Recombinant p35 from Bacteria Can Form Interleukin (IL-)12, but Not IL-35

The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.


Introduction
Cytokines were grouped into distinct families, mostly upon structural features and not upon homology among the amino acid sequences. These features include the protein fold or the usage of certain membrane-bound cytokine b-receptors, which are needed for signal transduction [1,2]. Members of the IL-6 and IL-12 families have pleiotropic functions and are critically involved in proliferation, apoptosis and differentiation of T cells. Interestingly, members of both families share cytokine subunits as well as cellular receptors, suggesting a yet only poorly understood cross-talk between IL-6 and IL-12 type cytokines [1].
Here, we show that IL-35, in contrast to IL-12 and IL-23, is not efficiently secreted from transfected cells, making the biochemical characterization of IL-35 signaling impossible. To enable the analysis of IL-35 signal transduction, we develop a protocol to purify recombinant biologically active p35 bac from E.coli. Combining p35 bac with p40 C197A , a solely monomeric form of p40, resulted in biologically active IL-12 that induced STAT1 and STAT3 phosphorylation as well as cytokine-dependent cellular proliferation. In contrast to this, recombinant p35 bac did not form biologically active IL-35 when combined with EBI3.

Plasmids
Expression plasmids for Hyper-IL-6, Hyper-IL-27 and Hyper-IL-30 have been described previously [17]. To clone the Hyper-IL-35 cDNA the pcEP-PU plasmid coding for Hyper-IL-27 was used as a template. Hyper-IL-27 consists of a signal peptide (METDTLLLWVLLLWVPGSTGD), mEBI3, a linker peptide (VPGVGVPGVG), mp28 and a myc-and His-tag. To insert a SmaI restriction site between linker and p28 by site directed mutagenesis this vector was digested with AflII and NotI and the sticky ends were filled up using Klenow fragment (Thermo Scientific, Schwerte, Germany) in order to subclone the fragment by blunt end ligation into the pCR-Script vector.
To test the influence of the linker on the secretion of overexpressed IL-35 we exchanged it by a 3xGGGGS-linker. To achieve this the double stranded oligonucleotide coding for the GGGGS-linker was inserted into pcR-Script-IL-35 through BamHI and SmaI excising EBI3 and the former linker. EBI was then amplified by PCR (Fwd-Primer: GATCAAGCTTTATG-GAGACAGACACACTCC, Rev-Primer: GATCGGATCCCT-TATGGGGTGCACTTT) inserting a HindIII site in 59 and a BamHI site in 39 of EBI3 and then cloned into the vector containing the GGGGS-linker and p35 through these restriction enzymes. The resulting cDNA was subloned into pcEP-PU through HindIII and XhoI. The cDNA coding for Hyper-IL-12 was cloned in analogy into pcEP-PU to generate a Hyper-IL-12 with myc and his-tag.

Expression, purification, and renaturation of murine p35 bac
For gene expression 1 l of LB medium supplemented with 100 mg/ml ampicillin was inoculated from an overnight starter culture and grown to on optical density at 600 nm (OD 600 ) of 0.6 at 37uC. Protein production was induced by addition of 0.1 mM isopropyl 1-thio-b-galactopyranoside. Following incubation at 37uC for additional 4 h bacteria were harvested by centrifugation (40006g, 4uC). Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0 containing 6 M guanidine hydrochloride and 10 mM imidazole). Cell lysis was carried out by sonification (Bandelin Sonoplus HD70). Lysates were cleared by centrifugation (100006g, 20 min, 4uC) and supernatants loaded on a 1 ml HisTrap HP column (GE Healthcare) equilibrated with lysis buffer. Bound proteins were eluted with lysis buffer containing 500 mM imidazole. Imidazole was removed from mp35 bac samples by ultrafiltration using Amicon Ultra-15 filters (Millipore) with a 10,000 Da molecular weight cut off. Denatured mp35 bac was refolded at a concentration of 1 mg/ml by dialysis (4uC) against 50 mM Tris-HCl (pH 8.0) containing 250 mM NaCl. Dialysed mp35 bac was cleared by centrifugation (10006g, 20 min, 4uC), concentrated by ultrafiltration and applied to a Superdex 75 10/300 GL (GE Healthcare) column connected to an Ä kta Purifier 10 system. Fractions containing mp35 bac were concentrated by ultrafiltration. Purified proteins were analyzed by SDS-PAGE.
Purification of CD4+ T cells 6-8 weeks old C57/BL6J mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). CD4 + T cells were enriched by positive selection using the CD4 (L3T4) MicroBeads (Miltenyi) from single cell suspended splenocytes and lymphnode cells following the manufacturer's instructions. 1610 5 cells per well were cultured on plates coated with 0.5 mg/ ml anti-CD3 (eBioscience) and 2 mg/ml soluble anti-CD28 (BD Pharmingen). 10% of the indicated cell culture supernatant was added and supernatant was harvested after three days to assess the concentration of IFN-c.

