Early Growth Response Gene-2 Controls IL-17 Expression and Th17 Differentiation by Negatively Regulating Batf

Tizong Miao, Meera Raymond, Punamdip Bhullar, Emma Ghaffari, Alistair L. J. Symonds, Ute C. Meier, Gavin Giovannoni, Suling Li and Ping Wang
J Immunol January 1, 2013, 190 (1) 58-65; DOI: https://doi.org/10.4049/jimmunol.1200868

Abstract

Early growth response gene (Egr)-2 is important for the maintenance of T cell homeostasis and controls the development of autoimmune disease. However, the underlying mechanisms are unknown. We have now discovered that Egr-2, which is induced by TGF-β and IL-6, negatively regulates the expression of IL-17, but not IL-2 or IFN-γ, in effector T cells. In the absence of Egr-2, CD4 T cells produce high levels of Th17 cytokines, which renders mice susceptible to experimental autoimmune encephalomyelitis induction. T cells lacking Egr-2 show increased propensity for Th17, but not Th1 or Th2, differentiation. Control of IL-17 expression and Th17 differentiation by Egr-2 is due to inhibition of Batf, a transcription factor that regulates IL-17 expression and Th17 differentiation. Egr-2 interacts with Batf in CD4 T cells and suppresses its interaction with DNA sequences derived from the IL-17 promoter, whereas the activation of STAT3 and expression of retinoic acid–related orphan receptor γt are unchanged in Th17 cells in the absence of Egr-2. Thus, Egr-2 plays an important role to intrinsically control Th17 differentiation. We also found that CD4 T cells from multiple sclerosis patients have reduced expression of Egr-2 and increased expression of IL-17 following stimulation with anti-CD3 in vitro. Collectively, our results demonstrate that Egr-2 is an intrinsic regulator that controls Th17 differentiation by inhibiting Batf activation, which may be important for the control of multiple sclerosis development.

Introduction

Thelper 17 cells differentiated from naive CD4 T cells provide protection in certain infections, but they play important roles in the immunopathology of autoimmune diseases (1), which has been demonstrated in several mouse models of autoimmune disease such as experimental autoimmune encephalomyelitis (EAE), inflammatory bowel disease, and collagen-induced arthritis, and they are also implicated in various human autoimmune diseases (2). Th17 differentiation is dependent on TGF-β, together with proinflammatory cytokines such as IL-6, IL-21, IL-23, and IL-1 (3). These cytokines induce expression and/or activation of the transcription factors retinoic acid–related orphan receptor (ROR)γt/RORα, STAT3, IFN regulatory factor-4, aryl hyrdrocarbon receptor, Iκbζ, and Batf, which individually or collectively drive expression of Th17 cytokines (4–10). The differentiation of Th17 cells and expression of Th17 cytokines are regulated to prevent excessive inflammation. However, little is known about the cell-intrinsic mechanisms that control Th17 differentiation and how they contribute to the control of autoimmune diseases.

Previously, we and others have shown that early growth response gene (Egr)-2 is induced in naive and tolerant T cells by Ag in vitro and in vivo (11–13). Egr-2 is a member of the Egr family of zinc finger transcription factors, which consists of four members: Egr-1, Egr-2, Egr-3, and Egr-4 (14). Egr-2 is essential for hindbrain development and myelination of the peripheral nervous system (15). Egr-1, Egr-2, and Egr-3 are expressed in thymocytes (16) and are involved in the development of double-negative thymocytes and in positive selection (17–20). Recently, expression of Egr-1 and Egr-2 has been found in NKT cells, which is required for the development of NKT cells (21, 22). In peripheral T cells, Egr-2 is involved in the induction of Fas ligand and the regulation of homeostasis (23, 24). RNA interference–mediated knockdown of Egr-2 in an established T cell line rendered the cells less susceptible to anergy induction (11), whereas overexpression of Egr-2 reduced T cell activation in vitro through induction of the E3 ligase Cbl-b (11, 12). Egr-2 has been found to be preferentially expressed in LAG3+ regulatory T cells, and forced expression of Egr-2 converted naive CD4 T cells into IL-10–expressing LAG3+ regulatory T cells (25), which further demonstrates the importance of Egr-2 in the regulation of immune homeostasis.

We have previously demonstrated that Egr-2 deficiency in T cells results in the development of autoimmune diseases in late life with hyperproliferation of effector phenotype T cells and accumulation of IFN-γ– and IL-17–producing CD4 T cells (26). However, despite this homeostatic disorder and development of autoimmune diseases, Egr-2 deficiency did not increase the activation or production of IL-2 by naive T cells induced by TCR ligation (26), suggesting that Egr-2 may be involved in the regulation of T cell subsets involved in the immunopathology of autoimmunity.

In this study, we show that Egr-2 regulates Th17 differentiation and expression of IL-17. CD2-specific Egr-2 deficiency resulted in the development of autoimmune diseases in later life, with accumulation of activated T cells and high levels of IL-17 expression in activated CD4 T cells (26). Egr-2 was induced by TGF-β and IL-6 in naive CD4 T cells. A defect in Egr-2 renders naive CD4 T cells highly prone to Th17 differentiation and increases production of Th17 cytokines. The Egr-2–deficient mice were highly susceptible to the induction of EAE. Importantly, we found that the expression of Egr-2 was reduced in activated T cells from multiple sclerosis (MS) patients. The reduced expression of Egr-2 was associated with increased expression of IL-17. We found that Egr-2 directly interacts with Batf, a Th17 inducer, and blocks its binding to the IL-17 promoter. Thus, Egr-2 induced during Th17 differentiation serves as a negative feedback inhibitor to control Th17-mediated inflammation.

