Longitudinal Analysis Over Decades Reveals the Development and Immune Implications of Type I Interferon Autoantibodies in an Aging Population

Abstract

Pre-existing autoantibodies (autoAbs) neutralizing type I interferons (IFN-Is: IFNα, IFNβ, IFNω) have recently been described as significant contributors to the severity of viral infectious diseases. Here, we explore the development and consequences of anti-IFN-I autoAbs at high-resolution using retrospective samples and data from 1876 well-treated individuals >65 years of age enrolled in the Swiss HIV Cohort Study, a nationwide, longitudinal cohort with up to 35 years of follow-up. Approximately 1.9% of individuals developed anti-IFN-I autoAbs, with a median onset age of ~63 years (range 45-80). Once developed, anti-IFN-I autoAbs persisted for life, and generally increased in titer over years. Most individuals developed distinct neutralizing and non-neutralizing anti-IFN-I autoAb repertoires at discrete times that selectively targeted various combinations of IFNα, IFNβ, and IFNω. Longitudinal analyses further revealed that emergence of neutralizing anti-IFNα autoAbs correlated with reduced IFN-stimulated gene (ISG) levels, indicating impairment of innate immunity. Patient data review suggested that prior recorded viral infections and autoimmune history influence the likelihood of mounting anti-IFN-I autoAbs. Indeed, systematic measurements in biobanked samples revealed significant enrichment of pre-existing autoreactivity against clinically relevant autoantigens in individuals who later developed anti-IFN-I autoAbs. In this context, we describe lifelong neutralizing anti-IFNα autoAbs (and impaired innate immunity), that manifested in an individual following IFNα therapy, and who was retrospectively found to have had pre-existing autoreactivity to β2-glycoprotein-I before IFNα treatment. Our decades-spanning longitudinal analyses illuminate the development and immune implications of anti-IFN-I autoAbs in an aging population, and support a 'two-hit' hypothesis whereby loss of self-tolerance prior to immune-triggering with endogenous or exogenous IFN-I may pose a risk for developing late-onset, lifelong IFN-I functional deficiency.

Highly-sensitive reporter cell line for detection of interferon types I-III and their neutralization by antibodies

“…Human plasma samples and ethics. Anonymized human plasma samples previously categorized as positive (n=5) or negative (n=5) for anti-IFNα autoAbs 10 were used to validate assays. The samples were originally derived from specimens stored in the Zurich center biobank of the Swiss HIV Cohort study (SHCS, 16 ) which covers their use in the current work.…”

“…Luciferase assays. The 293T cell-based luciferase assay for detection of IFN-Is has been described previously 6,10 . For detection of IFN activity by A549-IFN-reporter (AIR) cells, 30,000 cells per well were seeded in a 96-well white-bottomed tissue culture plate and stimulated 24h later with IFNα2 (Novusbio; NPB2-34971), IFNβ (pbl assay science; 11420-1), IFNω (Novusbio; NBP2-35893), IFNγ (Novusbio; NBP2-34992), IFNλ1 (Novusbio; NBP2-34996), IFNλ2 (R&D systems; 8417-IL), or IFNλ3 (R&D systems; 5259-IL) at the indicated concentrations.…”

“…Previously, neutralizing anti-IFN-I autoAbs have been detected by transfecting 293T cells with plasmids expressing Firefly luciferase under control of an ISG ( MX1 ) promoter, followed by stimulation with recombinant IFNs pre-incubated with patient plasmas. The ability of patient autoAbs to neutralize IFNs is then inferred from reduced levels of IFN-induced Firefly luciferase activity 16-24h later 6,10 . However, this system has several caveats: (i) transfection of cells adds 24-48h to assay duration and increases inter-batch variability (controlled by co-transfection of a constitutively-expressed reporter); (ii) relatively high IFN amounts are required to induce signaling, limiting detection of low titers of neutralizing autoAbs; (iii) peak IFN-induced Firefly luciferase requires 24h, extending assay duration; and (iv) 293T cells do not respond to IFN-IIIs 11 , prohibiting their use to study anti-IFN-III autoAbs.…”

“…2D ). Finally, we selected five previously characterized human plasmas that neutralize IFNα activity (and five negative plasmas) 10 , and demonstrated the ability of AIR cells to detect neutralizing activity in such specimens ( Fig. 2E ).…”