Cytokine stimulation of cells and cell lysis
For cytokine stimulation Ba/F3-gp130 or Ba/F3-gp130-IL-12Rb1-IL-12Rb2 cells were washed twice in PBS and subsequently starved for 3 h in serum-free DMEM. Conditioned supernatants were preincubated with recombinant p35 variants for 30 min at 37uC and added to 2610 6 cells at the concentrations indicated. The cells were stimulated for 15 min at 37uC, collected by centrifugation, and the pellet was lyzed in 60 ml of 2.5 x Laemmli-Buffer.

Pulldown assays
To assess binding of the different cytokine subunits to each other, COS7 cells were transiently transfected using TurboFect transfection reagent (Thermo Scientific, Schwerte, Germany) according to the manufacturer's instructions. After 48 h cells were washed in PBS and lyzed in 250 ml of immunoprecipitation buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM sodium orthovanadate and Complete Protease Inhibitor Cocktail Tablets (Roche, Mannheim, Germany)) for 1 h at 4uC. The lysates were centrifuged at 1000 rpm for 3 min and 100 ml of each of the supernatants were mixed and incubated overnight at 4uC. After adding 75 ml of Protein-Aagarose beads (Thermo Scientific, Schwerte, Germany) and incubation for 4 h at 4uC beads were spun down at 1000 rpm for 5 min at 4uC and 10% of the supernatant was used for Western blotting. The beads were washed five times with 500 ml immunoprecipitation buffer, boiled in 50 ml 1 x Laemmli Buffer and 50% were loaded on a 15% SDS-PAGE gel.

SDS-PAGE and Western Blotting
Lysates of COS7 cells with overexpressed cytokine subunits were prepared by suspending the cells in 250 ml of mild lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100 and Complete Protease Inhibitor Cocktail Tablets (Roche, Mannheim, Germany)), incubating them for 30 min on ice, centrifuging at 13,000 rpm for 5 min at 4uC and transferring the supernatant to a new tube. Total protein concentration was determined by applying the BCA protein assay kit (Thermo Scientific, Schwerte, Germany). 50 mg of total protein was separated by SDS-PAGE.
Semi-dry Western blotting was performed using the Trans-Blot Turbo Transfer System from Bio-Rad (München, Germany).

Proliferation assays
Proliferation of the different Ba/F3-gp130 cell lines was determined as described previously [22] using the Cell Titer Blue Cell viability assay reagent (Promega, Karlsruhe, Germany) following the manufacturer's protocol. The extinction was measured using a Tecan infinite M200 PRO reader (excitation 560 nm, emission 590 nm, gain 90, i-control 1.7 software, Tecan AG, Maennedorf, Switzerland). Normalization of relative light units (RLU) was achieved by subtraction of negative control values. All values were measured in triplicates per experiment.
The heterodimeric cytokines IL-12 and IL-27, but not IL-35, were secreted and biologically active To test if the protein fusion of mp35 and mEBI3 prevented secretion of biologically active IL-35, we sought to reconstitute heterodimeric IL-35 by co-transfection of two separate cDNAs coding for mp35 and mEBI3 ( Figure 3A). First, we tested the ability of the different subunits to form the other heterodimeric cytokines IL-12, IL-23 and IL-27. Co-transfection of mp19/mp40 ( Figure 3A Figure 3D, lane 6). These results suggest that EBI3 and p35 interact intracellularly, but cannot be efficiently secreted. These experiments confirmed our findings with the single-chain Hyper-IL-35 protein and are in line with previous findings that secretion of IL-35 is relatively poor, compared to that of IL-12 and IL-27 [2].

