Heterogeneity and Clinical Relevance of Group 2 Innate Lymphoid Cells Subsets in Nasal Polyps

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 (T2) inflammation with elevated IL-5 and IL-13. Although group 2 innate lymphoid cells (ILC2s) drive T2 inflammation, their subset diversity and clinical relevance in nasal polyps (NPs) remain unclear. Objective: To investigate cellular sources of T2 cytokines, subset heterogeneity of ILC2s and relationship between ILC2 subsets and clinical severity in CRSwNP. Methods: ILC2s and CD4⁺ T cells were isolated from NP tissue and analyzed for cytokine production. ILC2s from six NP and four peripheral blood (PB) samples underwent single-cell RNA sequencing. Differential gene expression, subset heterogeneity, and pseudotime trajectory analyses were performed. Flow cytometry validated ILC2 subsets and associations with clinical parameters. Results: Under ex vivo conditions, NP-derived ILC2s produced higher IL-5 and IL-13 levels than Th2 cells . NP-derived ILC2s showed downregulation of early developmental and trafficking genes (e.g. CD48, S1PR1) and upregulation of T2-associated cytokines (e.g. IL5, IL13), chemokines (e.g. XCL1, CXCL8), remodeling factors (e.g. AREG, TNFSF14), and METRNL. Four ILC2 subsets reflecting activation states were identified: migratory (tissue-homing with less activation), transitional (intermediate activation), inflammatory (high activation and T2 cytokines), and exhausted-like (expression of inhibitory receptors: e.g. TIGIT). The combined number of T2 cytokine-enriched subsets (transitional, inflammatory, and exhausted-like ILC2s) correlated with Lund-Mackay CT scores. Inflammatory and exhausted-like ILC2s were associated with clinical symptom severity. Conclusion: ILC2s are one of the important effector populations driving local T2 inflammation in NPs and include four subsets with specific clinical associations. Subset-level ILC2 profiling may clarify CRSwNP pathophysiology and inform clinical stratification.