The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells 1 – 3 . Considerable effort has been invested using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary “high affinity” receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ, and γ c 4 – 8 . Naïve T cells express only a low density of IL-2Rβ and γ c , and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ andγ c . Here, using in vitro evolution, we eliminated IL-2’s functional requirement for CD25 expression by engineering an IL-2 “superkine” (termed super-2) with increased binding affinity for IL-2Rβ. Crystal structures of super-2 in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, including a flexible helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in super-2 recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation T cells irrespective of CD25 expression. Compared to IL-2, super-2 induced superior expansion of cytotoxic T cells, leading to improved anti-tumor responses in vivo , and elicited proportionally less expansion of T regulatory cells and reduced pulmonary edema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.

Summary Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human IFN variants signal through the same cell surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique amongst the cytokine receptor superfamily, but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor-points" interspersed amongst ligand-specific interactions that ‘tune’ the relative IFN binding affinities, in an apparent extracellular ‘ligand proofreading’ mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.

Interleukin-15 (IL-15) and IL-2 possess distinct immunological functions despite both signaling through IL-2Rβ and the common cytokine receptor γ-chain, γc, We find that in the IL-15—IL-15Rα—IL-2Rβ—γc quaternary complex structure, IL-15 heterodimerizes IL-2Rβ and γc identically to the IL-2—IL-2Rα—IL-2Rβ—γc complex, despite differing receptor-binding chemistries. IL-15Rα dramatically increases the affinity of IL-15 for IL-2Rβ, and this allostery is required for IL-15 trans-signaling versus IL-2 cis-signaling. Consistent with the identical IL-2Rβ—γc dimer geometry, IL-2 and IL-15 exhibited similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induce similar signals, and the cytokine-specificity of IL-2Rα versus IL-15Rα determines cellular responsiveness. These results provide important new insights for specific development of IL-15-versus IL-2-based immunotherapeutics.