IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 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.

Typically, cytokines have defined binding sites and in many cases, single amino acid exchanges reduce or completely disturb cytokine:cytokine-receptor interaction 14 . This does not account for IL-35, because the binding of IL-12_p35 and EBI3 could not be interrupted by introduction of class-typical point mutations 5 . Therefore, binding of IL-12_p35 to EBI3 remains mysterious. Remarkably, in this study IL-35 was analyzed in cell lysates rather than in cell culture supernatants, mainly because IL-35 was very poorly if at all secreted 5 . Our own studies also failed to detect IL-35 (as single components and as Hyper-cytokine fusion protein) in cell culture supernatants and we were not able to stimulate Ba/F3 cells expressing IL-12Rβ2, WSX-1 and gp130 with purified, reconstituted recombinant IL-35 15 . Thus far, only one group succeeded to express and purify tiny amounts of recombinant IL-35 in insect cells, which was biologically active on murine primary T and B cells. Due to formation of p35:p35 and EBI3:EBI3 homodimers, the overall efficacy of p35:EBI3 heterodimer formation was very low 13 . Also commercially available IL-35 failed to induce proliferation of Ba/F3-cells expressing WSX-1, gp130 and IL-12Rβ2 (Fig. S1). After all, protocols to express and purify IL-35 and also IL-39 are still missing.
Due to these limitations, we developed an alternative strategy to analyze IL-35 signal transduction in the well-established, pre-murine B-cell-line Ba/F3, which was commonly used to investigate the signal transduction of cytokines of the IL-6/IL-12 family 17,18 . To this end we engineered shuffled IL-6/IL-12 type cytokine receptors that were responsive to extracellular IL-12/IL-23 stimulation and induced intracellular IL-35/IL-39-signal transduction. We refer to this strategy as synthetic signaling. Using this approach, we were able to confirm biological activity of all described IL-35-and the proposed IL-39-receptor complexes in Ba/F3 cells. Furthermore, we identified two additional receptor combinations of the IL-6/IL-12-type cytokine receptors.

Generation, expression and cell surface localization of synthetic IL-12 type receptor chimeras.
Cytokines of the IL-12 family signal via five different receptors and eight different receptor complexes, namely IL-12Rβ1:IL-12Rβ2 for IL-12, IL-23R:IL-12Rβ1 for IL-23, WSX-1:gp130 for IL-27 and IL-12Rβ2:gp130, IL-12Rβ2:IL-12Rβ2, gp130:gp130 and IL-12Rβ2:WSX-1 for IL-35, proposed IL-23R:gp130 for IL-39 (Fig. 1A). Typical analysis of signal transduction pathways by IL-35-and IL-39-stimulation of established cell lines with defined expression of the different receptor combinations is still missing. This is, at least in part, due to major problems to express and purify recombinant IL-35 13,15 and general lack of recombinant IL-39.
