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

Interferons (IFNs) are a critical component of innate immune defenses and limit viral disease severity. To advance studies on IFNs and their neutralization by pathogenic autoantibodies, we generated a Renilla luciferase-based reporter cell line capable of detecting the activities of IFN-Is, IFN-II, and IFN-IIIs. The reporter cell line exhibits a 125- to 2000-fold higher sensitivity to IFNs than a commonly used alternative biological reporter system and allows for a rapid and simple live-cell workflow for detecting low titer amounts of neutralizing anti-IFN antibodies. The human IFN system constitutes a critical component of innate immunity. Upon detection of infection, cells initiate the production and secretion of IFN cytokines (IFN-I, IFN-II, and IFN-III types) that trigger the expression of IFN-stimulated genes (ISGs), causing cells to enter an antiviral state. IFN-Is (e.g. IFN-α, IFN-β, and IFN-ω) and IFN-IIIs (e.g. IFN-λ1-4) are most important to first-line antiviral defenses and signal via type-specific receptors expressed on cell surfaces [1]. IFN system deficiencies can seriously exacerbate the severity of infections [2], and a functional IFN deficiency caused by autoantibodies (autoAbs) neutralizing IFN-Is promotes replication and disease caused by several viral pathogens [3-8]. In addition, IFN-II (IFN-γ) autoAbs have been described to promote susceptibility to some mycobacterial, fungal, and Gram-negative bacterial infections, and can increase the likelihood of herpes zoster [9]. While IFN-III autoAbs have also been identified, they have yet to be linked to a specific disease phenotype [10]. Identification of individuals harboring neutralizing anti-IFN autoAbs is critical to understand susceptibility to severe infections and requires highly sensitive and robust assays. Previously, the neutralizing capacity of anti-IFN-I autoAbs has been detected by transfecting 293T cells with plasmids expressing Firefly luciferase under the control of an ISG (MX1) promoter, followed by stimulation with recombinant IFNs preincubated with patient plasmas. Neutralization is then inferred from reduced levels of IFN-induced luciferase activity 16–24 h later [6, 11]. However, this system has several caveats: (1) transfection adds 24–48 h to assay duration and increases variability (controlled by co-transfecting a constitutively-expressed reporter); (2) relatively high IFN amounts are required, limiting assay sensitivity; (3) peak IFN-induced luciferase requires 24 h, extending assay duration; and (4) 293T cells are nonresponsive to IFN-IIIs, prohibiting their detection of anti-IFN-III autoAbs. We therefore developed highly sensitive reporter cells for the detection of IFN-Is, IFN-II, and IFN-IIIs based on A549s, which respond to all IFN types [12]. A549 cells were modified to harbor a gene cassette for monocistronic expression of Renilla luciferase and puromycin resistance downstream of the human ISG15 promoter (prISG15) (Fig. 1A)[13]. The ISG15 promoter was chosen for its inducibility by all IFNs, with ISG15 levels being maintained for at least 48 h [14]. Renilla luciferase was chosen as the reporter enzyme due to the availability of a live-cell substrate, allowing temporal monitoring of luciferase activity. To enhance sensitivity, Renilla luciferase was expressed with a C-terminal PEST (peptide rich in proline, glutamate, serine, and threonine) sequence that promotes protein degradation, reducing background luciferase from leaky transcription [15]. Single-cell clones were stimulated with 1000 international units (IU)/mL of IFN-α2 or IFN-λ1, and a clone with high responsiveness was selected for use (Fig. 1B), which was named the A549 IFN-reporter (AIR) cell line. First, we confirmed stable responsiveness of AIR cells to IFN-α2 or IFN-λ1 following passaging over 6 weeks (Fig. 1C). In addition, responsiveness to all 12 IFN-α subtypes, as well as IFN-β, IFN-ω, and IFN-γ, was confirmed (Fig. 1D). We then compared the IFN-sensitivities of AIR cell