List of primer sequences used in qPCR.

Interferon-lambdas (IFNλs) 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 IFNλs are constitutively expressed in pathogen-free environments, termed “basal” IFN expression. While intestinal epithelial cells constitutively express all basal IFNλ subtypes (IFNλ1, IFNλ2, and IFNλ3), their individual contributions to antiviral immunity remain poorly defined. Here, we systematically investigate the distinct roles of IFNλ1 and IFNλ2/3 in regulating intrinsic antiviral immunity using human intestinal epithelial T84 cells. Through genetic depletion of IFNλ1 or IFNλ2/3, we show that basal IFNλ2/3, but not IFNλ1, 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 IFNλ2/3 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 IFNλ2/3 reduced total STAT1 protein levels and blunted responsiveness to exogenous IFNλ, indicating compromised interferon signaling capacity. Furthermore, basal IFNλ2/3 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 IFNλ subtypes and establish IFNλ2/3 as the dominant, non-redundant regulators of epithelial immune readiness. Our study provides the first comprehensive analysis of basal IFNλ subtype functions in the gut epithelium and underscores the central role of basal IFNλ2/3 in maintaining mucosal antiviral defense.