PNAS PLUS
Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes Laure Ysebrant de Lendonck, Sandrine Tonon, Muriel Nguyen, Patricia Vandevenne, Iain Welsby, Valerie Martinet, Céline Molle, Louis-Marie Charbonnier, Oberdan Leo, and Stanislas Goriely1 Institute for Medical Immunology, Walloon Excellence in Life sciences and BIOtechnology (WELBIO), Université Libre de Bruxelles, B-6041 Charleroi-Gosselies, Belgium Edited by Kenneth M. Murphy, Washington University, St. Louis, MO, and accepted by the Editorial Board July 9, 2013 (received for review November 21, 2012)
transcription factor
| cytokine | Th17 | Tc17 | enhancer
P
olarization of CD4 T helpercells (Th) into distinct effector lineages is determined by the expression of master regulators, such as T-bet, Foxp3, GATA-3, or the orphan nuclear receptor RAR-related orphan receptor (ROR)γt, acting in close interaction with transcription factors from the signal transducers and activators of transcription (STAT) family. For example, STAT4 (activated by IL-12), STAT6 (activated by IL-4), STAT3 (activated by IL-6, IL-21, or IL-23), and STAT5 (activated by IL2) are directly implicated in Th1, Th2, Th17, or Treg development, respectively (1). Although CD8 T cells preferentially differentiate into cytotoxic effectors expressing high IFN-γ levels as a consequence of high T-bet and eomesodermin expression, these effector cells display functional plasticity both in vitro and in vivo and can, under the influence of polarizing factors, differentiate into IL-17–producing cells (Tc17), much as described for CD4 T lymphocytes. IFN regulatory factors (IRF) represent key components of the signaling pathways implicated in antimicrobial innate responses. There are multiple evidences that IRFs contribute to T-cell polarization by modulating the function of antigen-presenting cells (APCs). In addition, several IRFs are also expressed by T lymphocytes and contribute to their differentiation program (2). For example, IRF1 is required for proper expression of IL12Rβ1 and Th1 differentiation (3), whereas IRF4 is involved in both Th2 and Th17 differentiation (4, 5). Recent evidences also indicate that IRF8, expressed upon T-cell activation, represses Th17 programming (6). IRF3 is constitutively expressed in most cell types, including lymphocytes, often retained in the cytoplasm in an inactive form. Pathogen-associated molecular patterns (PAMPs), such as nucleic acids or LPS detected by intracellular sensors or Toll-like receptors (TLR) 3 and 4, activate IRF3 through the recruitment of adaptor molecules such as TIR-domain-containing adapterinducing interferon-β (TRIF), IFN-β promoter stimulator-1 (IPS-1), or STING (7). Subsequent activation of TANK-binding kinase 1 (TBK1) or IKKe leads to C-terminal phosphorylation of
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serine residues in the regulatory domain of IRF3, its dimerization, nuclear translocation, and association with coactivators. IRF3 is a central mediator of antiviral responses through binding to promoters of IFN-β, multiple IFN-stimulated genes (ISGs), and chemokines (7). In addition, this transcription factor also contributes to TLR-mediated IL-27 induction. This cytokine is produced by APCs and limits the development of Th17 responses through multiple mechanisms. This mechanistic link contributes to the propensity of IRF3−/− mice to develop severe inflammatory Th17 responses in the context of experimental autoimmune encephalomyelitis (EAE) (8). We hypothesized that IRF3 activation within T lymphocytes could also affect their polarization. Unexpectedly, we observed that IRF3 restrains IL-17 production by CD8 T cells, even in the absence of activation signals provided by exogenous PAMPs. We further demonstrate that IRF3 directly interacts with RORγt and interferes with its recruitment to regulatory regions. Results Poly(I:C) Represses IL-17 Production by CD8 T Cells in an IRF3Dependent Fashion. Because TLR3 is expressed on effectors and
memory T lymphocytes (9, 10), we used poly(I:C), a synthetic TLR3 ligand, to evaluate the impact of IRF3 activation on T-cell functions. To evaluate the contribution of IRF3 to T-cell responses independently of APCs, we used highly purified, FACSsorted CD4 or CD8 T lymphocytes from naïve WT mice as responder cells in an in vitro setting. Cells were cultured with plate-bound anti-CD3 and anti-CD28 in neutral conditions or in the presence of IL-6 and TGF-β for 3 d. As expected, this cytokine combination strongly supported IL-17 production by both CD4 and CD8 T cells while reducing IFN-γ production. Addition of poly(I:C) had no effect on IL-17 and IFN-γ production by CD4 T cells (Fig. 1A). In contrast, we observed a two- to threefold reduction in IL-17 production by CD8 T cells upon Significance Interferon regulatory factor (IRF) 3 is one of the key transcription factors implicated in innate antiviral responses. In recent years its role in shaping adaptive immune responses through activation of a specific transcriptional program in antigen-presenting cells has been appreciated. In this work we show that within CD8 T cells, IRF3 interacts with the transcriptional network that controls their polarization, thereby limiting the capacity of these cells to produce IL-17. These findings shed light on the functions of IRF3 in adaptive immune responses. Author contributions: L.Y.d.L., S.T., O.L., and S.G. designed research; L.Y.d.L., S.T., M.N., P.V., I.W., V.M., C.M., L.-M.C., and S.G. performed research; L.Y.d.L., S.T., and S.G. analyzed data; and L.Y.d.L., O.L., and S.G. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. K.M.M. is a guest editor invited by the Editorial Board. 1
To whom correspondence should be addressed. E-mail:
[email protected].