Materials and Methods

Mice

CD2-specific Egr-2−/− mice on the C57BL/6 background were reported in our previous publication (26). C57BL/6 mice were used as controls in all experiments. All mice were maintained in the Biological Services Unit, Brunel University, and used according to established institutional guidelines under the authority of a U.K. Home Office project license.

Abs, reagents, and flow cytometry

Cyclosporin A (CsA) was from Sigma-Aldrich. FITC-conjugated Abs to IFN-γ and IL-4, PE-conjugated Ab to IL-17A, and allophycocyanin-conjugated Ab to CD4 were obtained from BD Biosciences. Egr-1, Egr-2, and Egr-3 Abs were purchased from Covance and Santa Cruz Biotechnology. Ab to pSTAT3 was from Cell Signaling Technology, and anti-BATF was obtained from Santa Cruz Biotechnology. For flow cytometry analysis, single-cell suspensions were analyzed on an LSRII (BD Immunocytometry Systems) and the data were analyzed using FlowJo (Tree Star).

Microarray

Naive CD4 T cells were stimulated with or without anti-CD3 for 6 h. Total RNA was extracted and genome-wild transcriptional profiles were analyzed using MouseRef-8 v2.0 BeadChip expression array (Illumina). The probe labeling, array hybridization, and data processing were carried out at the Microarray Facility, Barts and London School of Medicine and Dentistry, according to the manufacturer’s instructions. We focused on genes that showed a difference of at least 3-fold between wild-type and Egr-2–deficient CD4+ T cells and excluded genes that had a detection p value >0.01. The differentially expressed genes were first grouped based on self-organizing maps (SOMs) by a clustering algorithm method. The SOM clusters were further clustered by a hierarchical clustering program to confirm the different profile (ArrayExpress accession E-MEXP-1698; http://www.ebi.ac.uk/arrayexpress).

T cell differentiation

Naive CD4+ T cells were isolated by a CD4+CD62L+ T cell isolation kit II (Miltenyi Biotec). The isolated cells were >95% CD4+CD62L+CD44lowCD25−. For differentiation, cells were stimulated with anti-CD3 and anti-CD28 supplemented with 20 ng/ml IL-12 and 20 μg/ml anti–IL-4 for Th1 or 50 ng/ml IL-4 and 10 μg/ml anti–IFN-γ or 2 ng/ml TGF-β, 20 ng/ml IL-6, 10 μg/ml anti–IFN-γ, and 20 μg/ml anti–IL-4 for 3 d. The cells were washed and cultured in Th condition medium in the absence of anti-CD3 and anti-CD28 for 2 d. The differentiated cells were restimulated with or without anti-CD3 and anti-CD28 for 6 h before analysis.

Experimental autoimmune encephalomyelitis

Induction of EAE was carried out using a myelin oligodendrocyte glycoprotein (MOG)35–55/CFA emulsion kit (EK-0111; Hooke Laboratories) according to the manufacturer’s instructions. The severity of EAE was scored on a scale of 0–4: 0, normal; 1, limp tail; 2, limp tail and weakness of hind legs; 3, limp tail with paralysis of one front and one hind leg; 4, limp tail, complete hind leg and partial front leg paralysis.

Thirty days after EAE induction mice were sacrificed and CNS tissues were fixed with 10% formalin in PBS and embedded in paraffin. Sections were stained with H&E. Histological examination of tissue sections was done in a blind manner. Additionally, CNS-infiltrating mononuclear cells were isolated and analyzed for IL-17– and IFN-γ–producing CD4 T cells by intracellular cytokine staining.

Patients and blood samples

Eleven relapsing and remitting MS patients in remission and five healthy donors were enrolled in this study. All MS patients were retrieved from the MS Center, Royal London Hospital, London. The study has been approved by our Ethics Committee. All patients were diagnosed as having relapsing and remitting MS according to the McDonald criteria (27), were drug naive in relation to disease-modifying therapy, and were in clinical remission. Blood was taken according to standard protocols, using heparinized tubes, after signed informed consent was obtained. CD4 T cells were isolated from blood lymphocytes by positive selection using MACS systems (Miltenyi Biotec) and stimulated in vitro for 48 h with anti-CD3 and anti-CD28 before restimulation for 6 h with the same stimuli. Total RNA extracted from these cells, and unstimulated cells, was analyzed for Batf, Egr-2, and IL-17A expression by real-time quantitative PCR (RT-qPCR). Patient characteristics are presented in Supplemental Table I.

Intracellular cytokine analysis

Splenocytes, CD4 T cells, and CNS-infiltrating mononuclear cells were stimulated with PMA (20 ng/ml) plus ionomycin (0.5 μg/ml) in the presence of brefeldin A for 5 h. After staining with cell surface markers, intracellular cytokine staining was performed with a fixation and permeabilization kit (eBioscience) and IFN-γ, IL-4, and IL-17A Abs in accordance with the manufacturer’s instructions.

