C57BL/6, BALB/c, and RAG1^−/− C57BL/6 mice were purchased from Harlan laboratories. OT-I and OT-II transgenic mice were maintained and bred in our own facility. All animals were kept and bred in a specific pathogen free animal facility. For all experiments, female, age-matched (6–8 weeks) mice were used. All animal experiments were performed after approval by the cantonal veterinary office (Service de la consommation et des affaires vétérinaires, Département du territoire et de l’environnement, Permission no. VD2490.1).

Murine tumor DC lines are derived from splenic tumors isolated from transgenic CD11c:SV40LgT C57BL/6 mice (8). Their phenotypical and functional characteristics have been thoroughly described by Fuertes Marraco et al. (9) and Duval et al. (10). A single-chain IL-35 construct, consisting of Ebi3 (Il27b) and p35 (Il12a), linked by (Gly₄Ser)₃ was amplified from mouse DCs, respectively, IL-12 expression vector (courtesy by B. Becher, Zürich) and cloned into pRRLSIN.cPPT.PGK-GFP.WPRE lentiviral expression vector (courtesy by D. Trono, Lausanne). The reading frame of Ebi3-(Gly₄Ser)₃-P35 was sequenced to exclude mutations. Lentiviral particles containing either an empty or an IL-35 expression vector were produced in 293T HEK cells. Wild-type MuTu DC line was stably transduced with either empty vector (mock DC) or IL-35 lentiviral particles (IL-35⁺ DC) (MOI = 20) and the cells maintained in vitro for maximally 15 passages. Transgene expression was confirmed after each transduction by RT-PCR using the following primers: Ebi3: 5′-ATGTCCAAGCTGCTCTTCCT-3′ (forward), 5′-AGAGGAGTCCAGGAGCAGTC-3′(reverse). P35: 5′-AAATGAAGCTCTGCATCCTGC-3′(forward), 5′-TCACCCTGTTGATGGTCACG-3′ (reverse). All MuTu DC lines were cultured in IMDM–Glutamax (GIBCO) supplemented with 10% heat-inactivated fetal calf serum, 10 mM HEPES (GIBCO), 50 µM β-mercaptoethanol (GIBCO), 50 U/ml of penicillin, and 10 µg/ml streptomycin (GIBCO) at 37°C in 5% CO₂ atmosphere.

Interleukin-35 was precipitated from cell lysate using IL-12p35-specific antibodies (R&D systems, clone #45806) and protein A/G beads (Santa Cruz Biotechnology). Western blotting was conducted as described previously. In brief, cell lysates were centrifuged at 12,000 rpm for 30 min at 4°C. Protein concentrations of cell lysates were determined with the BCA assay (Pierce) and 40 µg of proteins were loaded onto 7.5–15% SDS-polyacrylamide gels. The gels were transferred to nitrocellulose membrane (Amersham Pharmacia Biotech). The blot was blocked in blocking buffer (5% non-fat dry milk/1% Tween 20 in PBS) for 2 h at room temperature and then incubated with Ebi3-specific antibody (Shenandoah Biotechnology, clone V1.4H6.29) in TBST overnight at 4°C. The blot was incubated with HRP-coupled secondary antibody for 2 h at room temperature. Blot was visualized by ECL (Pierce Biotechnology).

B16.F0 melanoma and CMT93 colon carcinoma tumor cell lines were cultured in DMEM medium (GIBCO), supplemented with 10% FCS, and 50 U/ml of penicillin and 50 mg/ml streptomycin (GIBCO). Tumor cell lysate was generated by five consecutive freeze/thaw cycles in liquid nitrogen. Lysate was centrifuged at 1500 g for 15 min before use. Mock- or IL-35-transduced DCs were pulsed with respective lysate in a 1:4 lysate to DC ratio and 100 U/ml IFN-γ (eBioscience). Five and three days before and three days after tumor cell transfer, 2.5 × 10⁶ DCs were s.c. injected in the flank. Tumor was induced by s.c. injection of 2 × 10⁵ B16.F0 or 2 × 10⁶ CMT93 cells into the same flank. Tumor growth was followed by measuring length and width using a caliper. Tumor volume was measured using the formula V=π6⋅1.58⋅(length⋅width)32 as described by Wurzenberger et al. (12).