p35 interacts with EBI3
Next, we verified if p35 and EBI3 interacted with each other when expressed in mammalian cells via pulldown [2,11,25]. Fctagged EBI3, flag-tagged p35 and flag-tagged p19 were separately expressed in HEK293 cells. Since IL-35 was not secreted, cell lysates were used to check for interaction. The cell lysate containing EBI3 was mixed with lysate containing either p35 or p19 and incubated overnight to allow protein complex formation. Afterwards, EBI3 was precipitated with Protein-A-agarose beads via the Fc tag. As shown in Figure 4A, flag-tagged p35 was precipitated with Fc-tagged EBI3. Incubation of Protein-A-agarose beads with lysate containing only p35 did not reveal any p35 binding, demonstrating that p35 specifically interacted with EBI3. In contrast, EBI3 did not precipitate flag-tagged p19, underlining the specificity of the EBI3/p35 interaction ( Figure 4B). Again, p19 alone did also not bind to the Protein-A-agarose beads in the absence of EBI3 ( Figure 4B). These results show that the lack of secretion of heterodimeric IL-35 cannot be explained through impaired interaction of the two proteins within the cell. We cannot exclude that other, so far unknown, proteins are needed for IL-35 secretion that are not present in our in vitro setting, but facilitate IL-35 secretion in vivo.
The two cysteine residues that form the inter-molecular disulfide bond between p40 and p35 are dispensable for IL-12 action In contrast to IL-27, IL-12 is stabilized by an inter-chain disulfide bond between cysteine 92 of p35 and cysteine 197 of p40 (p35 C92 -p40 C197 ) ( Figure 5A and [28]). In human p35, the corresponding cysteine 74 forms an inter-chain disulfide bridge with cysteine 199 of p40. The p35 C74S mutant was still able to form complexes with p40 [28]. Consequently, Cys92 in p35 was mutated to alanine (p35 C92A ) or serine (p35 C92S ). In the absence of p35, IL-12p40 is secreted as an antagonistic disulfide-linked homodimer (p80) [4,5,6]. Gillessen et al. [4] have previously shown that treatment of p80 with reducing agents destroyed the p80 dimer and resulted in monomeric p40, suggesting that p80 is linked by an inter-chain disulfide bridge. To prevent p40 homodimerization, Cys197 of p40 was mutated to alanine (p40 C197A ). As expected, supernatant from transiently transfected HEK293 cells contained monomeric as well as dimeric p40 as demonstrated on a non-reducing gel ( Figure 5B). Mutation of Cys197 to Ala in p40 prevented dimer formation, as p40 C197A was solely monomeric ( Figure 5B). It was not known if this mutation still allows IL-12 formation. To answer this question, p35 C92A , p35 C92S and p40 C197A were expressed in transiently transfected HEK293 cells and analyzed by Western blotting ( Figure 5C). As shown before, transfection of p40 alone resulted in its secretion into the supernatant ( Figure 5C, lane 2). When co-expressed with p35, p35 C92A or p35 C92S , all three p35 variants were efficiently secreted in combination with p40 ( Figure 5C, lane 3-5). Next, we tested the ability of p40 C197A to facilitate secretion of p35, p35 C92A and p35 C92S . Whereas p40 C197A alone was again efficiently secreted ( Figure 5C, lane 6), we could only detect small amounts of p35 in the supernatant when co-expressed with p40 C197A (Figure 5C, lane 7). In contrast, co-expression of p40 C197A in combination with either p35 C92A or p35 C92S led to secretion of both IL-12 subunits ( Figure 5C, lane 8 and 9). We concluded from these results that the Cys92 of p35 and Cys197 of p40 were dispensable for IL-12 formation and secretion.
Taken together, these data show that the inter-chain disulfide bridge of p35 and p40 is dispensable for secretion and biological activity of IL-12.
In vitro reconstituted IL-12 composed of recombinant p35 from bacteria and p40 C192A from HEK293 cells is biologically active Since we did not obtain secreted p35 and IL-35 from cell culture supernatants of transfected mammalian cells, we decided to express p35 in bacteria and to combine this protein with p40 and EBI3-conditioned supernatant to in vitro reconstitute IL-12 and IL-35, respectively. Previously, we combined recombinant p28 from bacteria with conditioned cell culture supernatant containing EBI3 which led to in vitro reconstitution of IL-27 [17].
The cDNAs coding for mature murine p35 as well as p35 C92A were subcloned into the E.coli expression plasmid pet23a. p35 and p35 C92A were expressed in E.coli as inclusion bodies. After refolding, purification of monomeric p35 bac and p35 bac/C92A was completed by size-exclusion chromatography ( Figure 6A). Final yields of pure, monomeric p35 bac and p35 bac/C92A of about 15 and 35 mg/liter bacterial culture were obtained ( Figure 6B).