RT-qPCR

Total RNA was extracted from stimulated or unstimulated CD4+ T cells using an RNeasy kit from Qiagen and reverse transcribed using oligo(dT) primers (Amersham Biosciences). RT-qPCR was performed on a Rotor-Gene system (Corbett Robotics) using SYBR Green PCR Master mix (Qiagen). Primers were as follows: mouse Egr-2, sense, 5′-CTTCAGCCGAAGTGACCACC-3′, antisense, 5′-GCTCTTCCGTTCCTTCTGCC-3′; β-actin, sense, 5′-AATCGTGCGTGACATCAAAG-3′, antisense, 5′-ATGCCACAGGATTCCATACC-3′; IL-2, sense, 5′-GCATGTTCTGGATTTGACTC-3′, antisense, 5′-CAGTTGCTGACTCATCATCG-3′; IL-4, sense, 5′-CAAACGTCCTCACAGCAACG-3′, antisense, 5′-CTTGGACTCATTCATGGTGC-3′; IL-17A, sense, 5′-AGCGTGTCCAAACACTGAGG-3′, antisense, 5′-CTATCAGGGTCTTCATTGCG-3′; IL-17F, sense, 5′-AACCAGGGCATTTCTGTCCC-3′, antisense, 5′-TTTCTTGCTGAATGGCGACG-3′; IFN-γ, sense, 5′-CCATCAGCAACAACATAAGC-3′, antisense, 5′-AGCTCATTGAATGCTTGGCG-3′; IL-21, sense, 5′-ATCCTGAACTTCTATCAGCTCCAC-3′, antisense, 5′-GCATTTAGCTATGTGCTTCTGTTTC-3′; Egr3, sense, 5′-CGACTCGGTAGCCCATTACAATCAGA-3′, antisense, 5′-GAGATCGCCGCAGTTGGAATAAGGAG-3′; Egr1, sense, 5′-ACGACAGCAGTCCCATCTACTCGG-3′, antisense, 5′-GGACTCGACAGGGCAAGCATATGG-3′; BATF, sense, 5′-GAGCTGCGTTCTGTTTCTCC-3′, antisense, 5′-CCAGAAGAGCCGACAGAGAC-3′; human β-actin, sense, 5′-CCCAGCACAATGAAGATCAA-3′, antisense, 5′-ACATCTGCTGGAAGGTGGAC-3′; human Egr-2, sense, 5′-CTTTGACCAGATGAACGGAG-3′, antisense, 5′-CCCATGTAAGTGAAGGTCTG -3′; human Batf, sense, 5′-ACACAGAAGGCCGACACC-3′, antisense, 5′-CTTGATCTCCTTGCGTAGAGC-3′; human IL-17A, sense, 5′-CTCCTGGGAAGACCTCATTG-3′, antisense, 5′-GAGGACCTTTTGGGATTGGT-3′.

The data were analyzed using the Rotor-Gene software. All samples were run in duplicate, and relative mRNA expression levels were obtained by normalizing against the level of β-actin from the same sample under the same program using the following: relative expression = 2(Ctβ-actin − Cttarget) × 10,000, where Ct indicates threshold cycle.

EMSA

Probes corresponding to a Batf binding site in the IL-17 promoter (5′-TGGTTCTGTGCTGACCTCATTTGAGGATG-3′; nucleotides −155 to −187) (10) were labeled with α-[32P]dCTP using Ready-to-Go DNA labeling beads (Amersham Biosciences, Buckingham, U.K.) and used in binding reactions with nuclear extracts. Nuclear extracts were obtained as described in our previous report (26). For supershift reactions, anti-Batf was added after 10 min incubation. The samples were electrophoresed on 5% polyacrylamide gels in 0.5× TBE.

Reporter gene assay

Mouse Batf, Egr-1, Egr-2, and Egr-3 cDNAs were introduced into pcDNA expression vectors by PCR cloning approaches. The reporter plasmid IL-17-Luc (9) and the indicated expression plasmids were transfected into HEK293T cells using FuGENE 6 (Roche). After 36 h, a dual luciferase assay (Promega) was performed according to the manufacturer’s protocol.

Lentiviral transduction

The lentiviral constructs used were described previously (28). Egr-2 was introduced into these constructs by a PCR cloning approach. In addition to Egr-2, the construct carries an IRES-driven GFP that allows us to isolate transduced cells by fluorescence-activated cell sorting. Naive CD4 cells from K2/3 mice at 1 × 106 per well in a 24-well plate coated with anti-CD3 and anti-CD28 and supplemented with 2 ng/ml TGF-β, 20 ng/ml IL-6, 10 μg/ml anti–IFN-γ, and 20 μg/ml anti–IL-4 were infected with concentrated lentivirus at a multiplicity of infection of 50–100 (∼105–106 transducing units/ng) as previously described (28). Two days after infection, fresh medium supplemented with 2 ng/ml TGF-β, 20 ng/ml IL-6, 10 μg/ml anti–IFN-γ, and 20 μg/ml anti–IL-4 was added. The cells were harvested after 5 d and the GFP+ cells were isolated by cell sorting. The isolated cells were used for RT-qPCR analysis and analysis of IL-17–producing cells.

mRNA silencing

A specific small interfering (si) oligonucleotide (siRNA) targeting the mRNA sequence of Egr-2, siEgr-2-1 (5′-GCUGCUAUCCAGAAGGUAU-3′), was used. Irrelevant scrambled siRNA obtained from Qiagen (catalog no. 1027281) was used as a negative control. Primary naive CD4+ T cells isolated from 6- to 8-wk-old mice were transfected with 1 μM siRNA using an Amaxa Nucleofector according to the manufacturer’s instructions. Cells were stimulated with anti-CD3 and anti-CD28, which were supplemented with 2 ng/ml TGF-β, 20 ng/ml IL-6, 10 μg/ml anti–IFN-γ, and 20 μg/ml anti–IL-4, for 3 d before analysis of Egr-2 and IL-17 expression.

Statistical analysis

A Student unpaired t test was used to analyze the statistical significance of differences between the groups. Differences with a p value <0.05 were considered significant.

