Unpaired cysteine insertions favor transmembrane dimerization and induce ligand-independent constitutive cytokine receptor signaling.

Naturally occurring gain-of-function (GOF) mutants have been identified in patients for a variety of cytokine receptors. Although this constitutive activation of cytokine receptors is strongly associated with malignant disorders, ligand-independent receptor activation is also a useful tool in synthetic biology e.g. to improve adoptive cellular therapies with genetically modified T-cells. Balanced Interleukin (IL-)7 signaling via a heterodimer of IL-7 receptor (IL-7Rα) and the common γ-chain (γc) controls T- and B-cell development and expansion, whereas uncontrolled IL-7 signaling can drive acute lymphoid leukemia (ALL) development. The ALL-driver mutation PPCL in the transmembrane domain of IL-7Rα is a mutational insertion of the four amino acids proline-proline-cysteine-leucine and leads to ligand-independent receptor dimerization and constitutive activation. We showed here in the cytokine-dependent pre-B-cell line Ba/F3 that the PPCL-insertion in a synthetic version of the IL-7Rα induced γc-independent STAT5 and ERK phosphorylation and also proliferation of the cells and that booster-stimulation by arteficial ligands additionally generated non-canonical STAT3 phosphorylation via the synthetic IL-7Rα-PPCL-receptors. Transfer of the IL-7Rα transmembrane domain with the PPCL insertion into natural and synthetic cytokine receptor chains of the IL-6, IL-12 and Interferon families also resulted in constitutive receptor signaling. In conclusion, our data suggested that the insertion of the mutated PPCL IL-7Rα transmembrane domain is an universal approach to generate ligand-independent, constitutively active cytokine receptors.

Synthetic receptor platform to identify loss-of-function single nucleotide variants and designed mutants in the death receptor Fas/CD95.

Synthetic biology has emerged as a useful technology for studying cytokine signal transduction. Recently, we described fully synthetic cytokine receptors to phenocopy trimeric receptors such as the death receptor Fas/CD95. Using a nanobody as an extracellular-binding domain for mCherry fused to the natural receptor's transmembrane and intracellular domain, trimeric mCherry ligands were able to induce cell death. Among the 17,889 single nucleotide variants in the SNP database for Fas, 337 represent missense mutations that functionally remained largely uncharacterized. Here, we developed a workflow for the Fas synthetic cytokine receptor system to functionally characterize missense SNPs within the transmembrane and intracellular domain of Fas. To validate our system, we selected five functionally assigned loss-of-function (LOF) polymorphisms and included 15 additional unassigned SNPs. Moreover, based on structural data, 15 gain-of-function or LOF candidate mutations were additionally selected. All 35 nucleotide variants were functionally investigated through cellular proliferation, apoptosis and caspases 3 and 7 cleavage assays. Collectively, our results showed that 30 variants resulted in partial or complete LOF, while five lead to a gain-of-function. In conclusion, we demonstrated that synthetic cytokine receptors are a suitable tool for functional SNPs/mutations characterization in a structured workflow.

Synthetic mimetics assigned a major role to IFNAR2 in type I interferon signaling.

Type I interferons (IFNs) are potent inhibitors of viral replication. Here, we reformatted the natural murine and human type I interferon-α/ß receptors IFNAR1 and IFNAR2 into fully synthetic biological switches. The transmembrane and intracellular domains of natural IFNAR1 and IFNAR2 were conserved, whereas the extracellular domains were exchanged by nanobodies directed against the fluorescent proteins Green fluorescent protein (GFP) and mCherry. Using this approach, multimeric single-binding GFP-mCherry ligands induced synthetic IFNAR1/IFNAR2 receptor complexes and initiated STAT1/2 mediated signal transduction via Jak1 and Tyk2. Homodimeric GFP and mCherry ligands showed that IFNAR2 but not IFNAR1 homodimers were sufficient to induce STAT1/2 signaling. Transcriptome analysis revealed that synthetic murine type I IFN signaling was highly comparable to IFNα4 signaling. Moreover, replication of vesicular stomatitis virus (VSV) in a cell culture-based viral infection model using MC57 cells was significantly inhibited after stimulation with synthetic ligands. Using intracellular deletion variants and point mutations, Y510 and Y335 in murine IFNAR2 were verified as unique phosphorylation sites for STAT1/2 activation, whereas the other tyrosine residues in IFNAR1 and IFNAR2 were not involved in STAT1/2 phosphorylation. Comparative analysis of synthetic human IFNARs supports this finding. In summary, our data showed that synthetic type I IFN signal transduction is originating from IFNAR2 rather than IFNAR1.

Pro- and anti-apoptotic fate decisions induced by di- and trimeric synthetic cytokine receptors.

Synthetic strategies to activate cytokine receptors so far only address standard dimeric cytokine receptor assemblies. The 19 ligands of the tumor necrosis factor superfamily (TNFSF), however, form noncovalent trimers and receptor trimerization is considered to be essential for receptor activation. Synthetic TNFR1, TNFR2, and Fas/CD95 receptors were activated by synthetic trimeric ligands which induced NF-κB signaling or Caspase-induced apoptosis. Albeit dimeric receptor activation did not induce synthetic TNFR1 and TNFR2 signaling, dimeric FasL induced extenuated apoptosis. Simultaneous integration of dimeric Interleukin (IL-)6 receptor gp130 and trimeric Fas as synthetic cytokine receptors in one cell enabled binary cell fate decisions, gp130-mediated proliferation or Fas-mediated apoptosis. In summary, our modular fully synthetic cytokine signaling system allows precisely orchestrated cellular responses to selectively induce pro- and anti-apoptotic signaling via canonical dimeric receptors of the IL-6 family and non-canonical trimeric receptor complexes of the TNF superfamily.