The fluorochrome-conjugated antibodies used were specific to CD11c (clone N418, PECy7), GR1 (clone RB6-8C5, PE, or PerCP-PECy5.5), CD8a (clone 54-6.7, FITC, or APC-Cy7), DEC205 (clone 205yekta, PerCP-eF710), CD24 (cloneM1/69, PerCP-Cy5.5), Clec9A (clone 42D2, PE), CD11b (clone M1/70, APC), CD3 (clone 500A2, ef450), CD4 (clone RM4-5, APC, eF450), MHCI (clone AF6-88.5.5.3, APC), MHCII (clone M5/114.15.2, PE), CD40 (clone 1C10, APC), CD80 (clone 16-10A1, PECy5), CD86 (clone GL1, APC, or Alexa750), panNK (clone DX5, FITC), Ly6C (clone HK1.4, APC), and Ly6G (clone 1A8, PerCP-Cy5.5). For intracellular staining, the T cells were restimulated for 4 h with 10 ng/ml PMA and 500 ng/ml Ionomycin, in the presence of 10 µg/ml Brefeldin A. After extracellular staining, cells were fixed for 30 min in 4% paraformaldehyde and permeabilized in 0.5% Saponin buffer for 30 min. Intracellular staining was performed in Saponin buffer for 30 min. All antibodies were acquired from eBioscience or Biolegend. Flow cytometric analyses were performed on LSRII cytometer (Becton Dickinson) using FACS Diva6 (Becton Dickinson) and FlowJoX (Tree Star) software for data processing.

Adoptive transfer EAE was induced as described by Miller and Karpus (13). Briefly, myelin oligodendrocyte glycoprotein (MOG) reactive T cells were primed in donor C57BL/6 mice by two sub-cutaneous injections of MOG_35–55-peptide emulsified in complete Freud’s Adjuvant (Sigma). Intraperitoneal injection of 200 ng Pertussis Toxin (Sigma) was used to boost lymphocyte priming. In order to study the effect of tolerogenic molecules, MOG_35–55 peptide-pulsed DCs were intravenously injected 1 day before and 1 day after the immunization. Twelve days post immunization, inguinal, brachial, and axillary lymph nodes were isolated by digestion in 1 mg/ml Collagenase D (Roche) for 20 min at 37°C. Single cells suspension was prepared using a 40-µm cell strainer (Falcon). Lymph node cells were expanded in vitro for 3 days in the presence of MOG_35–55-pulsed DCs and 0.5 ng/ml recombinant IL-12p70 (eBioscience). Then, 5 × 10⁶ MOG-specific T cells were then transferred to C57BL/6 recipient mice by i.p. injection. Mice were i.v. injected with MOG_35–55-pulsed DCs 1 day after T cell transfer. Clinical EAE symptoms were scored daily in a blinded manner: 0—no obvious changes in motor functions; 1—decreased tail tonus; 2—abnormal gait (ataxia and/or impaired righting reflex); 3—partial hind limb paralysis; 4—complete hind limb paralysis; and 5—complete hind and fore limb paralysis.

Total RNA was isolated from the cells and purified using the RNeasy^® spin columns following the manufacturer’s protocol (Qiagen). Total RNA yields were quantified by Nanodrop spectrophotometry (Thermo Fisher Scientific). Synthesis of cDNA was performed using random nonamer primers and the Superscript II Reverse Transcriptase kit (Invitrogen). cDNA was purified using the PCR clean-up kit (Qiagen) and stored at −80°C.