Therefore, we decided to analyze signal transduction of the described IL-35 and potential IL-39 receptor complexes using synthetic cytokine receptors. Moreover, the synthetic cytokine receptors were used to generate additional thus far not described receptor complexes within the IL-12-type cytokine family. The synthetic cytokine receptors had a modular composition with the extracellular/membrane region (EXR) of one receptor fused to the intracellular region (IR) of another receptor, thereby enabling extracellular receptor activation by IL-12 or IL-23. In total we generated six synthetic receptor chimeras: IL-12Rβ2 EXR -gp130 IR , IL-12Rβ1 EXR -gp130 IR , IL-23R EXR -gp130 IR , IL-12Rβ1 EXR -IL-12Rβ2 IR , IL-12Rβ1 EXR -IL-23R IR , and IL-12Rβ1 EXR -WSX-1 IR (Fig. 1B). We verified the cell surface localization and expression of all synthetic receptor chimeras in Ba/F3 cells by flow cytometry and Western blotting (Fig. S2A,B). All receptor chimeras with the extracellular domains of IL-12Rβ1 and IL-12Rβ2 were as well expressed on the cell surface as their wild-type IL-12Rβ1 and IL-12Rβ2 receptors. The IL-23R EXR -gp130 IR variant was, however, much lower expressed on the cell surface as IL-23R. As cellular model system we used Ba/F3 cells to analyze signal transduction of synthetic IL-6/IL-12-type cytokine receptors. As described previously, Ba/F3 cells, stably expressing IL-12Rβ1:IL-12Rβ2 or IL-12Rβ1:IL-23R 18,19 , stimulated with IL-12 and IL-23 induced STAT1, STAT3 and Erk1/2 phosphorylation and cytokine-dependent cellular proliferation after stimulation with IL-12 and IL-23, respectively ( Fig. S3A-D). The transcription of the gene Pim-1 has been shown to be dependent on STAT3-activation 20 . Consequently, activation of IL-12 signal transduction resulted in the transcription of the STAT3 target gene Pim-1 (Fig. S2E). STAT4 phosphorylation was not analyzed, because STAT4 is not expressed in Ba/F3 cells 21 . Ba/F3 cells were stably transduced with a cDNA coding for human gp130 and responsive to Hyper-IL-6. Hyper-IL-6 (HIL-6) is a fusion protein of IL-6 and soluble IL-6R connected via a flexible peptide linker specifically inducing IL-6-trans-signaling via gp130 17 . Using these cells, Hyper-IL-6-induced proliferation and Pim-1 mRNA expression was observed for all cell lines and served as internal control (Fig. S3A,B,D,E). For induction of synthetic signal transduction the appropriate Hyper-cytokines for IL-12 and IL-23, HIL-12 and HIL-23, have been generated and expressed 18,19 (Fig. S3F).
The synthetic IL-35 receptor complexes consisting of IL-12Rβ2 and gp130 are biologically active. Next, we used our synthetic receptor chimeras to generate receptor combinations, which will be activated by IL-12 to induce IL-35 signal transduction.
Our results showed, that also hetero-dimerization of the intracellular regions of IL-12Rβ2 and WSX-1 which represents the fourth described IL-35 cytokine receptor complex resulted in signal transduction. As described previously, signal transduction of this receptor combination was canonical.

Analysis of IL-12-and IL-23-stimulation of synthetic receptor complexes mimicking IL-39 and
additional potential receptor complexes of the IL-12 family. Next, we used our synthetic cytokine receptor system to test the functional assembly of intracellular regions of IL-23R:gp130 for IL-39, IL-12Rβ1:gp130 for hypothetical IL-Y and IL-23R:IL-12Rβ2 for hypothetical IL-Z1 in Ba/F3 cells.
In conclusion, our combinatorial approach demonstrated that the combination of the intracellular regions of all tested intracellular receptor regions within the IL-12 family resulted in signal transduction which represent known (IL-39) and potential cytokine receptor combinations which have not yet been described to be functionally addressed by a cytokine of the IL-6/IL-12 family.

Discussion
In this study, we generated synthetic chimeric receptors of the IL-12/IL-6 type-cytokine family to mimic IL-35 and IL-39 signaling and to decipher additional potential receptor combinations within the IL-12 type cytokine family. Our receptor shuffling approach demonstrated that the IL-12 cytokine receptors are assembled as exchangeable, modular domain structures.
