TNF Receptor–Associated Factor 5 Limits IL-27 Receptor Signaling in CD4⁺ T Lymphocytes

The IL-6 family of cytokine IL-27, which is composed of IL-27p28 and EBV-induced gene 3 (EBI3), utilizes a receptor complex composed of WSX-1 (also known as IL-27Rα or TCCR) and gp130 (also known as CD130) (1–4). WSX-1 and gp130 are coexpressed by CD4⁺ T cells. IL-27 activates the JAK-STAT pathway via both WSX1 and gp130, and these receptors induce a more sustained phosphorylation of STAT1 as compared with STAT3 (5, 6). IL-27 combination with TCR signaling induces the proliferation of naive CD4⁺ T cells (7) and promotes the differentiation of both T-bet⁺ Th1 cells (8–11) and IL-10–producing T cells (12, 13).

TNF receptor–associated factor 5 (TRAF5) binds to gp130 and inhibits IL-6 receptor signaling in CD4⁺ T cells (14, 15), although TRAF5 was initially discovered as an activator of NF-κB via the receptors for TNF family molecules, lymphotoxin β receptor (LTβR) and CD40 (16, 17). The expression of TRAF5 is relatively higher in resting B and T lymphocytes. The constitutive binding between TRAF5 and gp130 restrains IL-6–driven differentiation of Th17 cells by decreasing STAT3 activity in activated naive CD4⁺ T cells, which limits the induction of neuroinflammation in experimental autoimmune encephalomyelitis (18).

Given that TRAF5 associated with gp130 restrains IL-6 receptor signaling in CD4⁺ T cells, TRAF5 may also inhibit IL-27 receptor signaling. In this study, we found that after exposure to IL-27 alone, Traf5^−/− CD4⁺ T cells rapidly expressed significantly higher levels of phosphorylated STAT1 and IL-27–regulated genes, Socs3 and Tbx21, when compared with Traf5^+/+ CD4⁺ T cells. IL-27 in combination with TCR triggering significantly promoted the induction of IL-10 and the proliferation in Traf5^−/− CD4⁺ T cells. Our results demonstrate, to our knowledge for the first time, that TRAF5 expressed in CD4⁺ T cells limits the signaling activity of the IL-27 receptor composed of gp130 and WSX-1.

Traf5^−/− mice with a C57BL/6 background were intercrossed to generate Traf5^+/+ and Traf5^−/− mice (14). All mice were bred and maintained under specific pathogen-free conditions, and experiments were in compliance with protocols approved by the University Committee for Animal Use and Care at the University of Toyama.

Anti-CD3ε (low endotoxin, azide free; 145-2C11, 100340), anti-CD28 (low endotoxin, azide free; 37.51, 102112), anti-CD40 (low endotoxin, azide free; HM40-3, 102907), anti–IFN-γ (low endotoxin, azide free; XMG1.2, 505834), anti–IL-2 (low endotoxin, azide free; JES6-1A12, 503704), anti–IL-4 (low endotoxin, azide free; 11B11, 504122), anti-GITR (low endotoxin, azide free; DTA-1, 126322), anti-p38 MAPK (Poly6224, 622403), anti-p38 MAPK phospho-Thr180/Tyr182 (A16016A, 690201), FITC-anti-CD4 (RM4-5, 100510), PE/Cyanine7-anti-CD4 (GK1.5, 100421), PE-anti-CD62L (MEL-14; 104407, 104408), allophycocyanin-anti-CD44 (IM7, 103011), PE-anti-CD45R/B220 (RA3-6B2, 103207), PE-anti–IL-10 (JES5-16E3, 505007), and recombinant mouse IL-27 (577404) were from BioLegend. FITC-anti–WSX-1 (263503, FAB21091F) was from R&D systems. Anti-STAT3 (4904), anti–phospho-STAT3 at Tyr⁷⁰⁵ (9145), anti-STAT1 (14995), and anti–phospho-STAT1 at Tyr⁷⁰¹ (7649) were from Cell Signaling Technology. Anti-cMyc (9E10, 017-21871), anti-FLAG (DYKDDDDK, 1E6, 018-22381), anti-PA (NZ-1, 016-25863), anti-V5 (6F5, 015-23594), recombinant human IL-6 (099-04631), recombinant human IL-6R (092-07301), and recombinant human TGF-β1 (209-16544) were from Fujifilm. Recombinant mouse IL-4 (214-14) was from PeproTech.