Real-time PCR (RT-PCR) was performed in technical triplicates using SYBR FAST qPCR mix (KapaBiosystems) and 5 ng RNA/reaction on a LightCycler480 machine (Roche Diagnostics). Relative expression levels were analyzed using qBase+ 1.2 data analysis software (BioGazelle). Gene expression for each individual sample was normalized to the housekeeping genes HPRT 5′-AGCCTAAGATGAGCGCC-3′ (forward) and 5′-TTACTAGGCAGATGGCCACA-3′ (reverse) and β-actin 5′-GCACAGCTTCTTTGCAGCTCCTTCG-3′ (forward) and 5′-TTTGCACATGCCGGAGCCGTTG-3′ (reverse).

Differences between groups were determined using either unpaired two-tailed Student’s t-test or Mann–Whitney U test where indicated. All statistical analyses were performed using Prism 6.0 (GraphPad). p-Values are indicated when considered statistically significant (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001).

A stable murine DC line, constitutively expressing a biologically functional, single-chain IL-35 construct (Supplementary Figure 1A) was generated by lentiviral transduction of the recently established CD8α⁺ MuTu DC line (thereafter named “wtDC”) (9). Il12p35 and Ebi3 transcripts were detected via quantitative PCR (Supplementary Figure 1B). Ebi3 protein expression was confirmed by western blot of the cell lysate of cultured IL-35-expressing DCs after immunoprecipitation using anti-IL-12p35 antibody (Supplementary Figure 1C). For the remainder of this manuscript, the IL-35-expressing MuTu DC line is referred to as “IL-35⁺ DC.”

Additionally, empty vector-transduced DCs (mock DCs) were generated and used as controls when appropriate. We could not observe any noticeable differences in the morphological characteristics or the proliferative capacity when comparing the wtDC line to the lentivirally transduced MuTu DC lines. Six independent transductions of MuTu DCs were conducted yielding IL-35-expressing DCs with comparable functional and phenotypic properties. The IL-35⁺ MuTu DC cell line was assessed for the expression of a panel of surface markers commonly used to characterize CD8α⁺ DCs by direct comparison to mock DCs. Flow cytometric analysis revealed a marked reduction of CD11c, CD8α, and DEC205 surface expression on IL-35⁺ DCs. By contrast, the expression of CD11b was drastically increased (Figure 1A). Furthermore, the IL-35-expressing DC line exhibited lower expression of MHC I and MHC II as well as costimulatory molecules CD40 and CD86 (Figure 1B). Activation of the control DCs with TLR3 and TLR9 ligands polyI:C and CpG led to increased surface expression of MHC II as well as costimulatory receptors CD40, CD80, and CD86 by control DCs. Conversely, IL-35⁺ DCs exhibited a complete unresponsiveness to TLR stimulation as indicated by the failure to upregulate any of the analyzed (co-)stimulatory molecules. Interestingly, IL-35⁺ DCs showed an increased CD80 expression already under resting condition when compared to mock DCs. A more than twofold reduction in IL-12p40 secretion could be detected by ELISA in the culture medium of IL-35⁺ DCs while the production of the immunoregulatory cytokine IL-10 was found to be increased significantly (Figure 1C).

In order to investigate whether the observed phenotypic change of IL-35⁺ DCs is provoked by an autocrine effect of the transgene, wild-type MuTu DCs were cultured for 4 days in IL-35 containing culture medium. As observed for IL-35-transduced MuTu DCs, IL-35-conditioned wild-type DCs expressed markedly higher levels of CD11b. On the contrary, the expression of CD11c, CD8α, and DEC205 was not significantly altered when compared to cells cultured in control medium (Figure 2A). Upon activation with TLR3 and TLR9 ligands, IL-35-conditioned but not control DCs exhibited a significantly impaired upregulation of MHCII and the costimulatory receptor CD40. Similar to IL-35⁺ DCs, IL-35 conditioning increased the expression of CD80 already under steady-state conditions and TLR stimulation did not further augment its surface expression (Figure 2B). Moreover, upon TLR stimulation, MuTu DCs cultured in IL-35 supplemented medium secreted significantly less IL-12p40 and IL-6 cytokine when compared to DC grown in control medium. In contrast to IL-35⁺ DCs, no increase in IL-10 secretion could be detected (Figure 2C).