Heterodimeric IL-12 induces IFN-c secretion in primary CD4 + T cells
We have generated IL-12 by three different approaches. First, we have fused p40 and p35 via a peptide linker (Hyper-IL-12), which we expressed in HEK293 cells. Second, we have transfected HEK293 cells with cDNAs individually coding for the IL-12 subunits p40 and p35. Last, we have purified and refolded p35 expressed in E. coli, which in combination with p40-containing supernatant from HEK293 cells gave rise to IL-12. All kinds of IL-12 were able to induce proliferation of Ba/F3-gp130-IL-12Rb1-IL-12Rb2 cells and phosphorylation of the transcription factor STAT3. To have a second physiological readout of IL-12, we isolated CD4 + T cells from mice, activated them with a-CD3/a-CD28 antibodies and stimulated them with supernatant from transfected HEK293 cells. As shown in Figure 7A, supernatant containing p40/p35, p40/p35 C92A and p40/p35 C92S induced the secretion of Interferon-c (IFN-c, black bars), whereas supernatant from cells transfected with eGFP or p40 alone did not (white bars). Supernatant containing Hyper-IL-12, p40 C197A /p35 C92A or p40 C197A /p35 C92S induced IFN-c as well (dark gray bars), whereas supernatant from cells transfected with p40 C197A alone did not (white bar). Supernatant containing p40 C197A /p35, which only induced a weak proliferative response and little STAT3 phosphorylation ( Figure 5D, E), induced IFN-c secretion, although to a lesser extent than the other p40/p35 combinations ( Figure 7A, light grey bar), underlining the reduced capacity of p40 C197A /p35 to form biologically active IL-12.
Finally, we asked whether bacterial produced p35 was able to induce IFN-c secretion when combined with p40 C197A . As shown in Figure 7B, supernatant from eGFP or p40 C197A transfected cells induced little IFN-c secretion (white bars). Addition of recombinant p35 bac/C92A induced IFN-c secretion on its own (light gray bar). However, combination of p35 bac/C92A together with supernatant containing p40 C197A drastically increased IFN-c production (dark gray bar), which was comparable to cells stimulated with Hyper-IL-12 containing supernatant (black bar).
In conclusion, we could show that the three differently produced forms of IL-12 are equally well able to induce IFN-c secretion from primary T cells, a hallmark of IL-12 activity.
p35 bac fails to form biological active IL-35 After the successful generation of IL-12 in vitro by our approach, we finally asked if in vitro reconstituted IL-35 was also biologically active and able to induce STAT phosphorylation. Since IL-35 is not stabilized through a disulfide bridge, p35 bac/ C92A should in principle be able to form active IL-35 with EBI3 ( Figure 8A). Unfortunately, we could not detect phosphorylation of STAT1 or STAT3 when Ba/F3-gp130/IL-12Rb1/IL-12Rb2 cells were stimulated with IL-35 ( Figure 8B). As control, stimulation of Ba/F3-gp130-IL-12Rb1-IL-12Rb2 with Hyper-IL-12 and IL-27 lead to robust STAT1 and STAT3 phosphorylation. Furthermore, in vitro reconstituted IL-35 did not induce proliferation of Ba/F3-gp130-IL-12Rb1-IL-12Rb2 or Ba/F3-gp130 cells ( Figure 8C, D). In conclusion, we describe a novel way to produce biologically active IL-12 via bacterially expressed p35, but fail to create IL-35 by this approach.