Vectors containing cDNA encoding mouse gp130 and TRAF5 were previously described (15). Based on cDNA sequence of mouse Il27ra (Wsx1, NM_016671.3), cDNA of the entire coding region was amplified with PCR using primers that added a 5′-BamHI site and a 3′-EcoRI site and ligated into a pcDNA3.1/V5-His A vector (Thermo Fisher Scientific). Mouse Ebi3 (NM_015766.2) and Il27 (p28, NM_145636.2) cDNA fragments were isolated by PCR. For construction of a plasmid encoding mouse single-chain IL-27 (Ebi3-p28) and Fc chimeric protein (IL-27-Fc), we inserted a signal sequence of Igκ (5′-ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC-3′), a fragment encoding the mature part of Ebi3 (Tyr¹⁹-Pro²²⁸), the (Gly₃Ser)₄ linker, and a fragment encoding the mature part of p28 (Phe²⁹-Ser²³⁴) with splicing donor signal (5-′GTAAGT-3′) into the pEF-Fc expression vector (19–21), which contains the Fc region (hinge, CH2, and CH3 domains) of human IgG1 genomic DNA. Additionally, the fragment encoding the entire Igk-Ebi3-p28-Fc sequence was amplified with PCR using primers that added a 5′-EcoRI site and a 3′-BglII site and cloned into the pCAGGS (LT727518.1) expression vector, and a PA-His tag (Gly-Val-Ala-Met-Pro-Gly-Ala-Glu-Asp-Asp-Val-Val−His-His-His-His-His-His) sequence was inserted at the C-terminal end of the fragment. For production of recombinant IL-27-Fc, the vector above was transfected into HEK293T cells by using polyethyleneimine (Merck, 408727) and then cultured in DMEM (Fujifilm) containing 2% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin for 5–7 d. Alternatively, a CHOK1 cell clone stably expressing IL-27-Fc was established to produce IL-27-Fc. Recombinant IL-27-Fc was purified from the culture supernatants by a HiTrap rProtein A FF column (Cytiva) and then concentrated with Amicon Ultra-4 (Merck Millipore). The concentration of IL-27-Fc was determined by ELISA using recombinant mouse IL-27 (577404, BioLegend) as a standard.

CD4⁺ T cells were purified from spleens of Traf5^+/+ and Traf5^−/− mice using CD4 (L3T4) microbeads (130-117-043, Miltenyi Biotec) or a naive CD4⁺ T cell isolation kit (130-104-453, Miltenyi Biotec). Resting B cells were purified from spleens of Traf5^+/+ and Traf5^−/− mice using CD43 (Ly-48) microbeads (130-049-801, Miltenyi Biotec). T cells were cultured in RPMI 1640 medium with 10% FCS, penicillin, streptomycin, 2 mM l-alanyl-l-glutamine, and 50 µM 2-ME. T cells were plated in 96-well culture plates and stimulated with 3 µg/ml plate-bound anti-CD3ε and 1 µg/ml soluble anti-CD28. For Th17 differentiation, naive cells were stimulated in the presence of 100 ng/ml IL-6-IL-6R, 0.1 ng/ml TGF-β, 1 µg/ml anti–IFN-γ, and 1 µg/ml anti–IL-4. For Th2 differentiation, naive cells were stimulated in the presence of 10 ng/ml IL-4 and 1 µg/ml anti–IFN-γ. Proliferation was assessed with a MTT assay (M2128, MilliporeSigma). The water-insoluble MTT formazan was solubilized with DMSO, and the absorbance was measured with a plate reader at 570 nm using FilterMax F5 (Molecular Devices).

The murine IL-3–dependent BAF/B03 pro–B cell line stably expressing exogenous mouse gp130 and TRAF5 was described previously (22). Endogenous expression of WSX-1 on the surface of BAF cells (23) was confirmed by staining the cells with anti–WSX-1 Ab. Cells were cultured with graded doses of IL-27 for 72 h, followed by addition of MTT to measure proliferative responses.

