Autoantibodies Neutralizing Type I IFNs in the Bronchoalveolar Lavage of at Least 10% of Patients During Life-Threatening COVID-19 Pneumonia

**Background:** Life-threatening COVID-19 pneumonia is associated with impaired type I interferon (IFN) immunity, either due to inborn errors or autoantibodies (auto-Abs) neutralizing type I IFNs. Circulating auto-Abs against type I IFNs are found in at least 15% of patients with critical COVID-19 and can neutralize high (10 ng/mL) or low (100 pg/mL) concentrations of IFNs. However, their presence and neutralizing activity in the alveolar space (bronchoalveolar lavage, BAL) had been assessed in only 11 individuals. This study aimed to determine the prevalence and functional impact of neutralizing auto-Abs against type I IFNs in the BAL of a large cohort of patients with life-threatening COVID-19 pneumonia.

**Methods:** The study enrolled 415 patients with life-threatening COVID-19 pneumonia from three university hospitals (La Pitié-Salpêtrière, Lariboisière in Paris, France; Amsterdam UMC, Netherlands). All patients were in the ICU on invasive ventilation. BAL was collected from all 415 patients, and plasma from a subset of 95 patients (within 24 hours of BAL). IgG auto-Abs against IFN-α2 and IFN-ω were detected using a high-throughput automated ELISA (Gyros). Neutralizing activity was assessed in HEK293T cells transfected with an ISRE-driven luciferase reporter, stimulated with IFN-α2, IFN-ω (at 10 ng/mL or 100 pg/mL), or IFN-β (10 ng/mL) in the presence of 1:10 diluted plasma or 1:5 diluted BAL. IgG purification confirmed that neutralizing activity was IgG-mediated. Urea and hemoglobin concentrations in BAL were measured to assess dilution and exclude hemorrhage. Concentrations of 59 biomarkers in BAL were measured by Luminex multiplex assay. Statistical analyses used Wilcoxon tests and principal component analysis (PCA).

**Key Results:** Among 415 patients (median age 60 years, 69% male, overall mortality 51%), auto-Abs neutralizing IFN-α2 and/or IFN-ω at low concentration (100 pg/mL) were detected in the BAL of 54 patients (13%). Specifically, 45 (11%) neutralized IFN-α2, 37 (9%) neutralized IFN-ω, and 28 (7%) neutralized both. Auto-Abs neutralizing IFN-β (10 ng/mL) were found in 5 patients (1%). In paired plasma samples (n=95), 16 (17%) had auto-Abs neutralizing low concentrations of IFN-α2 and/or IFN-ω, and 8 (8%) neutralized high concentrations (10 ng/mL). All patients with neutralizing auto-Abs in BAL also had them in plasma, except one patient with IFN-α2 neutralization only in plasma. Mortality was 60% in patients with auto-Abs in BAL vs. 50% in those without (p=0.19). Duration of invasive ventilation and ICU stay were similar between groups. PCA of 59 BAL biomarkers showed no clustering difference between patients with (n=11) and without (n=117) auto-Abs; only three biomarkers (MIP1α, MIP1β, TRAIL) were significantly higher in the auto-Ab group.

**Clinical Implications:** This study demonstrates that neutralizing auto-Abs against type I IFNs are present in the lower respiratory tract of at least 10% of patients with life-threatening COVID-19 pneumonia, likely reaching the alveolar space from the circulation. These auto-Abs impair local type I IFN immunity, facilitating viral spread and contributing to severe pneumonia. The similar inflammation profiles between patients with and without auto-Abs suggest that impaired type I IFN immunity is a common pathogenic mechanism. Screening for these auto-Abs in BAL or plasma could identify patients who might benefit from therapies that bypass type I IFN signaling, such as IFN-β or other antiviral strategies. The findings also highlight the autoimmune nature of a subset of severe COVID-19 cases.