Discussion
Our present study has three major findings. We describe for the first time a protocol for expression, purification and refolding of p35 from E.coli. In combination with p40, bacterially expressed p35 formed biologically active IL-12. Second, we identified the critical amino acid that links p40 monomers to form the IL-12/IL-23 inhibitor p80. Third, we show that bacterially expressed p35 was not able to form biologically active IL-35.
IL-12 (p35/p40) is stabilized by an inter-chain disulfide bond, linking p35 C92 and p40 C197 . We mutated both cysteins to alanine, which did not prevent IL-12 formation, but reduced its biological activity compared to wildtype IL-12. IL-12 signals via the receptor combination IL-12Rb1 and IL-12Rb2 [8]. We have previously shown that EBI3 in combination with recombinant p28 expressed in E.coli formed biologically active IL-27, showing that this is a valid approach to create composite cytokines [17]. We therefore produced recombinant p35 bac and the mutant p35 bac/C92A in E.coli and combined them analogous with supernatant containing p40 C197A . Biologically active IL-12 was formed, as demonstrated by cytokine-dependent proliferation of Ba/F3-gp130-IL-12Rb1-IL-12-Rb2 cells as well as phosphorylation of STAT1 and STAT3. It has to be noted, that bacterially expressed p35 was more than 100 fold less effective than recombinant Hyper-IL-12. In detail, at least 1 mg/ml bacterially expressed p35 in combination with p40 was needed to induce IL-12-dependent proliferation of Ba/F3-gp130-IL-12Rb1-IL-12-Rb2 cells. We hypothesize that this was due to ineffective refolding of p35 from inclusion bodies. Only about 20 mg monomeric p35 was refolded from 1 l bacterial culture. The minimal amounts of p35 prevented the characterization of p35 via e.g. circular dichroism (CD)-spectroscopy. It might be speculated that only a small percentage of the pure monomeric p35 fraction were correctly refolded and biologically active, whereas the majority was misfolded or partly unfolded but soluble and biologically inactive. Irrespective of the low biological activity of the refolded p35, we concluded that EBI3 as well as p35 bac were functional.    It was previously shown that mutation of C92 to serine within p35 did not prevent IL-12 formation and function, suggesting that the disulfide bond between p40 and p35 mediates stability, but is not needed for IL-12 activity [28]. We verified these results by introducing the C92S and a C92A point mutation in p35, which both were biologically active. Furthermore, we mutated the respective cysteine 192 in p40, which has not been done before. Co-expression of p40 C197A and p35 C92A in HEK293 cells gave rise to biologically active IL-12, albeit with lower activity compared to wildtype p35 and p40. Interestingly, p40 can be found in vivo as disulfide-linked p80 homodimer, and this makes up to one third of the total amount of p40 [7]. We show for the first time that Cys197 of p40 is needed for p80 formation, as p40 C197A is only found as a monomer. This means that p40 can either form IL-12 within the ER with p35 via p40 C197 -p35 C92 , or p80 via p40 C197 -p40 C197 .
One hallmark of IL-6-type cytokines is their ability to signal via the membrane-bound b-receptor gp130. Members of this family recruit specific, in part overlapping, gp130 homo-and heterodimers [1]. In all known cases, this leads to activation of certain downstream signaling cascades, whereas the Jak/STAT-pathway seems to be the major one. Among the seven known STAT proteins, STAT1 and STAT3 (and to a lesser degree STAT5) are phosphorylated after IL-6-type cytokine activation. The degree of STAT-activation between the individual cytokines seems to vary, as IL-27 predominantly activates STAT1 over STAT3. Nevertheless, it has been clearly demonstrated that in principal all IL-6 and IL-12 family cytokines activate the same pattern of STAT proteins. The only known exception to date is IL-35, which can signal via a gp130 homodimer, but solely induces phosphorylation of STAT1, not STAT3. In contrast, IL-6, IL-11 or Hyper-IL-6, which also recruit a gp130 homodimer, induce phosphorylation of both STAT1 and STAT3. To date, this finding is unique for IL-35, as it has not been seen by any other IL-6 type-cytokine. A molecular mechanism that explains this interesting finding is still missing.