Cells were lysed for 30 min in ice-cold radioimmunoprecipitation assay (RIPA) buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 50 mM NaF, 1 mM Na₃VO₄, 0.5% sodium deoxycholate, 0.1% NaDodSO₄, and 1 mM PMSF] with a protease inhibitor mixture (P8340; Sigma-Aldrich). Lysates were sonicated, and insoluble material was removed by centrifugation at 15,000 × g for 10 min. WSX-1 was immunoprecipitated from the lysates overnight at 4°C with anti-V5 Ab immobilized on Dynabeads protein G (10004D, Thermo Fisher Scientific). After being washed extensively with ice-cold RIPA buffer, beads were boiled for 10 min at 100°C in 4× Laemmli sample buffer [240 mM Tris-HCl (pH 6.8), 40% glycerol, 8% SDS, and 0.1% bromophenol blue]. The eluted sample was further reduced for 5 min at 100°C with 1% 2-ME for immunoblot analysis.

CD4⁺ T cells were cultured in the presence or absence of IL-27 in RPMI 1640. Cells were lysed for 30 min in ice-cold RIPA buffer with a protease inhibitor mixture. Insoluble debris was removed by centrifugation at 15,000 × g for 10 min. The supernatant was mixed with 4× Laemmli sample buffer and boiled for 5 min at 100°C. Samples were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes (IPVH00010, Merck), and analyzed by immunoblot with the appropriate Abs. The reaction was visualized with a chemiluminescence detection system using Western Lightning Plus-ECL (NEL105001EA; PerkinElmer) and LAS-4000mini (Fujifilm).

Cytokines in culture supernatants were assessed by a sandwich ELISA assay with anti–IL-2 (JES6-1A12, 503701) capture Ab, biotin-anti–IL-2 (JES6-5H4, 503803) detection Ab, anti–IL-10 (JES5-16E3, 505001) capture Ab, biotin-anti–IL-10 (JES5-2A5, 504905) detection Ab, anti–IL-17A (TC11-18H10.1, 506901) capture Ab, biotin-anti–IL-17A (TC11-8H4, 507001) detection Ab, anti–IL-27 p28 (MM27-7B1, 516901) capture Ab, biotin-anti–IL-27 (B02P6E6, 525903) detection Ab, anti–IFN-γ (R4-6A2, 505702) capture Ab, biotin-anti–IFN-γ (XMG1.2, 505804) detection Ab, HRP streptavidin (405210), and 3,3′,5,5′-tetramethylbenzidine (421101) from BioLegend. The absorbance was measured at 450 nm using FilterMax F5.

Total RNA was extracted from cells using Isogen II (Nippon Gene), and cDNA was synthesized with ReverTra Ace qPCR RT master mix with genomic DNA remover (Toyobo). Diluted cDNA was used for quantitative RT-PCR with Thunderbird SYBR qPCR mix (Toyobo) and MX3005p or MX3000p (Agilent Technologies). Each transcript was analyzed concurrently on the same plate with the gene encoding cyclophilin B (Ppib), and results are presented relative to the abundance of transcripts encoding Ppib. Primers were as follows: Ebi3 (forward primer, 5′-TCATTGCCACTTACAGGCTCG-3′; reverse primer, 5′-TGATGATTCGCTCAGCCACAA-3′), Fas (forward primer, 5′- GCGGGTTCGTGAAACTGATAA-3′; reverse primer, 5′- GCAAAATGGGCCTCCTTGATA-3′), Gata3 (forward primer, 5′-GGCAGAACCGGCCCCTTATC-3′; reverse primer, 5′-TGGTCTGACAGTTCGCGCAG-3′), Icam1 (forward primer, 5′- GTGATGCTCAGGTATCCATCCA-3′; reverse primer, 5′- CACAGTTCTCAAAGCACAGCG-3′), Ifng (forward primer, 5′-GGATGCATTCATGAGTATTGC-3′; reverse primer, 5′-CCTTTTCCGCTTCCTGAGG-3′), Il4 (forward primer, 5′-AGATCATCGGCATTTTGAACG-3′; reverse primer, 5′-TTTGGCACATCCATCTCCG-3′), Il6st (forward primer, 5′-TACATGGTCCGAATGGCCGC-3′; reverse primer, 5′-GGCTAAGCACACAGGCACGA-3′), Il10 (forward primer, 5′-TGCTCCTAGAGCTGCGGACT-3′; reverse primer, 5′-AGGCTTGGCAACCCAAGTAACC-3′), Il17a (forward primer, 5′-TTTAACTCCCTTGGCGCAAAA-3′; reverse primer, 5′-CTTTCCCTCCGCATTGACAC-3′), Il21 (forward primer, 5′-ACTCAGTTCTGGTGGCATGG-3′; reverse primer, 5′-GCTGATAGAAGTTCAGGATCCAAGT-3′), Il27 (forward primer, 5′-GGCCATGAGGCTGGATCTC-3′; reverse primer, 5′-AACATTTGAATCCTGCAGCCA-3′), Il27ra (forward primer, 5′-CAAGAAGAGGTCCCGT-3′; reverse primer, 5′-TTGAGCCCAGTCCACC-3′), Lta (forward primer, 5′-CCCATCCACTCCCTCAGAAG-3′; reverse primer, 5′-CATGTCGGAGAAAGGCACGAT-3′), Ppib (forward primer, 5′-CCTCTCGGAGCGCAATATGA-3′; reverse primer, 5′-TCGTCCTACAGATTCATCTCCAAT-3′), Rorc (forward primer, 5′-TCGACAAGGCCTCCTAGCCA-3′; reverse primer, 5′-CTTGGACCACGATGGGGTGG-3′), Socs3 (forward primer, 5′-AGGCCGGAGATTTCGCTTCG-3′; reverse primer, 5′-CGGGAAACTTGCTGTGGGTGA-3′), and Tbx21 (forward primer, 5′-GGTTGGAGGTGTCTGGGAAGC-3′; reverse primer, 5′-GCCACGGTGAAGGACAGGAAT-3′).