Results

Egr-2 controls expression of Th17, but not Th1 or Th2, cytokines in effector CD4 T cells

Previously, we found that, in association with the development of lupus like disease, Egr-2 deficiency induced high expression of IL-17 and IFN-γ in effector T cells in aged mice (26). To investigate the roles of Egr-2 in CD4 T cells, we carried out expression array analysis on unstimulated and anti-CD3– and anti-CD28–stimulated naive CD4 T cells from Egr-2 conditional knockout (cKO) mice. Among the genes associated with CD4 T cell function, IL-1, IL-17, and IL-21 were highly induced in Egr-2–deficient CD4 T cells whereas the expression of IL-2 and IFN-γ was unchanged in response to TCR stimulation (Fig. 1A, 1B). The selectively increased expression of Th17 cytokines in Egr-2–deficient CD4 T cells was confirmed by RT-qPCR (Fig. 1B). The expression of IL-2 and IFN-γ was effectively induced in both wild-type and Egr-2–deficient naive CD4 T cells in response to TCR stimulation (Fig. 1B), whereas the expression of Th17 cytokines, including IL-1, IL-17, and IL-21, was highly induced only in Egr-2–deficient CD4 T cells with minimal expression in CD4 naive T cells from wild-type mice (Fig. 1B). Consistent with this, the percentage of Egr-2–deficient CD4 cells that produced IL-17 was also increased compared with wild-type CD4 cells (Fig. 1C). Thus, Egr-2 is involved in the control of Th17 cytokine expression in effector T cells.

FIGURE 1.
FIGURE 1.

Egr-2 deficiency leads to enhanced expression of Th17 cytokines in naive CD4 T cells in response to TCR stimulation. (A) Altered expression of cytokine and cytokine receptor genes in Egr-2–deficient CD4 T cells as assessed by genome-wide transcriptional analysis. The SOM cluster of cytokines was further clustered by a hierarchical clustering program to show the genes with differential expression profiles. (B) Expression of cytokines in CD4 T cells as measured by RT-qPCR. (C) IL-17A–producing CD4 T cells were analyzed by intracellular cytokine staining after stimulation of splenocytes for 3 h with 100 ng/ml PMA/ionomycin in vitro. The PCR data are presented relative to the expression of β-actin mRNA. (B) and (C) are representative of three independent experiments. WT, Wild-type.

Egr-2 is induced in naive CD4 T cells by TGF-β and IL-6

Previously, we found that Egr-2 is effectively induced in naive T cells by Ag stimulation in vivo (13). The selective induction of Th17 cytokines in Egr-2–deficient CD4 T cells by TCR stimulation indicates that Egr-2 controls the expression of Th17 cytokines in T cells in response to mitogenic Ag stimulation. Th17 cytokines are normally induced in naive CD4 T cells in the presence of cytokines that can induce Th17 differentiation such as the well-defined Th17 inducers TGF-β and IL-6 (3). However, whether these stimuli can induce Egr-2 expression is unknown. We therefore examined the expression of Egr-2 in naive T cells following stimulation with different cytokines in vitro. The results showed that Egr-2 is rapidly induced in naive T cells following TGF-β and IL-6 stimulation (Fig. 2A). In contrast, IFN-γ and IL-12/IL-4, cytokines that can induce Th1 and Th2 differentiation, respectively, had little effects on Egr-2 expression in naive CD4 T cells (Fig. 2A). Egr-2 is induced in T cells by anti-CD3 stimulation and this induction is known to be inhibited by CsA (11). Consistent with this, we found that Egr-2 was strongly induced by anti-CD3 in naive T cells and the expression of Egr-2 was suppressed by CsA (Fig. 2B). However, the induction of Egr-2 by TGF-β and IL-6 was not suppressed by CsA (Fig. 2B), suggesting that Egr-2 is induced by different signaling pathways by TCR stimulation and TGF-β and IL-6. Although Egr-1 and Egr-3 share similar functions with Egr-2 in the regulation of thymocyte development (29), TGF-β and IL-6 did not induce expression of either of these transcription factors (Fig. 2C). These results further suggest that Egr-2 is selectively involved in the regulation of Th17 cytokines in effector CD4 T cells and Th17 differentiation.

FIGURE 2.
FIGURE 2.

Egr-2 controls Th17 differentiation. (A and B) Expression of Egr-2 in naive CD4 T cells from wild-type mice following stimulation with the indicated cytokines (A) or with 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28 or TGF-β/IL-6 in the absence or presence of 250 ng/ml CsA (B) for 6 h. (C) Expression of Egr-1, Egr-2, and Egr-3 in naive CD4 T cells from wild-type mice following stimulation with TGF-β, IL-6, or both. (D) Th differentiation of naive CD4 T cells from wild-type and Egr-2 cKO mice. Naive CD4 T cells were differentiated to Th cells as described in Materials and Methods. The differentiated CD4 cells were stained with the indicated Abs. (E) Expression of IL-17A, IL-17F, and IL-21 in Th17 cells differentiated from wild-type or Egr-2–deficient naive CD4 T cells. (F) Expression of Egr-1, Egr-2, Egr-3, and Batf in Th1, Th2, and Th17 cells stimulated for 6 h with or without 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28. Data from (A)–(C), (E), and (F) are presented relative to the expression of β-actin mRNA. Data are representative of three (A–C, E, F) or five experiments (D). WT, Wild-type.