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IMMUNOLOGY
IFN regulatory factor (IRF) 3 plays a key role in innate responses against viruses. Herein we assessed its contribution to T-cell activation. We observed that poly(I:C)-induced IRF3 activation in CD8 T cells represses IL-17 expression in a type I IFN-independent fashion. Even in the absence of poly(I:C), polyclonally activated naïve IRF3−/− CD8 T cells expressed high levels of IL-17 and IL-23R in comparison with wild-type cells. Furthermore, IRF3−/− OT1 cells adoptively transferred into wild-type hosts also produced higher IL-17 levels upon immunization than their wild-type counterparts. This phenotype could be reversed by ectopic expression of IRF3, confirming that this effect is intrinsic to T cells. We show that IRF3 directly interacts with RORγt in the cytoplasm through its IRF interaction domain and limits its ability to bind and transactivate the IL-17 promoter. These observations uncover an unexpected role of IRF3 in the control of CD8 T-cell polarization.
Fig. 1. Poly(I:C) represses Tc17 differentiation in an IRF3-dependent fashion. FACS-sorted CD4 T cells (A) and CD8 T cells (B) from WT or IRF3−/− mice were stimulated with αCD3-αCD28 in neutral conditions or in media supplemented with IL-6 and TGF-β for 3 d. When indicated, poly(I:C) (50 μg/mL) was added at the start of the culture. Cytokine production was quantified by ELISA. (C) T cells were activated either in the presence of αCD3-αCD28 or with Poly(I:C). Cell lysates were subjected to Western blotting using antibodies against phospho-IRF3 (ser396) or total IRF3. The data are representative of three independent experiments. (D) CD8 T cells were incubated for 4 h in the presence of poly(I:C) or stimulated with αCD3-αCD28. Expression of IFN-β, IRF7 ISG-54, and CXCL-10 mRNA was analyzed by quantitative RT-PCR. (E) CD8 T cells from WT, IRF3−/−, TRIF−/−, IPS-1−/−, and IFNAR−/− mice were activated in Tc17 skewing conditions for 3 d. When indicated, poly(I:C) was added at the start of the culture. Cytokine production was analyzed by ELISA. Data are expressed as mean ± SEM (n = 4) and is representative of three to five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
treatment with poly(I:C) (Fig. 1B). To assess the specific role of IRF3 in this process, we performed the same experiment with IRF3-deficient CD8 T cells. As shown in Fig. 1B, the repressive action of poly(I:C) on IL-17 production was found to be IRF3E3190 | www.pnas.org/cgi/doi/10.1073/pnas.1219221110
dependent. Strikingly, even in the absence of poly(I:C), CD8 T cells from IRF3−/− mice produced higher levels of IL-17 than their WT counterparts (Fig. 1B). Although differences were less marked, a similar trend was also observed for CD4 T cells. We Ysebrant de Lendonck et al.
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Fig. 2. IRF3 represses IL-17 production by CD8 T cells in a T-cell intrinsic fashion. (A) Sorted naïve (CD62LhighCD44low) and memory (CD62LlowCD44high) CD4 and CD8 T cells from WT or IRF3−/− mice were stimulated with αCD3-αCD28 for 3 d. Cytokines were quantified by ELISA. (B and C) Naïve WT or IRF3−/− CD8 T cells were polyclonally activated in complete media (Tc0) or in media supplemented with IL-6 and TGF-β (Tc17) or IL-6, TGF-β, and IL-23 (Tc17/IL-23) for 3 d. Cytokines produced were quantified by ELISA, and expression of IL-23R and granzyme B was assessed by quantitative RT-PCR. (D) Naïve CD8 T cells were activated in Tc17 skewing conditions. Expression of transcription factors was analyzed by quantitative RT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001. (E) CD8 T cells from IRF3−/− mice differentiated into Tc17 conditions were transduced with retroviral vectors encoding for GFP (empty-RV) or IRF3-IRES-GFP (IRF3-RV). Two days after infection, GFP-positive cells were sorted and IL-17A, IL-23R, RORC, and IFN-γ mRNA expression was analyzed by quantitative RT-PCR. (F–H) Activated CD8 T cells were transduced with the retroviral vectors as described above or with a retroviral vector encoding IRF3 S379A mutant (F and G) or truncated forms of IRF3 (H). GFP-positive cells were sorted and reactivated for 2 d in the presence of IL-23. ELISA (F) and quantitative RT PCR (G and H) were then performed. Data are expressed as mean ± SEM (n = 4) and are representative of three independent experiments. For H, data are expressed as mean ± SEM of experimental triplicates and are representative of two independent experiments.