Cells were stained with Abs for cell surface markers. For staining of intracellular IL-10, CD4⁺ T cells were stimulated for 3 h with 50 ng/ml PMA (Fujifilm, 162-23591) and 1 µg/ml ionomycin (Cayman Chemical, 10004974) in the presence of 2 µM monensin (BioLegend, 420701). After staining of surface CD4, cells were fixed and permeabilized with buffer solutions (BioLegend, 420801 and 421002). Intracellular staining for IL-10 was performed with a PE-IL-10 Ab or a PE-isotype control IgG Ab diluted in permeabilization wash buffer. FACS data were acquired on a FACSCanto II (BD Biosciences) and analyzed with Flowing Software (http://flowingsoftware.btk.fi/).

The delayed-type hypersensitivity (DTH) response was evaluated as previously described (24). Mice were immunized s.c. at the tail with 200 µl of 1.25 mg/ml methyl BSA (mBSA) (A1009, Merck) emulsified with CFA (F5881, Merck) on day 0. Mice were additionally administered 1 µg of IL-27-Fc or PBS via i.p. injection on days 0 and 2. Seven days after the immunization, mice were challenged s.c. in a footpad with 30 µl of 7 mg/ml mBSA in PBS. An equal volume of PBS was injected into another footpad as a control. One day after the challenge, footpad thickness was measured with a digital caliper (Shinwa Rules, Japan). The magnitude of the DTH response was determined as follows: [footpad swelling (%)] = ([footpad thickness of mBSA-injected footpad (mm)] – [footpad thickness of PBS-injected footpad (mm)]) ÷ [footpad thickness of PBS-injected footpad (mm)] × 100.

To evaluate the mBSA-specific T cell response, spleen cells were cultured in the absence or presence of mBSA for 72 h. Proliferation was assessed with MTT assay. Cytokines in culture supernatants were assessed by ELISA.

Statistical significance was assessed with a Student t test with two-sided distributions.

TRAF5 limits IL-6 receptor signaling through interacting with the common chain gp130, indicating that TRAF5 may also inhibit signals downstream of receptors for other IL-6 family cytokines that use gp130. In this study, we aim to clarify the activity of TRAF5 for the IL-27 receptor complex composed of gp130 and WSX-1. We first tested whether the expression of TRAF5 could inhibit IL-27 receptor signaling using BAF-gp130 cells, which express endogenous WSX-1 and exogenous gp130 (23). BAF cells with overexpressed TRAF5 showed significantly reduced proliferation in response to IL-27 when compared with BAF cells with control vector (Supplemental Fig. 1A), although these cells displayed a comparable proliferative response to IL-3 (22). This result indicates that TRAF5 negatively regulates IL-27 receptor signaling.