Egr-2 regulates Th17 differentiation

Egr-2 is induced in T cells in response to TCR stimulation (13); therefore, it is expressed in all types of Th cells. To investigate whether Egr-2 regulates the differentiation of CD4 T cells, naive CD4 T cells were differentiated into various Th subsets under specific polarization conditions. We found that Egr-2–deficient CD4 T cells displayed normal Th1 and Th2 differentiation (Fig. 2D). However, an Egr-2 defect rendered CD4 T cells more susceptible to differentiation into Th17 cells than naive CD4 T cells from wild-type mice (Fig. 2D). The expression of several Th17 cytokines, including IL-17 and IL-21, was increased in Th17 cells differentiated from Egr-2–deficient CD4 T cells (Fig. 2E). Although Egr-2, but not Egr-1 and Egr-3, was induced by TGF-β and IL-6, all three Egr molecules were induced in Th1, Th2, and Th17 cells in response to anti-CD3 stimulation (Fig. 2F). Interestingly, Egr-2 expression persisted in Th17 cells in the absence of anti-CD3 stimulation (Fig. 2F), further supporting the notion that Egr-2 is induced by both the Th17 environment and Ag receptor stimulation. To further define the role of Egr-2 in the control of IL-17 expression, we reconstituted Egr-2 expression in CD4 T cells from Egr-2 cKO mice before Th17 differentiation (Fig. 3A). The restoration of Egr-2 expression reduced the percentage of IL-17–producing CD4 T cells after Th17 differentiation (Fig. 3B, 3C). Conversely, knockdown of Egr-2 expression by siRNA in wild-type CD4 T cells enhanced the fraction of IL-17–producing CD4 T cells following Th17 differentiation (Fig. 3D–F). Thus, Egr-2 selectively controls Th17 differentiation and expression of Th17 cytokines, which is consistent with the selectively altered expression of Th17 cytokines in naive CD4 T cells from Egr-2 cKO mice in response to TCR stimulation (Fig. 1). This dysregulation of Th17 differentiation was not observed in Egr-3–deficient naive CD4 T cells (data not shown). Thus, Egr-2 plays an important role in the control of Th17 differentiation.

FIGURE 3.
FIGURE 3.

Regulation of Egr-2 expression alters Th17 differentiation. (A–C) CD4 T cells from Egr-2 cKO mice were transduced with Egr-2-lentivirus-IRES–GFP (Egr-2) or lentivirus-IRES–GFP (control) and differentiated into Th17 cells. The GFP+ cells were analyzed for the expression of Egr-2 and IL-17A by PCR (A, B) or cytokine producing cells by flow cytometry (C). (D–F) CD4 cells from wild-type mice were differentiated into Th17 cells after transfection with siRNAs against Egr-2 or control siRNA. The transfected cells were cultured under Th17 conditions for 3 d (see Materials and Methods) before analysis of Egr-2 and IL-17A expression (D, E) by PCR or cytokine producing cells by flow cytometry (F). Results are representative of two independent experiments.

Egr-2 interacts with Batf and blocks its binding to the IL-17 promoter

To investigate the mechanisms by which Egr-2 controls Th17 differentiation, we analyzed the major regulatory pathways involved in the differentiation of Th17 cells. Despite the increased Th17 differentiation, we did not find differences in the expression levels of RORγt/RORα, IFN regulatory factor-4, aryl hydrocarbon receptor, Iκbζ, or Batf or in the activation of STAT3 in Egr-2–deficient Th17 cells (data not shown). However, when we examined transcriptional activity, we found that the binding of Batf to DNA binding sites from the IL-17 promoters in Th17 cells was significantly increased in the absence of Egr-2 (Fig. 4A), suggesting that Egr-2 may control Batf function in the regulation of IL-17 expression. Consistent with previous reports (10), Batf was induced in Th1, Th2, and Th17 cells in response to anti-CD3 stimulation (Fig. 2F). Despite the increase in binding to Batf sites in the IL-17 promoter, we did not find increased expression of Batf in naive CD4 T cells or Th17 cells derived from Egr-2 cKO mice (Fig. 4B), suggesting that Egr-2 does not transcriptionally regulate Batf. To investigate whether Egr-2 antagonizes Batf function directly, we analyzed Batf immunoprecipitates from wild-type CD4 T cells after TCR stimulation. Egr-2 was discovered in the Batf precipitates (Fig. 4C), whereas Egr-1 and Egr-3 were not found (Fig. 4D). Thus, Egr-2 and Batf directly interact in CD4 T cells, raising the possibility that Egr-2 may regulate Batf by directly blocking its binding to promoters of Th17 cytokines. To further analyze the antagonistic function of Egr-2 in the regulation of Batf-mediated IL-17 expression, we measured the activity of an IL-17 promoter reporter gene (9) in the presence of Batf alone or together with Egr-1, Egr-2, or Egr-3. The results showed that Batf transactivated the IL-17 promoter, consistent with previous results (10), whereas Egr-2, but not Egr-1 or Egr-3, effectively suppressed Batf-mediated transactivation (Fig. 4E). Thus, Egr-2 directly suppresses Batf function in the induction of Th17 cytokines.

FIGURE 4.
FIGURE 4.

Egr-2 interacts with Batf and antagonizes its function in transactivation of the IL-17 promoter. (A) Interaction of Batf from Th17 cells with a DNA sequence derived from the IL-17 promoter. The differentiated Th17 cells were restimulated with or without anti-CD3 and anti-CD28 for 6 h before nuclear proteins were extracted and analyzed by EMSA. (B) Expression of Batf in Th17 cells from wild-type and Egr-2 cKO mice. The differentiated Th17 cells were restimulated with or without anti-CD3 and anti-CD28 for 6 h. The expression of Batf was analyzed by RT-qPCR and data are presented relative to the expression of β-actin mRNA. (C and D) Interaction of Batf with Egr-2, but not Egr-1 and Egr-3, in stimulated CD4 T cells. Naive CD4 T cells from wild-type or Egr-2 cKO mice were stimulated with or without anti-CD3 and anti-CD28 for 36 h and then restimulated with the same stimuli for 6 h. The nuclear proteins were extracted and used for immunoprecipitation with anti-Batf. The precipitates were blotted with anti–Egr-2 (C) or anti–Egr-1 and –Egr-3 (D). (E) IL-17 promoter reporter gene assay. The IL-17 promoter-reporter gene was cotransfected with the indicated genes into 293 cells. Cells were stimulated with PMA and ionomycin for 30 min before reporter gene activity was measured. The data are presented as fold increase above the background signal from reporter gene–transfected cells. The presented data are representative of two (A, C, D) and three (B, E) experiments. WT, Wild-type.

