# Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

## TL;DR

This study shows that IFNλ2 and IFNλ3 are crucial for keeping gut cells ready to fight viruses even before infection occurs.

## Contribution

The study identifies IFNλ2/3 as the dominant regulators of baseline antiviral immunity in gut epithelial cells.

## Key findings

- Basal IFNλ2/3, but not IFNλ1, is essential for restricting replication of multiple viruses in intestinal cells.
- IFNλ2/3 controls the expression of antiviral genes and components of the IFN signaling pathway.
- Loss of IFNλ2/3 reduces STAT1 levels and impairs antiviral readiness in neighboring cells.

## Abstract

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.

Interferon-lambdas (IFNλs) are antiviral molecules that help protect the surfaces of our body, such as the gut and lungs, from infection. While IFNλs are best known for being produced during viral infections, recent work has shown that some IFNλs are also made at low levels even when no pathogen is present. This “basal” IFN activity acts like a constant security system that keeps cells alert and ready to respond quickly when a virus arrives. However, the specific roles of the different flavors of IFNλ (i.e. IFNλ1, IFNλ2, and IFNλ3) in this baseline protection have remained unclear.

In this study, we investigated how each type of IFNλ (IFNλ1, IFNλ2, and IFNλ3)contributes to antiviral readiness in human intestinal epithelial cells. By selectively removing each IFNλ, we discovered that IFNλ2 and IFNλ3, but not IFNλ1, are essential for maintaining this built-in antiviral state. Cells lacking IFNλ2/3 became highly vulnerable to a wide range of viruses and lost the ability to activate key antiviral genes. Our findings reveal a previously unrecognized hierarchy among IFNλs and highlight IFNλ2/3 as critical guardians of gut antiviral defense, even before infection occurs.