IL-35 is produced by forkhead box P3 + Treg cells and activated B cells and has important roles in preventing autoimmunity, maintaining self-tolerance, and suppressing antitumor immune responses 9 . Some difficulties with IL-35-induced signal transduction have emerged, since this cytokine cannot be produced in bacterial expression systems 15 and only one group was able to produce very limited amounts in insect cells 13 . Secretion of IL-35 in eukaryotic cell culture supernatant was, however, not observed 15 . This was due to the retention of IL-35 and Hyper-IL-35 in the ER-golgi-system of producing cells. Other IL-6/IL-12-type Hyper-cytokines, such as Hyper-IL-6, Hyper-IL-12 and Hyper-IL-27 were secreted into cell culture supernatants and biologically active. Interestingly, even though in vitro reconstitution of biologically IL-12 was possible, using p40 from eukaryotic cells with p35 purified and refolded from E.coli, the same p35 did not form a biologically active complex with EBI3 obtained from eukaryotic cells 15 . This lack of recombinant, purified, biologically active cytokine has prevented the analysis of IL-35 signal transduction in typical cellular model systems, such as Ba/F3 cells 17,18 . the presence of 4 ng/ml HIL-12. Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. HIL-6-induced proliferation (10 ng/ml) was set to 100%. One representative experiment out of three is shown. Error bars represent SD. Statistical analysis used a Welch t test (n = 3; **p ≤ 0.01; ***p ≤ 0.001). (E) Schematic overview of IL-39-type signaling by IL-23-induced receptor activation of IL-12Rβ1 EXR -gp130 IR and IL-23R. (F) Representative histograms of IL-12Rβ1 EXR -gp130 IR (upper panel) and IL-23R (lower panel) surface expression of Ba/F3-gp130/IL-12Rβ1 EXR -gp130 IR /IL-23R cells (light solid lines). Gray-shaded areas indicate Ba/ F3-gp130 cells (negative control). (G) Analysis of STAT1/3 and Erk1/2 activation of Ba/F3-gp130/IL-12Rβ1 EXR -gp130 IR and Ba/F3-gp130/IL-12Rβ1 EXR -gp130 IR /IL-23R cells as described in (C). Western blot data show one representative experiment out of three. (H) Cellular proliferation of Ba/F3-gp130/IL-12Rβ1 EXR -gp130 IR and Ba/ F3-gp130/IL-12Rβ1 EXR -gp130 IR /IL-23R cells as described in (D). One representative experiment out of three is shown. Error bars represent SD. Statistical analysis used a Welch t test (n = 3; ***p ≤ 0.001). Ba/F3-gp130/IL-12Rβ2 EXR -gp130 IR and Ba/F3-gp130/IL-12Rβ1/IL-12Rβ2 EXR -gp130 IR cells were washed three times, starved, and stimulated with 4 ng/ml HIL-12 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT1/3/Erk1/2 and STAT1/3/Erk1/2. Western blot data show one representative experiment out of three. (D) Cellular proliferation of Ba/F3-gp130/IL-12Rβ2 EXR -gp130 IR and Ba/F3-gp130/ IL-12Rβ1/IL-12Rβ2 EXR -gp130 IR cells. Equal numbers of cells were cultured for 3 days in the presence of 4 ng/ ml HIL-12. Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. HIL-6-Here, the chimeric receptor strategy enabled the biologically active reconstitution of all described IL-35-induced receptor complexes, namely the two hetero-dimeric receptor complexes IL-12Rβ2/gp130 and IL-12Rβ2/WSX-1 and the two homo-dimeric receptor complexes of IL-12Rβ2/IL-12Rβ2 and gp130/gp130. Since the chimeric receptors contained the extracellular domains for IL-12Rβ1 and IL-12Rβ2, activation of IL-35 signaling was inducible by IL-12. The degree of STAT-activation between the individual cytokines seems to vary. Nevertheless, it has been clearly demonstrated that in principal all IL-6 and IL-12 family cytokines activate the same pattern of STAT proteins, mainly STAT1, STAT3, STAT4 and to a lesser extent also STAT5. The only known exception to date is IL-35. Previously, using primary T cells STAT1 and STAT4 phosphorylation was shown for IL-12Rβ2/gp130, only STAT4 phosphorylation for IL-12Rβ2/IL-12Rβ2 and only STAT1 phosphorylation for gp130/gp130, whereas Erk1/2 phosphorylation was not analyzed 11 . Homo-dimerization of gp130 without the need of a membrane-bound α-receptor such as IL-6R or IL-11R