We have previously demonstrated that the MuTu DC lines efficiently induce T cell proliferation in MHCI or MHCII restricted systems (9). IL-35 on the other hand, is described to inhibit CD4⁺ T cell proliferation (2, 14). We therefore tested whether the IL-35-producing DC line is able to affect T cell activation and function. An allogeneic MLR was performed using variable amounts of mock transduced or IL-35 expressing, C57BL/6-derived DCs as stimulators. Coculture with mock-transduced DCs resulted in a robust, DC number-dependent proliferation of BALB/c-derived CD4⁺ responder T cells. This was accompanied by an increased percentage of activated CD44⁺ CD62L⁻ T cells and IFN-γ-expressing T cells. On the contrary, target cell proliferation and activation were severely reduced when IL-35⁺ DCs were used as activator cells (Figure 3A). Analysis of target T cell RNA revealed a significant upregulation of IL-35-associated P35 and Ebi3, but not IL-12- or IL-27-specific P40, respectively, P28 transcripts. In contrast to IL-35⁺ DCs, culture with mock-transduced DC led to a significant upregulation with IL-12/23-associated P40 but not P35 or Ebi3 transcripts (Figure 3B). No single-chain IL-35 cDNA could be detected by PCR, excluding the possibility of a contamination of the RNA by IL-35⁺ DCs (Supplementary Figure 2). Similar results were obtained upon the cotransfer of OVA_323–339 peptide-pulsed mock or IL-35⁺ DCs and congenically marked OT-II CD4⁺ T cells into C57BL/6 mice. Four days after the transfer, IL-35-expressing but not control-transduced DCs were shown to reduce both the amount of detectable CD45.1⁺ OT-II T cells as well as their IFN-γ production (Supplementary Figure 3A).

There is only limited information on the effects of IL-35 on the proliferation and effector functions of CD8⁺ T cells available. Allogeneic MLR-culturing BALB/c-derived CD8⁺ T cells with C57BL/6 IL-35⁺ or mock-transduced MuTu DCs were therefore performed. We found that IL-35⁺ but not mock DCs significantly reduced proliferation, activation status, as well as IFN-γ expression by CD8⁺ T lymphocytes (Figure 3C). IL-35⁺ DCs furthermore induced the transcription of significantly higher levels of Ebi3 and P35 in CD8⁺ T cells than mock-transduced DCs (Figure 3D). Similarly, a drastic reduction in the abundance of CD45.1⁺ CD8⁺ T cells could be observed upon co-injection of OT-I CD8⁺ T cells together with OVA_323–339 peptide-pulsed IL-35-expressing but not mock-transduced DCs into C57BL/6 mice (Supplementary Figure 3B). No differences in cell number and in IFN-γ expression of host-derived CD45.2⁺ CD8⁺ T cells were observed (Supplementary Figure 3C).

Interleukin-35 cytokine was originally described to mediate infectious tolerance via the induction of a regulatory phenotype on naïve CD4⁺ T cells (14). We therefore tested whether the observed induction of P35/Ebi3 transcription by CD4⁺ T cells cocultured with IL-35⁺ DCs is accompanied with the acquisition of suppressive properties. Purified naïve wild-type CD4⁺ T cells were cocultured for 4 days with either IL-35⁺ DC or mock DCs and recombinant IL-2 and TGF-β. A classical suppression assay was then performed by culturing the conditioned T cells at different ratios with naïve CD4⁺ responder T cells in the presence of CD3/CD28 stimulation. We could measure a significantly reduced, ratio-dependent, responder cell proliferation when suppressor T cells were conditioned in the presence of IL-35⁺ DC or mock DC supplemented with IL-2 and TGF-β. No such effect was observed when cells were conditioned with mock DC alone. However, IL-35⁺ DC-conditioned T cells consistently proved to be more potent suppressors than TGF-β/IL-2-induced Tregs. Both in terms of maximal suppressive capability as well as the minimal ratio of suppressor T cells needed to observe an inhibitory effect. In accordance with literature, IL-35 induced regulatory cells did not express the classical Treg cell marker Foxp3. T cells conditioned for 4 days in IL-35 containing medium but in the absence of mock DC, did not exert any suppressive functions (Figure 4 and data not shown).

