**1. Introduction**

Interferon (IFN) β plays a critical role in the first line of defense against viruses, through its ability to induce a broad antiviral transcriptional response in virtually all cell types [1]. IFNβ also possesses key immunoregulatory functions that determine the outcome of the adaptive immune response against pathogens [1,2]. Over the years, in vitro and in vivo studies aimed at characterizing the mechanisms and the functional outcomes of IFNβ signaling were mostly performed in relation to single cytokine stimulation. This unlikely reflects physiological settings, as a plethora of cytokines are secreted in a specific situation. As a consequence, a cell rather simultaneously responds to a cocktail

of cytokines to foster the appropriate transcriptional program. Response to IFNβ is no exception and is very context-dependent, particularly regarding the potential crosstalk with other cytokines. Elevated levels of IFNβ and Tumor Necrosis Factor (TNF) are found during the host response to viruses. Aberrant increased levels of both cytokines is also associated with a number of autoinflammatory and autoimmune diseases such as Systemic Lupus Erythematosus (SLE), psoriasis, and Sjögren's syndrome [3]. While the cross-regulation of IFNβ and TNF is well documented [4–6], the functional crosstalk between these two cytokines remains poorly known.

IFNβ typically acts through binding to the IFNAR receptor (IFNAR1 and IFNAR2) leading to the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving JAK1- and Tyk2-mediated phosphorylation of STAT1 and STAT2, and to a lesser extent other STAT members in a cell-specific manner [7,8]. Phosphorylated STAT1 and STAT2, together with IFN Regulatory Factor (IRF) 9, form the IFN-stimulated gene factor 3 (ISGF3) complex that binds to the consensus IFN-stimulated response element (ISRE) sequences in the promoter of hundreds of IFN stimulated genes (ISGs) [9]. Formation of the ISGF3 complex is considered a hallmark of the engagemen<sup>t</sup> of the type I IFN response and, consequently, the requirement of STAT1 in a specific setting has become a marker of the engagemen<sup>t</sup> of type I IFN signaling [7,10]. However, in recent years, this paradigm has started to be challenged with accumulating evidence demonstrating the existence of non-canonical JAK-STAT signaling that mediates type I IFN responses [8,11].

Synergism between IFNβ and TNF was shown to enhance the antiviral response to Vesicular Stomatitis Virus (VSV), Myxoma virus, and paramyxovirus infections [12–14]. Gene expression analyses showed that IFNβ and TNF synergistically regulate hundreds of genes induced by individual cytokines alone, but also drive a specific delayed transcriptional program composed of genes that are either not responsive to IFNβ or TNF separately or are only responsive to either one of the cytokine [4,13]. The signaling mechanisms engaged downstream of the costimulation with IFNβ and TNF remained elusive, but it is implicitly assumed that the fate of the gene expression response requires that both IFNβ- and TNF-induced signaling pathways exhibit significant crosstalk. Analysis of the enrichment of specific transcription factors binding sites in the promoters of a panel of genes synergistically induced by IFNβ and TNF failed to give a clue about the specificity of the transcriptional regulation of these genes [13]. We previously showed that the *DUOX2* gene belongs to the category of delayed genes that are remarkably induced to high levels in response to the combination of IFNβ and TNF in lung epithelial cells [8]. We found that *DUOX2* expression required STAT2 and IRF9 but not STAT1, suggesting that STAT2 and IRF9 activities might segregate in an alternative STAT1-independent pathway that could be involved in gene regulation downstream of IFNβ and TNF [14].

In the present study, we aimed to fully characterize the transcriptional profile of the delayed response to IFNβ and TNF that occurs independently of STAT1 and evaluate the role of STAT2 and IRF9 in the regulation of this response. We found that the costimulation by IFNβ and TNF induces a broad set of antiviral and immunoregulatory genes in the absence of STAT1. We also report the di fferential regulation of distinct subsets of IFNβ− and TNF-induced genes by STAT2 and IRF9. While IFNβ and TNF act in part through the concerted action of STAT2 and IRF9, specific sets of genes were only regulated by either STAT2 or IRF9. Altogether, our findings uncovered non-canonical STAT2 and/or IRF9-dependent pathways that coexist to regulate distinct pools of antiviral and immunoregulatory genes in a context of IFNβ and TNF crosstalk.

### **2. Materials and Methods**

### *2.1. Cell Culture and Stimulation*

A549 cells (American Type Culture Collection, ATCC) were grown in F-12 nutrient mixture (Ham) medium supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% L-glutamine. The 2ftGH fibrosarcoma cell line and the derived STAT1-deficient U3A cell line, a generous gift from Dr. G. Stark, Cleveland, USA [15], were grown in DMEM medium supplemented with 10% HI-FBS or HI-Fetal Clone III (HI-FCl) and 1% L-glutamine. U3A cells stably expressing STAT1 were generated by transfection of the STAT1 alpha flag pRc/CMV plasmid (Addgene plasmid #8691; a generous gift from Dr. J. Darnell, Rockfeller University, USA [16,17]) and selection with 800 μg/ml Geneticin (G418). Monoclonal populations of U3A stably expressing STAT1 cells were isolated. A pool of two clones, referred to as U3A-STAT1, was used in the experiments to mitigate the clonal effects. U3A-STAT1 cells were maintained in culture in DMEM supplemented with 10% HI-FCl, 1% Glu, and 200 μg/mL G418. All cell lines were cultured without antibiotics except for the selection of stable cells. All media and supplements were from Gibco (Life Technologies, Grand Island, NY, USA), with the exception of HI-FCl, which was from HyClone (Logan, UT, USA). Mycoplasma contamination was excluded by regular analysis using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland). Cells were stimulated with IFNβ (1000 U/mL, PBL Assay Science, Piscataway, NJ, USA), TNF (10 ng/mL, R&D Systems, Minneapolis, MN, USA) or IFNβ (1000 U/mL) +TNF (10 ng/mL) for the indicated times.