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hypothesized that in the absence of exogenous PAMPs, IRF3 could be activated by endogenous signals, such as DNA released by dying cells or by cytokines (11). As demonstrated by Western blotting (Fig. 1C), treatment of CD4 or CD8 T cells with poly(I: C) induced phosphorylation of IRF3 on Ser396, a direct target of TBK1 (12). However, we were unable to detect consistent phosphorylation in response to CD3-dependent activation (Fig. 1C). Accordingly, poly(I:C) treatment induced mRNA expression of IFN-β and classic ISGs such as ISG54, IRF7, or CXCL10 in an IRF3-dependent fashion (Fig. 1D). Anti-CD3–dependent stimulation failed to induce expression of these ISGs, confirming the inability of T cell receptor-initiated signals to activate the canonical type I IFNs, IRF3-dependent transcriptional program. In an attempt to characterize the signaling pathway leading to the repression of IL-17 by IRF3, CD8 T cells from WT, IRF3−/−, TRIF−/−, IPS-1−/−, and IFNAR−/− mice were activated in Tc17skewing conditions (Fig. 1E). Inhibition of IL-17 production by poly(I:C) was found to be dependent on both TRIF and IPS-1 pathways. Importantly, repression of IL-17 was still observed in IFNAR−/− cells, indicating that this effect is not a consequence of autocrine type I IFN signaling. In contrast to IRF3−/− CD8 T cells, production of IL-17 by TRIF−/−, IPS-1−/−, or IFNAR−/− cells in absence of poly(I:C) was comparable to that of their WT counterparts. Production of IFN-γ in these conditions was reduced in the absence of IRF3 but not in other groups. Collectively, these results indicate that “classic” IRF3 activation by poly (I:C) in CD8 T cells inhibits IL-17 production. They also suggest that even in the absence of exogenous PAMPs or sign of activation by endogenous signals, IRF3 restrains the capacity of CD8 T cells to produce IL-17. IRF3 Represses IL-17 Production by CD8 T Cells in a T-Cell Intrinsic Fashion Independently of its C-Terminal Regulatory Domain. We
further investigated the mechanisms leading to increased IL-17 production by IRF3−/− CD8 T cells. Initial experiments were performed with sorted CD3+CD4+ or CD3+CD8+ T cells. We next compared the capacity of memory CD4 T or CD8 T cells from WT and IRF3−/− mice to produce IL-17 and IFN-γ in neutral conditions. As expected, activated CD4 and CD8 cells displayed distinct cytokine profiles, with CD4 T cells producing high levels of IL-17, whereas CD8 cells produced mostly IFN-γ (Fig. 2A). IRF3 deficiency favored IL-17 production from CD8 but not CD4 memory T cells, and it led to a lower level of IFN-γ compared with WT CD8 T cells (Fig. 2A). The negative influence of IRF3 on the expression of a typical Tc17 profile could be recapitulated upon in vitro activation of naïve CD8 T cells in Tc17-skewing conditions (IL-6 + TGF-β with or without IL-23) as shown in Fig. 2B. IRF3-deficient, Tc17 cells also expressed higher levels of IL-23R mRNA and IL-22 (Fig. 2 B and C), whereas production of IFN-γ and expression of granzyme B was reduced in these cells. Taken together, these results strongly suggest that IRF3−/− CD8 T cells are intrinsically biased to differentiate into Tc17 cells. We therefore assessed the impact of IRF3 on the expression of key transcription factors. Expression of Rorc, T-bet, eomesodermin, IRF4, and c-maf in the course of Tc17 differentiation of naïve CD8 T cells was found to be comparable in both groups (Fig. 2D). To demonstrate that IRF3 expression by CD8 T cells was responsible for these observations, CD8 T cells from IRF3−/− mice cultured under Tc17-skewing conditions were transduced with retroviral vectors encoding GFP alone (empty-RV) or GFP and IRF3 (IRF3-RV). As shown in Fig. 2E, ectopic expression of IRF3 inhibited IL-17 and IL-23R expression but caused increased IFN-γ mRNA accumulation. As previously observed, IRF3 did not affect Rorc mRNA expression. Next we generated a construct encoding a mutant form of IRF3 (IRF3S379A, corresponding to S386 in human IRF3) that cannot dimerize, translocate to the nucleus, or promote IFN-β transactivation in E3192 | www.pnas.org/cgi/doi/10.1073/pnas.1219221110
response to classic activation pathways (13). Production of IL-17 and IL-22 and expression of IL-23R mRNA were inhibited by transduction of both WT and mutated IRF3 constructs (Fig. 2 F and G). Conversely, IFN-γ production increased to a comparable extent with both constructs. We generated truncated forms of IRF3 lacking the C-terminal region. Deletion of the 367–427 (corresponding to the regulatory domain) or the 308–427 regions did not impair the capacity of IRF3 to repress IL-17 or IL-23R mRNA expression. In contrast, ectopic expression of a shorter (1–128) IRF3 construct, lacking the entire IRF association domain, was unable to repress either IL-17 or IL-23R expression (Fig. 2H). Altogether these results indicate that IRF3 expression within CD8 T cells represses Tc17 differentiation independently of its capacity to activate gene transcription. IRF3 Deficiency Results in Uncontrolled IL-17 Production by Effector Cells in Vivo. To evaluate the global impact of IRF3 on IL-17
production upon T-cell activation in vivo, we injected agonist anti-CD3 antibody to WT or IRF3−/− mice and measured cytokine levels in sera at different time points. We observed higher IL-17 levels in the sera of IRF3−/− mice compared with their WT counterparts, whereas induction of other cytokines, such as IFNγ, IL-13, and IL-10 was comparable between both groups (Fig. 3A). Increased IL-17 production in IRF3−/− mice in these conditions can be attributed to multiple mechanisms, involving both T cells and APCs. To assess the specific role of IRF3 within CD8 T cells upon antigenic stimulation, we adoptively transferred naïve WT, or IRF3−/− OT1 cells (both in a Rag1−/− background) into a congenic WT host. Mice were then immunized with SIINFEKL peptide formulated in complete Freund’s adjuvant (CFA). IRF3 did not impact proliferation of effector cells as the frequency of WT and IRF3−/− OTI cells in the draining lymph nodes at the peak of the response (day 7 after immunization) reached comparable levels. In both groups, activated OTI cells displayed a typical effector phenotype characterized by downregulation of CD62L and up-regulation of CD44 and KLRG1 expression (Fig. 3 B and C). Upon ex vivo stimulation with PMA/ ionomycin or in vitro restimulation in the presence of IL-23, we observed an increased proportion of IL-17–positive cells in IRF3−/− compared with WT OT1 cells (Fig. 3 D and E). In contrast, the proportion of IFN-γ–positive cells was comparable in both groups. Our results demonstrate that IRF3 expression by CD8 T cells limits the development of IL-17–producing effectors cells upon antigenic stimulation in vivo. IRF3 Interacts with RORγt and Limits Its Recruitment to the IL-17 CNS-2 Enhancer Region. We further explored the mechanisms