Egr-2 cKO mice are susceptible to EAE induction

Th17 cells are the major pathological T cells in the development of EAE, and Batf deficiency results in resistance to EAE induction (10). To examine whether the control of Th17 differentiation by Egr-2 can affect immune pathology in autoimmunity, EAE was induced in Egr-2 cKO mice by immunization with MOG peptides. Because Egr-2 cKO mice develop spontaneous autoimmunity in later life, mice aged 10–12 wk, well before the onset of autoimmunity, were used for the induction of EAE. The onset of the diseases was similar in wild-type and Egr-2 cKO mice (Fig. 5A). However, the disease was more severe in Egr-2 cKO mice compared with wild-type mice as demonstrated both by higher clinical scores and increased infiltration of mononuclear cells into inflammatory lesions in spinal cords (Fig. 5A, 5B). Furthermore, the percentage of spinal cord CD4 T cells that was positive for IL-17A was much higher in Egr-2 cKO mice than in wild-type mice (Fig. 5C). These results suggest that Egr-2 is important for the control of Th17 cell–driven pathology and for the control of the development and severity of autoimmune disease. Thus, consistent with the development of lupus-like autoimmune disease in aged Egr-2 cKO mice (26), these results demonstrate that the regulatory function of Egr-2 in the control of Th17 differentiation and cytokine expression is important for the control of autoimmunity.

FIGURE 5.
FIGURE 5.

Egr-2 deficiency renders mice more susceptible to EAE induction. (A) Clinical scores. Five 12-wk-old wild-type or Egr-2 cKO mice were immunized with MOG peptides. The clinical symptoms were scored according to standard protocols (see Materials and Methods). (B) H&E staining of CNS tissues from two (I and II) diseased mice 30 d after immunization. Original magnification ×10. (C) Mononuclear cells pooled from CNS tissues from five diseased mice in each group were stained with CD4, IFN-γ, and IL-17A. The data shown are after gating on CD4+ cells. The data are representative of three experiments with five mice for each group. WT, Wild-type.

Altered expression of Egr-2 and IL-17 in T cells from MS patients

It has been found that IL-17 is highly expressed in CNS-infiltrating T cells and glial cells and is associated with active disease in MS (30). The increased production of IL-17 in Egr-2–deficient CD4 T cells and the high susceptibility of Egr-2 cKO mice to EAE induction suggest that Egr-2 may be important for the control of MS development. Egr-2 is expressed at a basal level in naive T cells and is rapidly induced by TCR stimulation. To analyze the expression of Egr-2, Batf, and IL-17 in T cells from MS patients, we isolated CD4 T cells from PBLs of treatment-naive MS patients. The CD4 T cells were stimulated with anti-CD3 and anti-CD28 in vitro for 48 h or left unstimulated, and then the expression of Egr-2, Batf, and IL-17 was analyzed by RT-qPCR. Consistent with the expression patterns in murine naive T cells, the expression of Egr-2 and Batf in T cells from healthy donors was highly induced in response to TCR stimulation (Fig. 6A, 6C), whereas IL-17 was barely detected (Fig. 6B). However, in T cells from MS patients, although the levels of Batf induced in response to TCR stimulation were similar to those seen in healthy donors, the levels of Egr-2 expression were significantly lower (Fig. 6A, 6C). In contrast to the reduced expression of Egr-2, the expression of IL-17 was increased in T cells from MS patients compared with healthy controls (Fig. 6B). Although we do not know whether the downregulation of Egr-2 and increased expression of IL-17 are associated with uncontrolled Batf activity in T cells from MS patients, our results provide direct evidence that Egr-2 expression in effector T cells may be important for the control of Th17-mediated inflammation, which is thought to be part of the mechanism underlying MS development.

FIGURE 6.
FIGURE 6.

Altered expression of Egr-2 and IL-17 in CD4 T cells from MS patients. CD4 T cells were isolated from blood lymphocytes of 11 treatment-naive MS patients and five healthy donors and stimulated with anti-CD3 and anti-CD28 for 48 h. The cells were then restimulated for 6 h and the expression of Egr-2 (A), IL-17A (B), and Batf (C) was analyzed by RT-qPCR. The data are represented relative to the expression of mRNA of β-actin and from two experiments. **p < 0.01.

Discussion

Previously, we found that Egr-2 is induced in both tolerant and naive T cells following Ag stimulation in vivo, and deficiency of Egr-2 in T cells results in the development of lupus-like disease in later life with accumulation of activated T cells (26). However, although Egr-2 is highly induced in naive T cells following Ag stimulation (13), a defect in Egr-2 does not increase T cell activation in response to TCR stimulation (26). We have now demonstrated that Egr-2 deficiency results in uncontrolled production of Th17 cytokines in naive CD4 T cells in response to TCR stimulation and renders CD4 T cells prone to Th17 differentiation, which contributes to increased susceptibility to EAE induction. The importance of Egr-2 in the control of Th17-driven pathology is further supported by the defective induction of Egr-2 and increased expression of IL-17 in T cells from treatment-naive MS patients.

