Staphylococcal Superantigens-specific IgE Reveals Functional Superantigen Production Beyond Staphylococcus aureus in CRSwNP

• Metagenomic sequencing revealed S. aureus was present in T2 CRSwNP patients with negative S. aureus cultures, regardless of SAg-IgE status. • In T2 CRSwNP patients, the SAg-IgE-positive group showed significantly higher abundances of S. aureus and S. capitis compared to the SAg-IgE-negative group, and the Staphylococcus genus exhibited a strong relationship with SAg-IgE and IL-5. • SEA/SEC-positive S. capitis isolates produced functional SAgs that induced clonal expansion of SEA/SEC-specific TCRs and enhanced T2 inflammation via IL-33. Metagenomic sequencing revealed S. aureus was present in T2 CRSwNP patients with negative S. aureus cultures, regardless of SAg-IgE status. In T2 CRSwNP patients, the SAg-IgE-positive group showed significantly higher abundances of S. aureus and S. capitis compared to the SAg-IgE-negative group, and the Staphylococcus genus exhibited a strong relationship with SAg-IgE and IL-5. SEA/SEC-positive S. capitis isolates produced functional SAgs that induced clonal expansion of SEA/SEC-specific TCRs and enhanced T2 inflammation via IL-33. Staphylococcal superantigen-specific immunoglobulin E (SAg-IgE) correlates with disease severity in type 2 (T2) chronic rhinosinusitis with nasal polyps (CRSwNP) patients. Although Staphylococcus aureus (S. aureus) is recognized as a primary source of SAgs, SAg-IgE is detected even in patients with culture-negative S. aureus. background To identify the source of SAgs in SAg-IgE-positive T2 CRSwNP patients with culture-negative S. aureus. Methods: Metagenomic sequencing was conducted in T2 CRSwNP patients with repeatedly negative S. aureus cultures, stratified by SAg-IgE status. We screened clinical isolates for SAg genes and evaluated SAg functionality by measuring SAg-specific TCR repertoire expansion and T2 inflammatory responses in an ex vivo infection model. The SAg-IgE-positive group showed significantly higher abundances of Staphylococcus epidermidis, S. aureus, Lysinibacillus xylanilyticus, and Staphylococcus capitis (S. capitis) compared to the SAg-IgE-negative group. Interestingly, in all participants where S. aureus was detected, S. capitis was also present, albeit at low abundance. Redundancy analysis demonstrated clustering of Staphylococcus genus, SAg-IgE, and IL-5, supporting a potential link between Staphylococcus genus and SAg-driven immune responses. Notably, a clinical S. capitis isolate carried SEA and SEC genes and secreted functional SAgs, which triggered the clonal expansion of SEA/SEC-specific TCRs and exacerbated the T2 inflammatory response via IL-33 induction. results Metagenomic sequencing reveals S. capitis, beyond S. aureus, produces functional SAg to drive T2 response in SAg-IgE-positive CRSwNP patients when conventional cultures fail to detect S. aureus. Independent of culturable bacterial load, tissue SAg-IgE positivity reliably indicates bacterial colonization and SAg exposure in CRSwNP. conclusion