underlying the repressive action of IRF3 on IL-17 expression. As previously mentioned, Rorc expression was found to be comparable in WT and IRF3−/− CD8 T cells (Fig. 2D). We thus hypothesized that IRF3 could act by interfering with the capacity of RORγt to activate downstream targets. We therefore assessed potential interactions between these two proteins in coimmunoprecipitation experiments. Overexpression of FLAG-tagged IRF3 and T7-tagged RORγt in HEK293T cells indicated that IRF3 is able to interact with RORγt (Fig. 4A). As a control for the specificity of this interaction, we used FLAG-tagged IRF5. Overexpression of truncated forms of IRF3 indicates that the IRF association domain (amino acids 197–357) is necessary and sufficient to interact with RORγt (Fig. 4B). To localize the interaction between IRF3 and RORγt, we performed coimmunoprecipitation (co-IP) experiments after separation of nuclear and cytoplasmic fractions. The interaction with IRF3 occurred exclusively in this latter fraction (Fig. 4C). We observed that although ectopic RORγt was predominantly located in the nucleus (as visualized by confocal microscopy; Fig. 4D), it was also expressed in the cytoplasm. Importantly, we observed that in Ysebrant de Lendonck et al.
PNAS PLUS
Th17 and Tc17 cells, endogenous RORγt also displayed both nuclear and cytoplasmic locations (Fig. 4E). We next performed co-IP experiments with lysates from CD4 T cells differentiated in Th0 or Th17-skewing conditions. They confirmed that endogenous IRF3 and RORγt proteins interact in this physiological context (Fig. 4F). We reached the same conclusions in CD8 T cells differentiated in Tc17-skewing conditions. Taken together, these data suggest that in the course of Tc17, and to a lesser extent Th17 differentiation, IRF3 could repress IL-17 expression by forming a complex with RORγt. To study the impact of IRF3 on the IL-17 promoter activity we performed reporter luciferase assay in the EL4 cell line. Consistent with previous reports (14, 15), we observed that these cells constitutively expressed RORγt (Fig. 5A). We transfected a reporter construct consisting of a 6-kb promoter region upstream of the transcription start site with an IRF3 expression vector. IL17 promoter activity was not influenced by IRF3 overexpression, which could be because EL4 cells already express high levels of endogenous IRF3. However, overexpression of IRF3(5D) strongly repressed both basal and inducible transcriptional activities of the IL-17 promoter (Fig. 5B). To define the cis-acting regions that were required to mediate this effect, we tested the impact of IRF3(5D) on a series of luciferase constructs containing 5′ deletions within the promoter region. Inhibition of Ysebrant de Lendonck et al.
promoter activity by IRF3(5D) required the region located between nucleotides −6021 and −3636 (Fig. 5C). This region encompasses the conserved noncoding sequence (CNS)-2 enhancer (16). We then generated reporter constructs with a minimal proximal promoter (−301/+37) linked to different portions of this critical region. Luciferase activity of the (−301/+37) construct was increased upon addition of the (−3636/−5254) fragment or of a 688-bp fragment corresponding to the CNS-2 region. This enhancer activity was inhibited by IRF3(5D) cotransfection (Fig. 5 E and F). In agreement with previous reports (17), the CNS-2 fragment strongly enhanced the responsiveness of the minimal promoter construct to RORγt overexpression (Fig. 5F). This effect was partially inhibited by IRF3(5D). On the basis of co-IP and reporter experiments, we reasoned that IRF3 could interfere with RORγt binding to the CNS-2 enhancer region. To test this hypothesis we performed ChIP experiments in WT or IRF3−/− CD8 T cells differentiated in Tc17-skewing conditions. In WT cells, we detected RORγt binding to the CNS-2 enhancer but not to the proximal promoter region (Fig. 5G). This recruitment was further increased in the absence of IRF3. We conclude that IRF3 represses IL-17 expression by interfering with RORγt recruitment to the CNS-2 enhancer region. PNAS | Published online August 5, 2013 | E3193
IMMUNOLOGY
Fig. 3. IRF3 deficiency results in increased IL-17 production by effector cells in vivo. (A) WT or IRF3−/− mice were injected with agonist anti-CD3 antibodies. Cytokines produced in sera were quantified at different time points as indicated. Data are present as mean ± SEM (n = 5 for each timepoint) and are representative of three independent experiments. (B–D) Naïve WT or IRF3−/− OT-1 T cells (CD90.2+) were injected in recipient mice (CD90.1+) followed by s.c. immunization with Ova257-264 peptide (SIINFEKL) mixed with CFA. Mice were killed at day 7, and draining lymph nodes cells were collected. (B) Frequency of CD90.2+ OT-1 cells among total CD8 was determined by flow cytometry. (C) OT-1 effector T cells were analyzed for CD62L, CD44, and KLRG-1 expression by flow cytometry. (D and E) Draining lymph nodes cells were either stimulated with PMA/ionomycin for 4 h (D) or incubated 3 d with Ova257-264 peptide (SIINFEKL) and IL-23 before restimulation with PMA/ionomycin for 4 h (E). IL-17 and IFN-γ production of donor-derived antigen-specific T cells was assessed by flow cytometry. Data are expressed as mean ± SEM (n = 7) and are representative of three independent experiments. Representative FACS plots for intracellular IFN-γ and IL-17 stainings in CD90.2+ OT1 cells are shown. *P < 0.05, **P < 0.01.