Spleen cells from Traf5^+/+ (wild-type) and Traf5^−/− (knockout) naive mice expressed equivalent amounts of Il27 (p28) and Ebi3 mRNA (Supplemental Fig. 1B), suggesting comparable basal levels of IL-27 in both groups. CD4⁺ T cells from Traf5^+/+ and Traf5^−/− mice express similar amounts of IL-6R-gp130 (14). In this study, we measured the levels of WSX-1 protein and Il6st (gp130) and Il27ra (Wsx1) mRNA, and we confirmed that CD4⁺ T cells from Traf5^+/+ and Traf5^−/− mice had similar amounts of these receptors (Fig. 1A, 1B).

Upon stimulation with IL-27 alone for 1 h, Traf5^−/− CD4⁺ T cells displayed significantly higher levels of IL-27–regulated genes Socs3 and Tbx21 than did Traf5^+/+ CD4⁺ T cells (Fig. 1C), showing that TRAF5 limits IL-27 receptor signaling in CD4⁺ T cells.

The level of Traf5 mRNA in CD4⁺ T cells was about half that expressed by B cells, whereas the level of Il6st mRNA in CD4⁺ T cells was nearly 6-fold that expressed by B cells (14). B cells from Traf5^+/+ and Traf5^−/− mice expressed comparable amounts of WSX-1 protein (Supplemental Fig. 1C). These results imply that IL-27 receptor signaling in B cells may be different from that in CD4⁺ cells. Upon stimulation with IL-27 alone, Traf5^−/− B cells displayed significantly higher levels of Tbx21 than did Traf5^+/+ CD4⁺ T cells, whereas no statistical significance was observed for Socs3 (Supplemental Fig. 1D). These results suggest that TRAF5 regulates IL-27 receptor signaling in B and T lymphocytes in both similar and distinct ways.

IL-27 plays a dominant role for inducing IL-10 and IL-21 from differentiating CD4⁺ T cells (12, 13). Thus, we decided to evaluate whether the Traf5 deficiency increases the IL-27–mediated induction of Il10 and Il21 mRNA in an early phase of naive CD4⁺ T cell activation. After stimulation with anti-CD3/CD28 plus IL-27 for 48 h, Traf5^−/− CD4⁺ T cells showed significantly higher levels of Il10 and Il21 mRNA when compared with Traf5^+/+ CD4⁺ T cells (Fig. 2A). Although we failed to detect IL-21 at the protein level, T cell activation in the presence of IL-27 produced significantly higher IL-10 protein in Traf5^−/− CD4⁺ T cells (Fig. 2B).

Because IL-27 works as a growth factor for CD4⁺ T cells (7), we next examined whether the Traf5 deficiency promotes the cell growth of naive CD4⁺ T cells. Indeed, IL-27–dependent proliferation was significantly enhanced in Traf5^−/− CD4⁺ T cells when compared with Traf5^+/+ CD4⁺ T cells (Fig. 2C).

IL-27 inhibits IL-2 production mediated by anti-CD3/CD28, and SOCS3 plays a critical role for the inhibition (25). Traf5^−/− CD4⁺ T cells cultured with IL-27 displayed a significantly decreased production of IL-2 mediated by anti-CD3/CD28 when compared with Traf5^+/+ CD4⁺ T cells (Fig. 2D). Collectively, these results support the hypothesis that TRAF5 negatively regulates IL-27 receptor signaling in CD4⁺ T cells.

IL-27 receptor signaling can influence the differentiation of Th1, Th2, and Th17 cells (11, 26, 27). To evaluate how TRAF5 downstream of the IL-27 receptor regulates the development of CD4⁺ T cells, naive CD4⁺ T cells from Traf5^+/+ and Traf5^−/− mice were cultured in neutral or polarizing cytokine conditions in the presence or absence of IL-27 in vitro. In the neutral condition, IL-27 increased the induction of Ifng and Tbx21, and Traf5 deficiency significantly enhanced the expression of these genes (Fig. 3A), indicating that TRAF5 limits the differentiation of Th1 cells mediated by IL-27. In the Th17 condition, IL-27 strongly suppressed the induction of Il17a and Rorc, whereas Traf5 deficiency enhanced the induction of Il17a in the absence of IL-27 (Fig. 3B). This result suggests that the inhibitory effect of IL-27 for Th17 differentiation dominates over the stimulatory effect of IL-6. In the Th2 condition, IL-27 displayed a marginal suppression for Gata3 but not for Il4 (Fig. 3C), suggesting that IL-4 confers resistance to IL-27–mediated suppression on Th2 differentiation (28).

