Crystal structure and immune single-cell atlas provide insights into the functional divergence of type I IFNs in fish.

Type I interferons (IFNs) are central antiviral cytokines in vertebrates, yet the mechanisms underlying their functional diversification in early vertebrates remain unclear. Teleost fish, whose IFN repertoires expanded through whole-genome duplication, provide a powerful model to address this question. Here, we systematically characterize grass carp (Ctenopharyngodon idella) IFNd to elucidate the mechanisms underlying its functional divergence from IFNa. IFNd used the same receptors of IFNa to activate JAK-STAT pathway, albeit with significantly lower antiviral potency than IFNa. Structural analysis revealed significant differences in F helix length and key receptor-binding residues between IFNa and IFNd. Furthermore, single-cell RNA sequencing demonstrated high heterogeneity among immune cell subpopulations responding to IFNa and IFNd, Notably, the signaling pathways enriched by differentially expressed genes in monocytes and macrophages were distinct for the 2 cytokines. Together, our findings link ligand structural conformation and receptor-binding composition to IFN signaling strength and immune cell specificity, providing mechanistic insight into the functional diversification of type I IFNs in lower vertebrates.