Fig. 4. IRF3 physically interacts with RORγt. (A) Immunoprecipitation with αT7 on lysates of HEK 293 transfected with expression vectors for T7-RORγt, FlagIRF3, Flag-IRF5, or empty vector followed by immunoblot analysis with αFlag antibody. *Ig heavy chain. (B) Immunoprecipitation of T7-Rorγt and FLAG-tagged IRF3 mutants. The corresponding IRF3 deletions are depicted. *Ig light chain. (C) Immunoprecipitation with αFLAG on nuclear and cytoplasmic fractions of HEK 293 transfected with expression vector for T7-RORγt, Flag-IRF3, or empty vector followed by immunoblot analysis with αT7. (D) HEK 293 cells were transiently transfected with T7-Rorγt and Flag-IRF3 expression vectors. Forty-eight hours after transfection, cells were fixed and stained with anti-T7 (Alexa 594) and anti-Flag (Alexa 488) antibodies followed by confocal microscopic analysis. (Scale bar, 10 μm.) (E) CD4 or CD8 cells were cultured for 72 h under neutral (Th0) or Th17/Tc17-polarizing conditions. Cells were then fixed and stained with anti-RORγt antibody. Nuclei were counterstained with DAPI, followed by confocal microscopy. (Scale bar, 10 μm.) (F) CD4 T cells (Upper) or CD8 T cells (Lower) were cultured under neutral (Th0) or Th17/Tc17-polarizing conditions for 72 h. Cell lysates were then immunoprecipitated with the indicated antibody for co-IP experiments. Data are representative of two independent experiments with similar results.
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Discussion In addition to their central role in regulating innate immune responses, TLR signaling pathways also influence T-cell differentiation via direct or indirect mechanisms. Indeed, several TLRs are expressed by T cells and may directly promote proliferation and cytokine production. For example, TLR2 signaling in CD4 T cells enhances Th17 differentiation and is required for their pathogenic potential in the EAE model (18). In CD8 T cells, TLR2 costimulation promotes T-bet expression and effector functions (19). However, several reports indicate that poly (I:C) directly activates mouse or human CD8 T cells (9, 10). In the present study we show that by activating IRF3 in CD8 T cells, poly(I:C) limits their capacity to produce IL-17. The sequence of Ysebrant de Lendonck et al.
events implicated in IRF3 activation is well understood. In response to classical activators such as LPS, dsRNA, or intracellular DNA, specific serine residues in the C-terminal (regulatory) region of IRF3 are phosphorylated, which causes nuclear translocation. A two-step phosphorylation model by TBK1 has been suggested. First, phosphorylation at Ser396 to Ser405 in site 2 alleviates autoinhibition to allow interaction with the coactivator cAMP response element-binding protein (CREB)-binding protein (CBP). CBP facilitates the phosphorylation of Ser385 or Ser386 at site 1, which then allows for IRF dimerization (7). Thus, the holocomplex containing dimerized IRF3 and coactivators such as CBP or p300 is formed in the nucleus and then binds to target IFN-stimulated response element (ISRE) DNA PNAS | Published online August 5, 2013 | E3195
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Fig. 5. IRF3 inhibits RORγt recruitment to the IL-17 CNS-2 enhancer region. (A) Western blot analysis of RORγt expression in EL4 cells and WT or IRF3−/− CD8 T cells cultured in nonpolarizing or Tc17 conditions for 3 d. (B) Luciferase activity of EL4 cells transiently transfected with the (−6021/+37)IL17Luc reporter construct and IRF3 or IRF3(5D) expression vectors. (C) Luciferase activity of EL4 cells transfected with the indicated reporter constructs and IRF3(5D) expression vector or its control. Data were normalized for each construct against promoter activity in presence of the empty expression vector. (D) Representation of the (−5254/−3636) region of the mouse IL-17 locus containing the CNS-2 region and the different fragments that were generated. (E) Luciferase activity of EL4 cells transfected with the indicated reporter constructs and IRF3(5D) expression vector or its control. Data were normalized against (−301/+37)Luc construct with empty expression vector. (F) Cotransfection experiments. The indicated reporter constructs were transfected with RORgt and/or IRF3(5D) expression vectors. Data were normalized against (−301/+37)Luc construct with empty expression vector. (G) ChIP experiments; binding of endogenous RORγt on IL17 promoter and IL17 enhancer regions in WT or IRF3−/− CD8 T cells differentiated in Tc17 condition for 4 d before resting and restimulation with αCD3/αCD28 overnight. Data are represented as mean ± SEM of triplicates and are representative of at least three independent experiments. R.L.U., relative light units; R.U., relative units.
