Autocrine responses to interleukin-6 family cytokines are insensitive to extracellular gp130 signaling receptor blockade.

Cytokines of the IL-12 family show structural similarities but have distinct functions in the immune system. Prominent members of this cytokine family are the pro-inflammatory cytokines IL-12 and IL-23. These two cytokines share cytokine subunits and receptor chains but have different functions in autoimmune diseases, cancer and infections. Accordingly, structural knowledge about receptor complex formation is essential for the development of new therapeutic strategies preventing and/or inhibiting cytokine:receptor interaction. In addition, intracellular signaling cascades can be targeted to inhibit cytokine-mediated effects. Single nucleotide polymorphisms can lead to alteration in the amino acid sequence and thereby influencing protein functions or protein–protein interactions. To understand the biology of IL-12 and IL-23 and to establish efficient targeting strategies structural knowledge about cytokines and respective receptors is crucial. A highly efficient therapy might be a combination of different drugs targeting extracellular cytokine:receptor assembly and intracellular signaling pathways.

The interleukin (IL)-12–type cytokines IL-12 and IL-23 are involved in T-helper (Th) 1 and Th17 immunity, respectively. They share the IL-12 receptor β1 (IL-12Rβ1) as one component of their receptor signaling complexes, with IL-12Rβ2 as second receptor for IL-12 and IL-23R for IL-23 signal transduction. Stimulation with IL-12 and IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase tyrosine kinase (Tyk) 2 associates with IL-12Rβ1, whereas Jak2 binds to IL-23R and also to IL-12Rβ2. Receptor association of Jak2 is mediated by Box1 and Box2 motifs located within the intracellular domain of the receptor chains. Here we define the Box1 and Box2 motifs in IL-12Rβ1 and an unusual Jak2-binding site in IL-23R by the use of deletion and site-directed mutagenesis. Our data show that nonfunctional box motifs abolish IL-12– and IL-23–induced STAT3 phosphorylation and cytokine-dependent proliferation of Ba/F3 cells. Coimmunoprecipitation of Tyk2 by IL-12Rβ1 and Jak2 by IL‑23R supported these findings. In addition, our data demonstrate that association of Jak2 with IL-23R is mandatory for IL-12 and/or IL-23 signaling, whereas Tyk2 seems to be dispensable.

IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor β1 (IL-12Rβ1). Complex formation was hypothesized to follow the "site I-II-III" architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rβ1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rβ1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rβ1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rβ1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rβ1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rβ1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.

IL-35 and IL-39 are recently discovered shared members of the IL-6- and IL-12–type cytokine family with immune-suppressive capacity. IL-35 has been reported to induce the formation of four different receptor complexes: gp130:IL-12β2, gp130:gp130, IL-12β2:IL-12β2, and IL-12β2:WSX-1. IL-39 was proposed to form a gp130:IL-23R receptor complex. IL-35, but not IL-39, has been reported to activate non-conventional STAT signaling, depending on the receptor complex and target cell. Analyses of IL-35 and IL-39 are, however, hampered by the lack of biologically active recombinant IL-35 and IL-39 proteins. Therefore, we engineered chimeric cytokine receptors to accomplish synthetic IL-35 and IL- 39 signaling by shuffling the extra- and intracellular domains of IL-6/IL-12–type cytokine receptors, resulting in biological activity for all previously described IL-35 receptor complexes. Moreover, we found that the proposed IL-39 receptor complex is biologically active and discovered two additional biologically active synthetic receptor combinations, gp130/IL-12Rβ1 and IL-23R/IL-12Rβ2. Surprisingly, synthetic IL-35 activation led to more canonical STAT signaling of all receptor complexes. In summary, our receptor shuffling approach highlights an interchangeable, modular domain structure among IL-6- and IL-12–type cytokine receptors and enabled synthetic IL-35 and IL-39 signaling.