center B-cells (p < 0.0002) and reduced regulatory T-cells (p < 0.0001) in higher AHI. IHC showed increased proliferation (Ki-67, p < 0.0001) and B-cell counts (CD20, p < 0.01) in higher AHI, with non-significant trends for aSMA, CD3, and fibrosis. These differences may reflect IHC resolution limits. To clarify these findings, six additional tonsils were analyzed, focusing on Low (1.9–10) and High (30–40) AHI groups while controlling for BMI. Future analyses will include FoxP3 (regulatory T-cells), CD19 (B-cells), and germinal center comparisons to refine the relationship between AHI, cell dynamics, and tonsillar hypertrophy.

Conclusions: These results highlight the role of effector T-cells, regulatory T-cells, and B-cells in tonsillar hypertrophy associated with OSA severity. Further analysis will elucidate cellular interactions underlying this pathology.