sequences within the promoters of type I IFN and other cytokine/chemokine genes. In the context of its function in innate immune responses, IRF3 also interacts with other transcription factors, coactivators, and repressors, including other IRFs, NFκB p65, Maf-B, or β-catenin (20, 21). We show that even in the absence of exogenous PAMPs, constitutively expressed IRF3 limits Tc17 differentiation without affecting RORγt expression. We observed that IRF3 binds to RORγt through its IRF association domain and that this interaction takes place in the cytoplasm. Localization and function of RORγt are dynamically regulated by the PI3K–Akt–mammalian target of rapamycin complex 1 (mTORC1) axis (22). It is therefore possible that IRF3 interferes with the shuttling of RORγt from the cytoplasm to the nucleus and hence its recruitment to DNA-binding sites. Our results also indicate that classic IRF3 activation by poly(I:C) or the use of an IRF3(5D) phosphomimetic construct further potentiates this repressive effect on RORγt. Although this effect is independent of type I IFNs, we cannot exclude that other inducible mediators (such as other IRFs or autocrine IFN-λ) contribute to the inhibition of IL-17 production in these settings. Our data support a unique regulatory function of IRF3 in CD8 T cells. The role of IRF3 in Th17 differentiation is less marked than in Tc17, suggesting qualitative or quantitative differences in the transcriptional network involved in the differentiation of both cell types. A physiological role for Tc17 cells is emerging. These cells are critical for immunity against invading fungi in immune-deficient hosts and protect against lethal influenza infection in mouse and humans (23, 24). In mouse models of lymphocytic choriomeningitis virus (LCMV), CD8 T cells lacking T-bet and eomesodermin massively differentiated into Tc17, leading to live-threatening multiorgan infiltration of neutrophils (25). Tc17 have also been involved in human pathologies, including primary biliary cirrhosis, immune thrombocytosis, cutaneous acute graft-vs.-host disease, psoriasis, or allergic contact dermatitis (26–29). Recent studies demonstrated that Tc17 massively infiltrate carcinoma and could promote progression through the recruitment of myeloid-derived suppressor cells (30, 31). Hence, developing strategies to manipulate Tc17 differentiation could be of great interest to treat human diseases. Materials and Methods Mice. IRF3-deficient (IRF3−/−) on C57BL/6 background mice were obtained from the Riken BioResource Center with the approval of T. Taniguchi (University of Tokyo, Tokyo, Japan). RAG1-deficient, OT1 TCR transgenic, Thy1.1 congenic, and TRIF-deficient (Lps2) mice on C57BL/6 background were obtained from the Jackson Laboratory. IPS-1–deficient and IFNAR-deficient mice on C57BL/6 background were kindly provided by T. Kawai (Osaka University, Osaka, Japan) and A. Randrup Thomsen (University of Copenhagen, Copenhagen, Denmark). Mice were housed and bred in our specific pathogen-free animal facility. All animal studies were approved by the institutional animal care committee and local committee for animal welfare. T-Cell Purification. Naïve and memory CD8+ T cells were isolated from spleen by flow cytometry (FACSAria; BD Biosciences) after staining with anti-CD8 (53-6.7), anti-CD62L (MEL-14), anti-CD44 antibodies (IM7). For experiment with Poly(I:C), total CD8 and CD4 T cells were sorted by flow cytometry after staining with anti-CD8 (53-6.7), anti-CD4 (RM4-5), and anti-CD3 (145-2C11). Naïve and memory CD4+ T cells were isolated from spleen by flow cytometry (FACSAria) after staining with anti-CD4 (RM4-5), anti-CD62L (MEL-14), and anti-CD44 antibodies (IM7). All antibodies were purchased from BD Biosciences. For co-IP, Western blotting, confocal microscopy, and ChIP experiments, total CD8 T cells were purified by magnetic-activated cell sorting (CD8α microbeads, mice; MACS Miltenyi Biotec). T-Cell Cultures. All cultures were performed in RPMI with 10% (vol/vol) FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 40 μM β-mercaptoethanol, 100 U/mL penicillin, and 100 U/mL streptomycin (all from Lonza). Total, naïve, or memory CD8 T cells were cultured with plate-bound anti-CD3 (5 μg/mL) and soluble anti-CD28 (1 μg/mL) for 3 d in culture medium without cytokines (Tc0) or with recombinant human
