Basal IFN?2/3, but not IFN?1, signaling is sufficient to control virus infection in intestinal epithelial cells

Interferon-lambdas (IFN?s) serve as critical mediators of antiviral defense at mucosal surfaces. Beyond their established role in regulating innate immune responses during infection, recent evidence demonstrates that IFN?s are constitutively expressed in pathogen-free environments, termed "basal" IFN expression. While intestinal epithelial cells constitutively express all basal IFN? subtypes (IFN?1, IFN?2, and IFN?3), their individual contributions to antiviral immunity remain poorly defined. Here, we systematically investigate the distinct roles of IFN?1 and IFN?2/3 in regulating intrinsic antiviral immunity using human intestinal epithelial T84 cells. Through genetic depletion of IFN?1 or IFN?2/3, we show that basal IFN?2/3, but not IFN?1, is essential for restricting replication and spread of diverse viruses, including vesicular stomatitis virus (VSV), mammalian orthoreovirus (MRV), rotavirus (RV), and vaccinia virus (VV). Transcriptomic profiling revealed that IFN?2/3 selectively controls the basal expression of interferon-stimulated genes (ISGs), including key antiviral effectors and components of the IFN signaling machinery (e.g., STAT1, STAT2, IRF9). Loss of IFN?2/3 reduced total STAT1 protein levels and blunted responsiveness to exogenous IFN?, indicating compromised interferon signaling capacity. Furthermore, basal IFN?2/3 was required for activating paracrine JAK/STAT signaling and ISG induction in neighboring bystander cells, thereby amplifying antiviral protection across the epithelial layer. These findings reveal a functional hierarchy among IFN? subtypes and establish IFN?2/3 as the dominant, non-redundant regulators of epithelial immune readiness. Our study provides the first comprehensive analysis of basal IFN? subtype functions in the gut epithelium and underscores the central role of basal IFN?2/3 in maintaining mucosal antiviral defense. Overall design: To define the distinct roles of IFN? subtypes in maintaining immune homeostasis and antiviral defense at basal levels in human intestinal epithelial cell, we generated T84 IFN?1 KO, IFN?2/3 KO and IFNLR KO cells. T84 WT and KO cells were seeded and harvested at two days post-seeding, and sent for RNA sequencing.