Th17 cells, one of the major Th subsets, can be generated by differentiation of naive CD4 T cells in response to Ag and Th17 mediators such as IL-6 and TGF-β (1, 2). However, in contrast to Th1 and Th2 cytokines, such as IFN-γ and IL-4, which are highly induced in naive T cells in response to TCR ligation, the induction of Th17 cytokines, such as IL-17 and IL-21, was barely detected in activated naive T cells from wild-type mice in the absence of Th17 mediators, suggesting that the expression of Th17 cytokines in effector T cells is tightly regulated. A number of regulatory factors have been discovered for inhibiting expression of Th17 cytokines and regulating Th17 differentiation (1, 2, 4–10). Most of them act by inhibiting the expression or antagonizing the function of RORγt, such as Runx1 and T-bet, or by inhibiting STAT3 activation, such as SOCS3, or by promoting Foxp3 expression, a counter-activator essential for regulatory T cell development (4). We have found that Egr-2 interacts with Batf to suppress Th17 cytokine expression. However, although deficiency of Egr-2 and Egr-3 in lymphocytes results in much more severe inflammatory autoimmune diseases than does Egr-2 single deficiency (30), we could not detect Egr-1 and Egr-3 in anti-Batf precipitates (data not shown), suggesting that EGR-2 is important for the control of Batf-mediated Th17 differentiations (30). Although it works in synergy with RORγt to induce Th17 differentiation, Batf regulates the expression of Th17 cytokines and Th17 differentiation independently of RORγt (10). In contrast to the suppression of RORγt expression in Th1 and Th2 cells, Batf is induced in all types of effector CD4 T cells in response to TCR ligation (10), suggesting that its Th17 promoting function is suppressed in effector T cells to avoid unwanted Th17 differentiation. TCR stimulation induces expression of both Egr-2 and Batf in all types of effector Th cells. The induction of Th17 cytokines, such as IL-17 and IL-21, in Egr-2–deficient T cells following TCR stimulation suggests that Egr-2 plays an important role in the control of Th17 cytokine expression in CD4 T cells in response to mitogenic Ag stimulation. The antagonistic function of Egr-2 on Batf in the regulation of Th17 cytokines in effector T cells may be essential for the control of Th17-mediated immunopathology during an adaptive immune response. Although Egr-2 and Batf are induced by TCR stimulation in all types of Th cells, we found that Egr-2 is selectively induced by TGF-β and IL-6, but not by IL-4, IL-12, or IFN-γ, indicating a feedback mechanism involving Egr-2 that serves to control Th17 differentiation. Although the mechanisms for the induction of Egr-2 expression by TGF-β and IL-6 are unknown, the increased expression of Egr-2 in Th17 cells indicates that the induction of Egr-2 expression and Th17 differentiation by TGF-β and IL-6 likely occurs via similar mechanisms that have yet to be fully investigated.

The importance of Egr-2 in the control of immune pathology in autoimmune diseases is highlighted by the susceptibility of Egr-2 cKO mice to the induction of EAE. The increased severity of disease and enhanced infiltration of inflammatory cells in Egr-2 cKO mice is associated with high levels of IL-17–producing CD4 T cells. The high susceptibility of Egr-2 cKO mice to EAE induction is in stark contrast to the reduced EAE induction in Batf-deficient mice (10), indicating that the repression of Batf function by Egr-2 contributes to the development of EAE.

The defective expression of Egr-2 and its association with increased production of IL-17 in CD4 T cells from treatment-naive MS patients is consistent with previous reports (31) and further indicates that impaired expression of Egr-2 in activated T cells may contribute to MS development. Egr-2 expression was found to be important for T cell tolerance, and its expression in anergic T cells is regulated by NFAT (11, 12). The dysregulated expression of Egr-2 and IL-17, but normal induction of Batf, in CD4 T cells from MS patients resembles the expression profile of these molecules in CD4 T cells from Egr-2 cKO mice, suggesting that uncontrolled Batf activity may contribute to the increased IL-17 production and immunopathology in MS.

Taken together, we have demonstrated a previously unknown function of Egr-2 in the control of Th17 cytokine expression and Th17 differentiation by inhibition of Batf function. The susceptibility of Egr-2 cKO mice to EAE induction and the reduced induction of Egr-2 in T cells from MS patients after TCR stimulation further indicate the importance of Egr-2 in the control of autoimmunity and Th1-mediated inflammation.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Julie Walker (Biological Science, Brunel University) and Alicia Rosello (Blizard Institute of Cell and Molecular Science, Barts and London School of Medicine and Dentistry) for excellent technical support.

Footnotes

  • This work was supported by Arthritis Research U.K.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    cKO
    conditional knockout
    CsA
    cyclosporine A
    EAE
    experimental autoimmune encephalomyelitis
    Egr
    early growth response gene
    MOG
    myelin oligodendrocyte glycoprotein
    MS
    multiple sclerosis
    ROR
    retinoic acid–related orphan receptor
    RT-qPCR
    real-time quantitative PCR
    si
    small interfering
    SOM
    self-organizing map.

  • Received March 20, 2012.
  • Accepted October 24, 2012.