has besides IL-35 previously been observed for viral IL-6 23 or IL-6 in complex with the soluble IL-6R 24 . However, in both cases phosphorylation of STAT1 and STAT3 was detected, whereas IL-35 solely activates STAT1 11 . Using Ba/F3 cells, STAT1, 3 and Erk1/2 phosphorylation was detected for gp130 containing receptor complexes IL-12Rβ2/gp130 and gp130/gp130. Interestingly, STAT3 phosphorylation, weak Erk1/2 phosphorylation and no STAT1 phosphorylation were detected for IL-12Rβ2 homo-dimers. IL-35 signaling in primary B cells was mediated by IL-12Rβ2 and WSX-1 and resulted in STAT1, 3 and 5 phosphorylation. IL-35 also activates primary T cells via IL-12Rβ2 and WSX-1 where it induced STAT1, 3 and 4 phosphorylation. STAT5 and STAT6 phosphorylation was not detected 13 . In Ba/F3 cells, however, the chimeric IL-35-mimicking IL-12Rβ2:WSX-1-receptor complex resulted in STAT1 and 3 phosphorylation but no Erk1/2-activation. Since Ba/F3 cells lack expression of STAT4, we cannot exclude that this might influence the signaling behavior of some chimeric receptors. Interestingly, STAT proteins appear to be at least to some extent interchangeable. The lack of STAT3 in murine embryonic fibroblasts shifted IL-6-signaling to an IFNγ-like response mediated by compensatory STAT1 activation 25 . The data presented here demonstrate that IL-35 signaling has the potential to induce almost canonical signaling as would be expected from a novel member of the IL-6/ IL-12 type cytokine family. To this end, it is not clear why IL-35 induces different signaling patterns in the other studies 11,13 . One explanation might be that primary T and B cells have imprinted specialized signaling pathways. In a cellular model system, such as Ba/F3 cells, such imprinting is not present and the IL-35-mimicking receptors were able to execute more canonical signal transduction.
Recently, IL-39, which is composed of IL-23_p19 and EBI3, was supposed to induce signal transduction via IL-23R and gp130 9 . IL-39 is produced by activated B cells and induced differentiation and/or expansion of neutrophils, and contributes to lupus-like diseases in MRL/lpr mice 6,22 . Production of IL-39 by keratinocytes appeared to contribute to wound healing by dampening inflammatory responses 26 . The formation of IL-39 was, however, only demonstrated in culture supernatants by immune-precipitation 6 , and recombinant production and purification of biologically active IL-39 was not accomplished to date. Using chimeric receptors, the IL-23R:gp130 heterodimer was shown to induce STAT1 and STAT3 phosphorylation, which is on good agreement with the published STAT phosphorylation profiles of IL-39 6 . Interestingly, IL-12-and IL-23-induced IL-39 signaling resulted in comparable STAT3 but different STAT1 activation, which demonstrate that not only the intracellular domains determine the signaling strength but also the extracellular assembly. Recently a similar effect was described for the EPO-receptor. A point mutation in EPO was shown to reduce EPO receptor dimerization, which resulted in reduced STAT1 and STAT3 but not in reduced STAT5 activation 27 . In addition we also demonstrated Erk1/2 activation.
In a final step, two additional potential receptor combinations IL-12Rβ1:gp130 and IL-12Rβ2:IL-23R were shown to form biologically active receptor complexes, which might be activated by thus far unknown cytokines, IL-Y and IL-Z1, respectively. Albeit, we have not functionally tested the two remaining potential combinations of IL-6/IL-12 receptors, the WSX-1:IL-12Rβ1 alternative for IL-Y and WSX-1:IL-23R for IL-Z2, from our data we strongly assume that these combinations would also result in active receptor complexes.
In conclusion, all tested combinations of cytokine receptors of the IL-12 type cytokine family were biologically active, which illustrates the combinatory potential among this family. Interestingly, even though non-conventional STAT activation pattern were described for IL-35, we clearly demonstrate that these combinations induce more conventional STAT activation patterns.