As demonstrated above, IL-35⁺ DCs as well as wild-type DCs cultured in IL-35 containing medium did not adequately upregulate MHCII and costimulatory receptors upon activation, but remained in a rather immature state with increased CD80 expression. Such immature DC populations have been described to induce and maintain T cell tolerance (15). We therefore considered the possibility that the observed effects on T cells were not only caused directly by the secreted IL-35, but indirectly through the modulation of the immunogenic potential of the conditioned DCs. In order to test this hypothesis, wild-type MuTu DCs were conditioned for 3 days in IL-35 containing or control culture medium. Upon removal of IL-35, OVA_323–339-pulsed IL-35-conditioned DCs exhibited a significantly impaired potential to stimulate OT-II CD4⁺ T cell proliferation, activation, and IFN-γ production when compared to control medium-conditioned MuTu DCs. The inhibitory effect on CD4⁺ T cells by IL-35-conditioned DCs was found to be comparable to OT-II CD4⁺ T cells stimulated with IL-35⁺ DCs (Figure 5A). In contrast to coculture with IL-35-expressing DCs, no induction of Ebi3 transcription and only a moderate induction of IL-12p35 were observed by OT-II CD4⁺ T cells incubated with IL-35-conditioned wtDC. Interestingly, IL-35-conditioned DCs as well as IL-35⁺ DCs transcribed significantly less of the IL-27-associated p28 chain than control DCs (Figure 5B).

Taken together, the inhibitory effect on T cell proliferation and function in vitro and in vivo demonstrated that IL-35 transgenic DC line efficiently interfered with proliferation of both, CD4⁺ and CD8⁺ T cells. Furthermore, IL-35⁺ DC impaired the function of CD4⁺ T cells. Our findings further indicate that IL-35 secretion alone does not only directly affect T cell proliferation and function but also indirectly via the induction of a tolerogenic phenotype on MuTu DCs.

Interleukin-35 has been shown to promote tumor progression by interfering with tumor-directed immune responses in several human cancers (6, 16, 17). In order to determine whether IL-35⁺ DCs affect T lymphocyte-dependent antitumor responses in vivo, C57BL/6 mice (n = 5 per group) were vaccinated a total of three times with tumor cell lysate pulsed, IFN-γ-stimulated DCs. Five days after first vaccination 2 × 10⁵ B16.F0 melanoma cells were transferred subcutaneously into the flank of the mice (Figure 6A). B16.F0 tumor volume increased progressively and withdrawal criteria were reached in median within 20.5 days in PBS-injected mice but only 14 days for mice vaccinated with IL-35⁺ DCs. Mice that were vaccinated with control DCs exhibited a slight, but not significant delay in tumor growth when compared to PBS-injected animals (Figure 6B). Flow cytometric analysis of tumor tissue showed drastic reduction of tumor infiltration by CD3⁺ T cells in IL-35⁺ DC-vaccinated mice. In addition, infiltrating CD4⁺ and CD8⁺ T cells exhibited a significant reduction in IFN-γ production. However, no differences in Foxp3⁺ Treg cells infiltration could be observed (data not shown). IL-35⁺ DC transfer did not significantly affect the percentage or composition of infiltrating CD11b⁺ myeloid cells (Figure 6C). We next sought to determine whether IL-35-conditioned DCs contribute to the accelerated increase in tumor volume. B16.F0 growth was compared upon vaccination with either unpulsed or tumor lysate pulsed, IFN-γ-activated IL-35⁺ DCs. Only the transfer of tumor lysate loaded IL-35⁺ DCs but not unpulsed IL-35⁺ DCs caused a significant increase in tumor volume (Figure 6D).