An important caveat here is whether TRAF5 positively regulates TNF receptor signaling in lymphocytes. It has been demonstrated that the expression of TRAF5 supports GITR cosignaling in CD4⁺ T cells (29, 30) and CD40 signaling in B cells (31). Indeed, anti-GITR promoted the proliferation and IL-2 production mediated by anti-CD3 in Traf5^+/+ CD4⁺ T cells, and these responses were significantly decreased in Traf5^−/− CD4⁺ T cells (Supplemental Fig. 2A, 2B). Additionally, the expression levels of CD40-induced genes, such as Icam1, Fas, and Lta, were significantly decreased in Traf5^−/− B cells when compared with Traf5^+/+ B cells (Supplemental Fig. 2C). Thus, we confirmed that TRAF5 works as a positive regulator in GITR and CD40 signaling in lymphocytes, and these results suggest that TRAF5 regulates cytokine receptor signaling both in positive and negative ways.

IL-27 activates both STAT1 and STAT3 pathways, and these STATs differentially regulate the proliferation, differentiation, and survival of CD4⁺ T cells (9, 11, 32–34). IL-27 supports more prolonged STAT1 activation than does IL-6 (5, 6). Thus, it would be important to understand how TRAF5 controls IL-27–mediated activation of STAT1. IL-27 alone promoted the active tyrosine phosphorylation of STAT1 in CD4⁺ T cells in both a time- and dose-dependent manner. Importantly, the phosphorylation of STAT1 in Traf5^−/− CD4⁺ T cells was significantly higher than that of Traf5^+/+ CD4⁺ T cells (Fig. 4A–C, Supplemental Fig. 3). Phosphorylation of p38 MAPK mediated by IL-27 was also increased in Traf5^−/− CD4⁺ T cells as compared with Traf5^+/+ CD4⁺ T cells (Supplemental Fig. 3B). In contrast, the phosphorylation of STAT3 was reduced in Traf5^−/− CD4⁺ T cells as compared with Traf5^+/+ CD4⁺ T cells (Supplemental Fig. 3B). These results indicate that TRAF5 limits the activation of STAT1 mediated by IL-27 and suggest that TRAF5 exhibits a more dominant effect on STAT1. To evaluate whether TRAF5 binds to gp130 of the IL-27 receptor, we transfected HEK293T cells to express c-Myc–tagged gp130, V5-tagged WSX-1, and FLAG-tagged TRAF5 and evaluated the binding of TRAF5 to WSX-1 by immunoprecipitation. The amount of TRAF5 associated with WSX-1 and gp130 increased after stimulation with IL-27 (Fig. 4D). Collectively, these results suggested that binding of TRAF5 to gp130 of the IL-27 receptor was inhibitory for phosphorylation of STAT1 mediated by IL-27.

WSX-1–deficient mice display reduced Th1 responses. IL-27 receptor signaling is required for the initial mounting of Th1 responses (35, 36). In DTH, classified as type IV hypersensitiveness, Th1 cells play a dominant role for inducing inflammation via promoting recruitment of inflammatory cells to inflammatory sites. IFN-γ from Ag-specific Th1 cells enhances the development of DTH responses. Thus, the Traf5 deficiency may promote IL-27–driven DTH responses mediated by Th1 cells.

To evaluate this possibility, Traf5^+/+ and Traf5^−/− mice were immunized and challenged with mBSA to induce DTH responses. IL-27-Fc was additionally administered into mice at early stages of CD4⁺ T cell development to evaluate the role for TRAF5 in IL-27 receptor signaling (Fig. 5A). We found that Traf5^−/− mice injected with IL-27-Fc displayed significantly augmented DTH responses as determined by footpad swelling (Fig. 5B). Spleen cells from Traf5^−/− mice injected with IL-27-Fc vigorously proliferated and produced significantly higher amounts of IFN-γ in response to mBSA (Fig. 5C). This result indicates that TRAF5 limits the development of Th1 cells mediated by IL-27 in vivo.

Taken together, our study demonstrates that TRAF5 acts as a negative regulator in IL-27 receptor signaling in CD4⁺ T cells and suggests that the function of TRAF5 in restricting IL-6–related cytokine receptor signaling is independent from that in promoting TNF receptor family signaling.