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TGF-β (2.5 ng/mL) and recombinant mouse IL-6 (10 ng/mL) either in the absence (Tc17) or presence of recombinant mouse IL-23 (25 ng/mL) (Tc17/IL-23). All cytokines were purchased from R&D Systems and antibodies from BD Biosciences. After stimulation, supernatants were collected for cytokines quantification by ELISA (Duoset, R&D Systems). Restimulation and Intracellular Staining. Purified CD8 T cells or draining lymph node cells were stimulated for 4 h with phorbol myristate acetate (PMA) (25 ng/mL; Sigma) and ionomycin (500 ng/mL; Sigma), with the addition of brefeldin A (5 μg/mL, Sigma) for the last 2 h. Cells were first stained for surface antigens and then treated with Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s directions. Intracellular cytokine staining was performed using anti-IL-17 APC (BD Biosciences, TC11-18H10) and anti-IFN-γ phycoerythrin (PE) (BD Biosciences, XMG1.2). In Vivo T-Cell Activation. For in vivo polyclonal T-cell stimulation, mice were injected i.p. with anti-CD3 (50 μg, 145 2C11). For adoptive transfer, 106 WT or IRF3 OTI T cells (CD90.2+) were adoptively transferred into congenic C57/BL6 recipient mice (CD90.1+). Twenty-four hours later, recipient mice were immunized with 20 μg of Ova257-264 peptide (SIINFEKL) mixed with an equal volume of CFA (Sigma-Aldrich). Seven days after immunization recipient mice were killed, and draining lymph node cells were either immediately stimulated with PMA/ionomycin or stimulated with 200 nM of Ova257-264 peptide (SIINFEKL) for 48 h before restimulation with PMA/ionomycin. Donor-derived antigen-specific T cells were identified by surface staining with anti CD90.2 (53-2.1) and anti-CD8 (53-6.7). RNA Purification and Real-Time RT-PCR. Total RNA from cells was extracted using a MagnaPure LC RNA-High Performance Isolation Kit (Roche Diagnostics). RT and real-time PCR reactions were carried out using a Taqman RNA amplification kit (one-step procedure) on a Lightcycler 480 Real-Time PCR system (Roche Diagnostics). The sequences of primers and probes will be provided upon request. Retroviral Transduction. The mouse IRF3 coding sequence was PCR amplified, subcloned in TA-TOPO vector (Invitrogen), and cloned into pMXs-IRES-GFP (pMIG) retroviral vector downstream of the internal ribosomal entry site (IRES)-GFP coding sequence as an EcoRI fragment. The IRF3S379A mutant was derived by site-directed mutagenesis using the Quikchange II kit (Agilent Technologies) according to the manufacturer’s instructions. Truncated IRF3 constructs were generated by PCR. Plat-E cells were transfected with the indicated plasmids using the phosphate calcium technique. Viral supernatant was collected and supplemented with 10 μg/mL polybrene (Sigma). CD8 T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 in Tc17-skewing conditions. Twenty-four hours after activation, cells were centrifuged at 6,000 × g with retrovirus and then cultured for an additional 48 h under Tc17 conditions. GFP+ cells were sorted and stimulated for 2 d without cytokines or with IL-23. Western Blotting and Co-IP Experiments. HEK293 cells were transiently transfected via Fugene-6 (Roche) with empty vectors or vectors encoding for FLAG-IRF3 or a FLAG-tagged truncated form of IRF3 or FLAG-IRF5 and T7Rorγt. Then, 48 h after transfection, cells were lysed in lysis buffer [0.5% Nonidet P-40, 50 mM Tris (pH 7.4), 200 mM NaCl, 10% (vol/vol) glycerol 1 mM DTT, and protease and phosphatase inhibitors). For co-IP of endogenous IRF3 and Rorγt, CD8 T cells were differentiated with IL-6 and TGF-β for 3 d and were lysed in the lysis buffer described above. Phospho-specific IRF3 (Ser396) and total IRF3 antibodies were purchased from Cell Signaling and Invitrogen, respectively. Whole-cell lysates were incubated overnight at 4 °C with rabbit polyclonal anti-Rorγt (H190; Santa Cruz Biotechnology), polyclonal rabbit anti-IRF3 (ZM3; Invitrogen), rabbit polyclonal anti-T7 (MBL), or monoclonal mouse anti-FLAG (M2; Sigma) covalently linked to protein G magnetic-activated beads (Dynabeads Protein G, Invitrogen). Nuclear and cytoplasmic fractions were separated using nuclear extract buffer [10 mM Tris·HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40]. Nuclei were lysed using cellular extract buffer [50 mM Tris (pH 8.0), 280 mM NaCl, 0.5% octylphenoxypolyethoxyethanol (IGEPAL), 0.2 nM EDTA, 2 mM EGTA, 10% (vol/vol) glycerol, and 1 mM DTT) containing protease and phosphatase inhibitors. Immunofluorescence Microscopy. HEK 293T cells were cultured on poly-lysine treated coverslips. T cells were centrifuged onto glass slides using a Shandon Eliott Cytocentrifuge (3 min, 37 × g). The cells were fixed in 4% (vol/vol) paraformaldehyde (10 min at room temperature) and washed twice in PBS,
Ysebrant de Lendonck et al.