Receptor plasticity is a well-documented phenomenon for IL-6type cytokines [1]. One example is CNTF which usually binds to the non-signaling CNTFRa and recruits a b-receptor heterodimer of gp130/LIFR [29]. Additionally, CNTF can use the IL-6R as alpha-receptor, and this complex also engages gp130/LIFR for signal transduction, showing the use of different a-receptors by the same cytokine [30]. Interestingly, plasticity is not limited to the level of the a-receptor. Oncostatin M (OSM), another IL-6-type cytokine, does not need an a-receptor for signaling, but in contrast directly activates a b-receptor heterodimer of either gp130/LIFR or gp130/OSMR. Since OSM is the only known cytokine that signals through OSMR, the distinct expression of either OSMR makes a cell responsive to OSM alone, whereas LIFR expression allows signal initiation by other cytokines, including OSM, LIF and CT-1 [1]. Another example is IL-30 (IL-27p28), which can signal in combination with EBI3 as IL-27 via gp130/WSX-1, but has been shown to have signaling capacities on its own, as either in combination with cytokine-like factor (CLF) or alone is able to initiate signaling via the IL-6R [17,31,32]. In contrast to IL-27, IL-30/IL-6R recruits a gp130 homodimer, and revealed plasticity on the level of the aas well as the b-receptor [17].
IL-35 has been shown to engage four different b-receptor complexes [10,12]. Homodimerization of gp130 without the need of a membrane-bound a-receptor has besides IL-35 been seen by viral IL-6 [33] or IL-6 in complex with the soluble IL-6R [16,34]. However, in both cases phosphorylation of STAT1 and STAT3 was detected, whereas IL-35 solely activates STAT1 [10]. This finding suggests a different, yet unsolved mechanism by which IL-35 engages gp130 homodimerization, that is clearly different by all other known cytokines like IL-6, IL-11, IL-30, Hyper-IL-6 or viral IL-6. and EBI3 (blue). C92 of p35, which is not connected via a disulfide bond with EBI3, is highlighted with a red circle. (B) Equal amounts of Ba/F3-gp130-IL-12Rb1-IL-12Rb2 cells were incubated with 50% conditioned supernatant containing EBI3 with or without 2 mg/ml p35 bac/C92A for 15 min. Phosphorylation of STAT1 and STAT3 was determined per Western blotting. Total amounts of STAT1 and STAT3 were visualized as internal loading control. (C) Equal amounts of Ba/F3-gp130-IL-12Rb1-IL-12Rb2 cells were incubated with 10 ng/ml Hyper-IL-6 or 10% conditioned supernatant containing EBI3, either with or without 4 mg/ml recombinant p35 bac . Cellular proliferation was determined 48 h later as described in Materials and Methods. (D) The experiment was performed as described under panel (C), but with Ba/F3-gp130 instead of Ba/F3-gp130-IL-12Rb1-IL-12Rb2 cells. Cellular proliferation was determined 48 h later as described in Materials and Methods. The Western blots shown as well as the proliferation assays are representative of three different experiments with similar outcomes. doi:10.1371/journal.pone.0107990.g008 However, we were not able to verify this specific STAT1 activation by IL-35. Hyper-IL-35 was the only cytokine tested that was not secreted from cells. Furthermore, unlinked EBI3/p35 was not detected in the cell supernatant. From our experiments we concluded that EBI3 (in combination with p28 bac [17]) and p35 bac in combination with p40 were correctly folded and biologically active. However, when p35 and EBI3 were combined, for unknown reasons no active IL-35 was formed. One possibility is that IL-35 needs additional, yet unidentified factors, for efficient biological activity and secretion that were not included in our assays.
Receptor plasticity and cross-talk within the IL-6 and IL-12 families complicates the functional investigation and assignment of individual cytokines, for example in knock-out models. Bacterial expression and refolding of specific cytokines or cytokine subunits especially of poorly secreted species as demonstrated here for p35 can help to circumvent these limitations and thus enhance the understanding of these cytokines.
In summary, our study shows novel aspects in IL-12 biology and highlights that IL-35 differs in several aspects from the other IL-6/ IL-12 family cytokines.