TRAF family molecules show multifunctional activity downstream of various cytokine receptors in T cells (37, 38). The aim of our study is to clarify the general principle of the inhibitory function of TRAF5 for IL-6 family cytokine receptors and to distinguish the inhibitory effects from the stimulatory effects on TNF receptor family molecules. In this study, we found that TRAF5 reciprocally regulated IL-27 and TNF receptor signaling in lymphocytes, which suggests that TRAF5 controls signal transduction in a diverse array of cytokine receptors and plays important roles in inflammatory responses mediated by lymphocytes.

It is unclear why TRAF5 exhibited its inhibitory activity for STAT1 but not for STAT3 after stimulation with IL-27 alone. IL-6 and IL-27 receptors share gp130 and activate both STAT1 and STAT3. Upon IL-27 binding, WSX-1 together with gp130 predominantly activates STAT1. In contrast, upon IL-6 and IL-6R binding, gp130 predominantly activates STAT3 (5, 6). There is a binding site for STAT1 in the C-terminal region in WSX-1, which is closely related to a STAT1-binding site in IFN-γ receptor (23). TRAF5 constitutively associates with a cytoplasmic region in gp130 (aa 774–798) via the TRAF-C domain. After stimulation with IL-27 alone, Traf5^−/− CD4⁺ T cells showed an enhanced phosphorylation of STAT1 but not STAT3, as compared with Traf5^+/+ CD4⁺ T cells. This intriguing observation suggests that TRAF5 associated with gp130 negatively regulates STAT1 activation in the IL-27/WSX-1/gp130 receptor complex. Although the mechanism is unknown, it is tempting to speculate that TRAF5 may restrain the close proximity of gp130 and WSX-1 receptors and alter the configuration needed to optimally activate the JAK-STAT signaling pathway.

TRAF5 may also restrain IL-27–mediated STAT3 signaling in activated CD4⁺ T cells. IL-27 induces proliferation of naive CD4⁺ T cells (7). STAT3 is required for IL-27–mediated CD4⁺ T cell proliferation, and the IL-27/gp130/STAT3 axis is responsible for the induction of c-Myc and Pim-1 that promote cell proliferation (33, 39). We showed in this study that the expression of TRAF5 limits IL-27– and TCR-mediated proliferation of naive CD4⁺ T cells. Thus, we speculate that TRAF5 also negatively regulates IL-27–driven STAT3 activity in CD4⁺ T cells, although current results do not support this assumption.

Induction of T-bet and SOCS3 by IL-27 is negatively controlled by TRAF5 in CD4⁺ T cells. Upon stimulation with IL-27, the Traf5 deficiency resulted in upregulation of Tbx21 and Socs3 and promoted the suppression of IL-2 production in CD4⁺ T cells. Consistent with our results, T-bet and SOCS3 were rapidly induced by IL-27 alone in naive CD4⁺ T cells (40). Although proposed mechanisms are inconsistent, the molecular pathway regulated by T-bet and SOCS3 is critical for inhibiting IL-2 production from CD4⁺ T cells (25, 41, 42). These results well explain how Traf5 deficiency augments the signaling pathway downstream of the IL-27 receptor.

Induction of IL-10 mediated by IL-27 is negatively controlled by TRAF5 in CD4⁺ T cells. As previously described (40), a short-term stimulation with IL-27 alone does not result in IL-10 induction in naive CD4⁺ T cells. Both STAT1 and STAT3 activities induced by IL-27 plus anti-CD3/CD28 can drive the production of IL-10 from CD4⁺ T cells (12). IL-27 promotes IL-21 via Maf, and IL-21 further supports the expansion of IL-10⁺ CD4⁺ T cells in an autocrine manner (13). We observed a significant upregulation of Maf by IL-27 but found no significant difference between groups (data not shown). Thus, it is reasonable to speculate that TRAF5 regulates both STAT1 and STAT3 activity mediated by the IL-27 receptor and TCR/CD28.

Collectively, the results presented in this study demonstrate, to our knowledge for the first time, that TRAF5 limits the signaling activity of the IL-27 receptor in CD4⁺ T cells. TRAF5 controls different signaling pathways via the shared gp130 chain and the TNF receptor family molecules in a cell. Therefore, we propose that the balance between positive and negative activities of TRAF5 regulated by diverse cytokine receptors would critically determine the final outcome of the CD4⁺ T cell response.

We thank the Life Science Research Center, University of Toyama and the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine for technical support.

The authors have no financial conflicts of interest.