Multimodal single cell analysis of the paediatric lower airway reveals novel immune cell phenotypes in early life health and disease

Respiratory disease is a major cause of morbidity and mortality in children worldwide. Many childhood respiratory diseases are characterised by chronic inflammation, however, the immune landscape of the paediatric airway remains uncharacterized. This is due to difficulties obtaining tissue-specific samples in early life as well as limited application of technologies that permit deep profiling from small sample volumes. Here, we employ multiomic single-cell sequencing to generate the first immune cell atlas of the paediatric lower airway with more than 44,900 cells across 12 preschool aged children. By integrating transcriptome-wide gene expression, assessment of 154 surface proteins, and functional pathway analysis, we extensively characterised 41 immune and epithelial cell populations present in the bronchoalveolar lavage of 11 children with cystic fibrosis and an age-matched healthy control. Paired spectral flow cytometry analysis of over 256,000 cells revealed high correlation in cell subset proportions and protein expression across the two techniques. We further revealed that paediatric alveolar macrophages consist of 13 functionally distinct sub populations, including previously undescribed populations enriched for IFN-α/β signalling, markers of vesicle production, and regulatory/repair function. Other novel immune cell populations not observed in previous studies of the adult lung include CD4 T cells expressing inflammatory signalling genes. Further, whilst we show no significant difference in overall cell proportions between CF and healthy lung, we observed significant differential gene expression in the alveolar macrophage population, including genes associated with lung inflammation (IL33, CCL15) and fibrosis (RBMS3, COL4A1, SPP1). Our work provides a comprehensive cellular analysis of the paediatric lower airway, reveals key immune signatures of early life lung disease, and provides a reference for investigations of respiratory immunity in children. ### Competing Interest Statement The authors have declared no competing interest.