Correction: Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands.

[This corrects the article DOI: 10.1371/journal.pone.0230804.].

Synthetic interleukin 22 (IL-22) signaling reveals biological activity of homodimeric IL-10 receptor 2 and functional cross-talk with the IL-6 receptor gp130.

Cytokine signaling is transmitted by cell-surface receptors that function as biological switches controlling mainly immune-related processes. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of GFP and mCherry nanobodies fused to transmembrane and intracellular domains of cytokine receptors that phenocopy cytokine signaling induced by nonphysiological homo- and heterodimeric GFP-mCherry ligands. Interleukin 22 (IL-22) signals via both IL-22 receptor α1 (IL-22Rα1) and the common IL-10R2, belongs to the IL-10 cytokine family, and is critically involved in tissue regeneration. Here, IL-22 SyCyRs phenocopied native IL-22 signal transduction, indicated by induction of cytokine-dependent cellular proliferation, signal transduction, and transcriptome analysis. Whereas homodimeric IL-22Rα1 SyCyRs failed to activate signaling, homodimerization of the second IL-22 signaling chain, SyCyR(IL-10R2), which previously was considered not to induce signal transduction, led to induction of signal transduction. Interestingly, the SyCyR(IL-10R2) and SyCyR(IL-22Rα1) constructs could form functional heterodimeric receptor signaling complexes with the synthetic IL-6 receptor chain SyCyR(gp130). In summary, we have demonstrated that IL-22 signaling can be phenocopied by synthetic cytokine receptors, identified a functional IL-10R2 homodimeric receptor complex, and uncovered broad receptor cross-talk of IL-22Rα1 and IL-20R2 with gp130.

Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors.

Interleukin (IL)-12 and IL-23 belong to the IL-12 type family and are composite cytokines, consisting of the common ß subunit p40 and the specific cytokine α subunit p35 and p19, respectively. IL-12 signals via the IL-12Rß1·IL-12Rß2 receptor complex, and IL-23 uses also IL-12Rß1 but engages IL-23R as second receptor. Importantly, binding of IL-12 and IL-23 to IL-12Rß1 is mediated by p40, and binding to IL-12Rß2 and IL-23R is mediated by p35 and p19, respectively. Previously, we have identified a W157A substitution at site 3 of murine IL-23p19 that abrogates binding to murine IL-23R. Here, we demonstrate that the analogous Y185R site 3 substitution in murine and Y189R site 3 substitution in human IL-12p35 abolishes binding to IL-12Rß2 in a cross-species manner. Although Trp157 is conserved between murine and human IL-23p19 (Trp156 in the human ortholog), the site 3 W156A substitution in hIL-23p19 did not affect signaling of cells expressing human IL-12Rß1 and IL-23R, suggesting that the interface of murine IL-23p19 required for binding to IL-23R is different from that in the human ortholog. Hence, we introduced additional hIL-23p19 substitutions within its binding interface to hIL-23R and found that the combined site 3 substitutions of W156A and L160E, which become buried at the complex interface, disrupt binding of hIL-23p19 to hIL-23R. In summary, we have identified substitutions in IL-12p35 and IL-23p19 that disrupt binding to their cognate receptors IL-12Rß2 and IL-23R in a murine/human cross-species manner.

Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands.

Cytokine signaling is transmitted by cell surface receptors which act as natural biological switches to control cellular functions such as immune reactions. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of green fluorescent protein (GFP)- and mCherry-nanobodies fused to the transmembrane and intracellular domains of cytokine receptors. Following stimulation with homo- and heterodimeric GFP-mCherry fusion proteins, the resulting receptors phenocopied signaling induced by physiologically occurring cytokines. GFP and mCherry fusion proteins were produced in E. coli or CHO-K1 cells, but the overall yield and stability was low. Therefore, we applied two alternative multimerization strategies and achieved immunoglobulin Fc-mediated dimeric and coiled-coil GCN4pII-mediated trimeric assemblies. GFP- and/or mCherry-Fc homodimers activated synthetic gp130 cytokine receptors, which naturally respond to Interleukin 6 family cytokines. Activation of these synthetic gp130 receptors resulted in STAT3 and ERK phosphorylation and subsequent proliferation of Ba/F3-gp130 cells. Half-maximal effective concentrations (EC50) of 8.1 ng/ml and 0.64 ng/ml were determined for dimeric GFP-Fc and mCherry-Fc, respectively. This is well within the expected EC50 range of the native cytokines. Moreover, we generated tetrameric and hexameric GFP-mCherry-Fc fusion proteins, which were also biologically active. This highlighted the importance of close juxtaposition of two cytokine receptors for efficient receptor activation. Finally, we used a trimeric GCN4pII motif to generate homo-trimeric GFP and mCherry complexes. These synthetic cytokines showed improved EC50 values (GFP3: 0.58 ng/ml; mCherrry3: 0.37 ng/ml), over dimeric Fc fused variants. In conclusion, we successfully generated highly effective and stable multimeric synthetic cytokine receptor ligands for activation of synthetic cytokine receptors.