Mechanisms of Differential Signal Transduction by IFNLR1 Variants.

Background: Lambda interferons bind the interferon lambda receptor-1 (IFNLR1) and IL10RB heterodimer to induce interferon stimulated genes (ISGs) and impart antiviral immunity. We previously showed that protein variants derived from distinct IFNLR1 splice isoforms uniquely influence gene expression and HBV replication in stem cell-derived hepatocytes (iHeps). Here, we evaluated the molecular mechanisms of signal transduction by full-length canonical IFNLR1 (variant 1) and a noncanonical variant missing a portion of the Box1 and Box2 JAK1-interacting motifs (variant 2). Methods: We used HEK293T cells, wild-type (WT), and IFNLR1 knock-out (KO) iHeps that stably express doxycycline-inducible, FLAG-tagged IFNLR1 variants to evaluate function. Cellular responses to IFNL were measured using the Duolink proximity ligand assay, ImageStream flow cytometry, western blotting of JAK-STAT proteins, susceptibility to JAK1 and TYK2-specific inhibitors, and gene expression profiling. Results: While each IFNLR1 variant colocalized with IL10RB after IFNL treatment, variant 1 was more rapidly and extensively internalized than variant 2. In WT iHeps with intact endogenous IFNLR1, expression of variant 2 enabled higher JAK1 and TYK2 phosphorylation than variant 1, yet contrarily, variant 1 enabled greater STAT1 and STAT2 phosphorylation that resulted in broader and higher expression of ISGs. Select ISGs exhibited differential constitutive expression in IFNL-untreated variant-expressing iHeps but were IFNL-inducible through both variants. In iHeps expressing variant 1, WT-iHeps were more resistant than KO-iHeps to TYK2-inhibition of antiviral ISG expression yet conversely were more susceptible to TYK2 inhibition of proinflammatory ISG expression, suggesting noncanonical variants made from endogenous IFNLR1 transcripts influence the TYK2-dependence of IFNL signaling. Conclusions: IFNLR1 variants promote differential utilization of signaling mediators to influence IFNL-induced gene expression patterns, indicating a putative role in pathway regulation.