assays with those of the standard 293T transfection-based system. Both reporter systems were stimulated with a concentration range of IFN-Is or IFN-IIIs, and IFN-induced luciferase activities were determined 24 h later. Sensitivity was defined as the lowest IFN dose that induced activity >fivefold above the nonstimulated condition. The 293T system detected 2.5 ng/mL IFN-α2, 150 pg/mL IFN-β, and 2.5 ng/mL IFN-ω (Fig. 1E). By comparison, the AIR system detected as low as 1.2 pg/mL IFN-α2 or IFN-β, and 2.4 pg/mL for IFN-ω (Fig. 1F). While 293T cells did not respond to stimulation with IFN-IIIs (Fig. 1G), AIR cells responded to 195 pg/mL IFN-λ1 or IFN-λ2, and 97.5 pg/mL IFN-λ3 (Fig. 1H). Additionally, AIR cells responded to IFN-γ stimulation, although with a lower sensitivity (3.1 ng/mL) (Fig. 1I), which may be a limitation of these cells. Thus, AIR cells display a 125- to 2000-fold higher sensitivity to IFNs than 293Ts, and universally react to all three IFN types. Next, we compared IFN-induced luciferase kinetics between the AIR cell system and the 293T-based system. Using a single dose of each IFN (10 ng/mL IFN-α or IFN-ω, 1.21 ng/mL IFN-β, 50 ng/mL IFN-λ1-3), the earliest robust luciferase induction (>fivefold over nonstimulated) in 293Ts occurred 6–8 h poststimulation, and peaked at 24–30 h (Fig. 2A). In contrast, the earliest luciferase signal in AIR cells was detectable after 1 h of stimulation, and peaked after 8 h (Fig. 2B). Importantly, this signal remained stable for 30 h, providing a rapidly-induced, long-lasting experimental window compared to the lengthy transfection steps, slow reactivity, and cell lysis requirements in transfection-based 293T assays. Finally, we assessed the sensitivity of AIR cell assays to detect neutralizing anti-IFN antibodies. We initially used the lowest amount of IFN-I which resulted in a 10-fold induction of luciferase signal over nonstimulated cells (200 pg/mL IFN-β for 293T cells, 3.6 pg/mL IFN-β for AIR cells). These IFNβ amounts were incubated with a dilution series of a neutralizing anti-IFN-β antibody for 1 h prior to stimulation of each cell system. With a neutralization threshold of 75% reduction, the 293T assay detected 10 µg/mL neutralizing antibody, while AIR cell assays had a 250-fold improved sensitivity, detecting neutralization with only 39 ng/mL antibody (Fig. 2C). Similar assays demonstrated that AIR cells can be used to detect neutralizing anti-IFN-II and IFN-III antibodies (Fig. 2D and E). Lastly, we selected previously characterized human plasmas shown to neutralize low doses of IFN-α (200 pg/mL), IFN-β (40 pg/mL), or IFN-ω (200 pg/mL) using the 293T assay [11] and demonstrated the improved ability of AIR cells to detect the neutralizing activity of these patient samples against IFN-I concentrations as low as 10 pg/mL (Fig. 2F). In summary, the high sensitivity of AIR cells to respond to all IFN types permits their application to detect low titer neutralizing anti-IFN autoAbs. The authors thank Didier Trono and Jeremy Wilusz for gifting plasmids via Addgene. We are also grateful to the Swiss HIV Cohort Study (SHCS) and Beat M. Frey (Zurich Blood Transfusion Service of the Swiss Red Cross) for permitting the use of anonymized human plasma samples. The graphical abstract and Fig. 1A were created with BioRender.com. Financial support for this study was provided by the Academy of Medical Sciences (grant no. SBF003/1028 to DJH), the Wellcome Trust Institutional Strategic Support Fund (to DJH), and the Novartis Foundation for Medical-Biological Research (grant no. 23A069 to BGH). Open access funding provided by Universitat Zurich. The authors declare no financial or commercial conflict of interest. The peer review history for this article is available at https://publons.com/publon/10.1002/eji.202451325. The data that support the findings of this study are available from the corresponding author upon reasonable request. Detailed methods can be found in the Supporting Information file. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.