Single-cell profiling of paired nasal brushing and tissue samples reveals distinct cellular landscapes and immune phenotypes

Background: Since the COVID-19 pandemic, local immune responses in the nasal mucosa have become an area of growing research interest. In this context, studies using noninvasive nasal brushing (NB) samples have increased markedly. However, it remains unclear whether NB samples accurately reflect the immune landscape of the nasal tissue (NT). Objective: To directly compare the cellular composition and immune cell responses of NB and NT samples. Methods: Paired NB and NT samples were collected from the same anatomical site. The frequency, phenotype, and effector functions of epithelial and immune cells were analyzed using single-cell RNA sequencing and flow cytometry. Results: NB samples contained a significantly higher proportion of epithelial cells than NT samples, while fibroblasts, endothelial cells, and B cells were significantly less abundant. Within the epithelial compartment, NB showed an enrichment of ciliated and secretory cells, whereas basal cells were less frequent and glandular basal/secretory cells were rarely detected. Additionally, CD103+ tissue-resident memory T cells and CD56bright NK cells were more abundant in NB samples than in NT samples. Functional analyses revealed distinct T-cell effector profiles between the two sample types. Notably, SARS-CoV-2–specific T cells were significantly less frequent in NB samples than in NT. Conclusions: NB is well-suited for sampling cells located near the epithelial surface but captures fewer cells from deeper mucosal layers. Additionally, NB samples display distinct T-cell effector profiles and virus-specific T cell frequencies compared to NT. These findings highlight the importance of selecting sampling methods that align with the specific objectives of a study. Overall design: To collect nasal brushing (NB) samples, flocked swabs (Pap brush; SGS, Daejeon, Korea) were inserted into the nasal cavity of the donors and rotated 10–20 times. The swabs were then placed in tubes containing Dulbecco's Modified Eagle's Medium/F-12 (1:1) (1X) media (Gibco, Grand Island, NY, USA). The tubes were vigorously vortexed to dislodge the cells and pelleted via centrifugation. Nasal tissue (NT) was mechanically dissociated, and single-cell suspensions were prepared using a Tumor Dissociation Kit (Miltenyi Biotec, Auburn, CA, USA) in combination with a gentleMACS dissociator (Miltenyi Biotec) and 70-µm cell strainers (SPL Life Sciences, Gyeonggido, Korea).