Similarly, subcutaneously transferred CMT93 carcinoma cells showed progressive tumor growth in IL-35⁺ DC-vaccinated mice and tumor volume was significantly bigger when compared to control animals (Supplementary Figure 4A). In accordance to our observations in the B16.F0 model, flow cytometric analysis of CMT93 tumor infiltrating leukocytes revealed a markedly decreased accumulation of total CD3⁺ T lymphocytes in IL-35⁺ DC-treated mice. Both CD4⁺ and CD8⁺ T cells produced significantly less IFN-γ. In contrast to the B16.F0 model, vaccination with IL-35⁺ DCs was furthermore accompanied with an increased infiltration by CD11b⁺ Gr1⁺ myeloid-derived suppressor cells when compared to control DC-vaccinated animals (Supplementary Figure 4B).

In order to assess the potential of IL-35⁺ DCs to abrogate autoimmune T cell activity in vivo, adoptive transfer EAE was induced as described in Section “Animals and Methods.” Adoptive transfer allowed us to introduce IL-35⁺ DCs into the experimental system either already during priming of the encephalitogenic lymphocytes (experimental groups 2 + 3) or only in already diseased recipient mice (experimental groups 4 + 5) (Figure 7A). After in vivo priming, T cells were restimulated in vitro as depicted in Figure 7A. T cells restimulated with myelin oligodendrocyte glycoprotein-35–55 (MOG_35–55)-pulsed IL-35⁺ DCs (experimental group 2) exhibited a less activated phenotype and produced less IFN-γ than T cells restimulated in the presence of MOG_35–55-pulsed mock DCs (experimental group 1) (Figure 7B). After in vitro restimulation, 5 × 10⁶ T cells were transferred into wild-type C57BL/6 mice. The animals were surveilled daily for the development of EAE-related symptoms. Recipient mice receiving MOG_35–55 reactive T cells that were primed in the presence of mock DCs (experimental group 1) reproducibly exhibited EAE symptoms within 9 days after T cell transfer. A maximum disease score of three, corresponding to a complete paralysis of the hind limbs, could be observed between 14 and 18 days after T cell transfer. The animals subsequently recovered and no disease symptoms could be observed 25 days after the transfer of immunogenic T cells. By contrast, mice that received T cells primed and restimulated in the presence of MOG_35–55 peptide loaded IL-35⁺ DCs exhibited a drastically reduced disease severity (experimental group 2). Animals injected with unpulsed IL-35⁺ DCs (experimental group 3) exhibited an intermediate disease course: EAE symptoms were less vigorous when compared to the mock DC control group but were significantly more severe than in MOG_35–55-pulsed IL-35⁺ DC injected mice (Figure 7C). We further assayed whether the therapeutic transfer of IL-35⁺ DCs is sufficient to limit the function of MOG-primed lymphocytes. Both, in vitro restimulation with MOG_35–55-pulsed IL-35⁺ DCs (experimental group 4) or the injection of IL-35⁺ DCs into recipient animals 6 and 8 days after the transfer of MOG-primed T cells (experimental group 5) slightly delayed the disease onset and led to a significant reduction of EAE symptoms.

The brains were collected at the peak of disease. Histological analysis of sections of the cerebral cortex revealed focal inflammatory lesions within the myelin rich regions with a preferential perivascular accumulation of CD3⁺ cells in diseased control animals. No formation of such lesions, but only few isolated T cells could be observed in the IL-35 experimental group (Figure 7D).