Chromatin Immunoprecipitation. Total CD8 T cells were differentiated in Tc17skewing conditions (5 d) and were stimulated for 4 h with PMA/ionomycin. Cells were then fixed for 10 min at room temperature with 1% formaldehyde, and glycine was added to a final concentration of 0.125 M. Cells were washed twice with ice-cold PBS, resuspended in lysis buffer, and sonicated to obtain chromatin fragments 200–500 bp in length with a bioruptor device (Diagenode). Chromatin fragments were diluted with ChIP dilution buffer and precleared with protein G magnetic-activated beads (Millipore) for 1 h. Chromatin was then incubated overnight at 4 °C with polyclonal rabbit antiRorγt (H190; Santa Cruz Biotechnology) or rabbit polyclonal IgG (ab27472; Santa Cruz Biotechnology) and protein G magnetic-activated beads (Millipore). Beads were washed three times and were incubated overnight with RNase at 65 °C. DNA was purified with the QIAquick kit according to the
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PNAS PLUS
manufacture instructions (Qiagen). Quantitative PCR was performed with primers encompassing the CNS-2 or the proximal promoter regions. Transient Transfection, Luciferase Assays, and Constructs. EL4 cells were transfected using FuGENE-6 (Roche Diagnostics). Promoter activities were analyzed 48 h after transfection using the Dual-Glo Luciferase Reporter Assay system (Promega). Promoter activities were then normalized to Renilla luciferase activities. The (−6021/+37) IL17A-luc construct (into pGV-basic2 backbone) and its 5′ deletions (15) were kindly provided by A. Yoshimura (Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan). Enh#1–5 fragments were generated by PCR and inserted into the SacI restriction site upstream of the (−301/+37) minimal promoter region. Flag-tagged pCMV2-IRF3 expression vector and its derivatives were kindly provided by R. Lin (Lady Davis Institute for Medical Research, McGill University, Montreal, QC, Canada). pCMVTRORγt-T7 expression vector was kindly provided by A. Yoshimura (Keio University School of Medicine, Tokyo, Japan). Statistical Analysis. Statistical analysis was performed using Student’s t test. ACKNOWLEDGMENTS. The Institute for Medical Immunology is sponsored by the government of the Walloon Region and GlaxoSmithKline Biologicals. This study was supported by the Fonds National de la Recherche Scientifique (FRS-FNRS, Belgium) and an Interuniversity Attraction Pole of the Belgian Federal Science Policy. L.Y.d.L. is supported by a grant from the Télévie. L.Y.d.L., C.M., V.M., and S.G. are supported by the FRS-FNRS.
17. Zhang F, Meng G, Strober W (2008) Interactions among the transcription factors Runx1, RORgammat and Foxp3 regulate the differentiation of interleukin 17-producing T cells. Nat Immunol 9(11):1297–1306. 18. Reynolds JM, et al. (2010) Toll-like receptor 2 signaling in CD4(+) T lymphocytes promotes T helper 17 responses and regulates the pathogenesis of autoimmune disease. Immunity 32(5):692–702. 19. Geng D, et al. (2010) When Toll-like receptor and T-cell receptor signals collide: A mechanism for enhanced CD8 T-cell effector function. Blood 116(18):3494–3504. 20. Honda K, Taniguchi T (2006) IRFs: Master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol 6(9):644–658. 21. Yang P, et al. (2010) The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a beta-catenin-dependent pathway. Nat Immunol 11(6): 487–494. 22. Kurebayashi Y, et al. (2012) PI3K-Akt-mTORC1-S6K1/2 axis controls Th17 differentiation by regulating Gfi1 expression and nuclear translocation of RORgamma. Cell Rep 1(4): 360–373. 23. Nanjappa SG, Heninger E, Wüthrich M, Gasper DJ, Klein BS (2012) Tc17 cells mediate vaccine immunity against lethal fungal pneumonia in immune deficient hosts lacking CD4+ T cells. PLoS Pathog 8(7):e1002771. 24. Hamada H, et al. (2009) Tc17, a unique subset of CD8 T cells that can protect against lethal influenza challenge. J Immunol 182(6):3469–3481. 25. Intlekofer AM, et al. (2008) Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin. Science 321(5887):408–411. 26. Oo YH, et al. (2012) CXCR3 dependent recruitment and CCR6 mediated positioning of Th-17 cells in the inflamed liver. J Hepatol 57(5):1044–1051. 27. Hu Y, et al. (2011) Increased number of Tc17 and correlation with Th17 cells in patients with immune thrombocytopenia. PLoS ONE 6(10):e26522. 28. Zhao Y, Yang J, Gao YD (2011) Altered expressions of helper T cell (Th)1, Th2, and Th17 cytokines in CD8(+) and γδ T cells in patients with allergic asthma. J Asthma 48(5):429–436. 29. Res PC, et al. (2010) Overrepresentation of IL-17A and IL-22 producing CD8 T cells in lesional skin suggests their involvement in the pathogenesis of psoriasis. PLoS ONE 5(11):e14108. 30. Zhuang Y, et al. (2012) CD8(+) T cells that produce interleukin-17 regulate myeloidderived suppressor cells and are associated with survival time of patients with gastric cancer. Gastroenterology 143(4):951–962, e8. 31. Li J, et al. (2011) Distribution, characterization, and induction of CD8+ regulatory T cells and IL-17-producing CD8+ T cells in nasopharyngeal carcinoma. J Transl Med 9:189.
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then permeabilized in PBS containing 0.1% Triton X-100 for 10 min at room temperature. After three washes in PBS, the cells were blocked for 30 min in PBS-BSA 10% (wt/vol) and incubated (1 h, room temperature) with the primary antibodies [rabbit anti-T7 (MBL), 1/500; mouse anti-Flag M2 (Sigma), 1/ 500; anti-RORγt AFKJS-9 (eBioscience), 1/200] in PBS-Tween 20 0.1%. The slides were then washed 3 × 10 min (PBS-Tween) and incubated (1 h, room temperature) with the appropriate secondary antibodies (goat anti-mouse Alexa 488, goat anti-rabbit 594, goat anti-rat 488; Invitrogen, 1/1000), washed 3 × 10 min in PBS-Tween, rinsed rapidly in dH2O, and mounted in Fluorescence Mounting Medium (Dako) containing DAPI. The slides were visualized on a Zeiss LSM 710 confocal microscope.