References

  1. ↵
    1. Hirota K.,
    2. B. Martin,
    3. M. Veldhoen
    . 2010. Development, regulation and functional capacities of Th17 cells. Semin. Immunopathol. 32: 3–16.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Ghoreschi K.,
    2. A. Laurence,
    3. X. P. Yang,
    4. K. Hirahara,
    5. J. J. O’Shea
    . 2011. T helper 17 cell heterogeneity and pathogenicity in autoimmune disease. Trends Immunol. 32: 395–401.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Zhou L.,
    2. M. M. Chong,
    3. D. R. Littman
    . 2009. Plasticity of CD4+ T cell lineage differentiation. Immunity 30: 646–655.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Hirahara K.,
    2. K. Ghoreschi,
    3. A. Laurence,
    4. X. P. Yang,
    5. Y. Kanno,
    6. J. J. O’Shea
    . 2010. Signal transduction pathways and transcriptional regulation in Th17 cell differentiation. Cytokine Growth Factor Rev. 21: 425–434.
    OpenUrlCrossRefPubMed
    1. Ivanov I. I.,
    2. B. S. McKenzie,
    3. L. Zhou,
    4. C. E. Tadokoro,
    5. A. Lepelley,
    6. J. J. Lafaille,
    7. D. J. Cua,
    8. D. R. Littman
    . 2006. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126: 1121–1133.
    OpenUrlCrossRefPubMed
    1. Harris T. J.,
    2. J. F. Grosso,
    3. H. R. Yen,
    4. H. Xin,
    5. M. Kortylewski,
    6. E. Albesiano,
    7. E. L. Hipkiss,
    8. D. Getnet,
    9. M. V. Goldberg,
    10. C. H. Maris,
    11. et al
    . 2007. Cutting edge: an in vivo requirement for STAT3 signaling in TH17 development and TH17-dependent autoimmunity. J. Immunol. 179: 4313–4317.
    OpenUrlAbstract/FREE Full Text
    1. Brüstle A.,
    2. S. Heink,
    3. M. Huber,
    4. C. Rosenplänter,
    5. C. Stadelmann,
    6. P. Yu,
    7. E. Arpaia,
    8. T. W. Mak,
    9. T. Kamradt,
    10. M. Lohoff
    . 2007. The development of inflammatory TH-17 cells requires interferon-regulatory factor 4. Nat. Immunol. 8: 958–966.
    OpenUrlCrossRefPubMed
    1. Veldhoen M.,
    2. K. Hirota,
    3. A. M. Westendorf,
    4. J. Buer,
    5. L. Dumoutier,
    6. J. C. Renauld,
    7. B. Stockinger
    . 2008. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature 453: 106–109.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Okamoto K.,
    2. Y. Iwai,
    3. M. Oh-Hora,
    4. M. Yamamoto,
    5. T. Morio,
    6. K. Aoki,
    7. K. Ohya,
    8. A. M. Jetten,
    9. S. Akira,
    10. T. Muta,
    11. H. Takayanagi
    . 2010. IkappaBzeta regulates TH17 development by cooperating with ROR nuclear receptors. Nature 464: 1381–1385.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Schraml B. U.,
    2. K. Hildner,
    3. W. Ise,
    4. W. L. Lee,
    5. W. A. Smith,
    6. B. Solomon,
    7. G. Sahota,
    8. J. Sim,
    9. R. Mukasa,
    10. S. Cemerski,
    11. et al
    . 2009. The AP-1 transcription factor Batf controls TH17 differentiation. Nature 460: 405–409.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Harris J. E.,
    2. K. D. Bishop,
    3. N. E. Phillips,
    4. J. P. Mordes,
    5. D. L. Greiner,
    6. A. A. Rossini,
    7. M. P. Czech
    . 2004. Early growth response gene-2, a zinc-finger transcription factor, is required for full induction of clonal anergy in CD4+ T cells. J. Immunol. 173: 7331–7338.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Safford M.,
    2. S. Collins,
    3. M. A. Lutz,
    4. A. Allen,
    5. C. T. Huang,
    6. J. Kowalski,
    7. A. Blackford,
    8. M. R. Horton,
    9. C. Drake,
    10. R. H. Schwartz,
    11. J. D. Powell
    . 2005. Egr-2 and Egr-3 are negative regulators of T cell activation. Nat. Immunol. 6: 472–480.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Anderson P. O.,
    2. B. A. Manzo,
    3. A. Sundstedt,
    4. S. Minaee,
    5. A. Symonds,
    6. S. Khalid,
    7. M. E. Rodriguez-Cabezas,
    8. K. Nicolson,
    9. S. Li,
    10. D. C. Wraith,
    11. P. Wang
    . 2006. Persistent antigenic stimulation alters the transcription program in T cells, resulting in antigen-specific tolerance. Eur. J. Immunol. 36: 1374–1385.
    OpenUrlCrossRefPubMed
  14. ↵
    1. O’Donovan K. J.,
    2. W. G. Tourtellotte,
    3. J. Millbrandt,
    4. J. M. Baraban
    . 1999. The EGR family of transcription-regulatory factors: progress at the interface of molecular and systems neuroscience. Trends Neurosci. 22: 167–173.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Topilko P.,
    2. S. Schneider-Maunoury,
    3. G. Levi,
    4. A. Baron-Van Evercooren,
    5. A. B. Chennoufi,
    6. T. Seitanidou,
    7. C. Babinet,
    8. P. Charnay
    . 1994. Krox-20 controls myelination in the peripheral nervous system. Nature 371: 796–799.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Shao H.,
    2. D. H. Kono,
    3. L. Y. Chen,
    4. E. M. Rubin,
    5. J. Kaye
    . 1997. Induction of the early growth response (Egr) family of transcription factors during thymic selection. J. Exp. Med. 185: 731–744.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Carter J. H.,
    2. J. M. Lefebvre,
    3. D. L. Wiest,
    4. W. G. Tourtellotte
    . 2007. Redundant role for early growth response transcriptional regulators in thymocyte differentiation and survival. J. Immunol. 178: 6796–6805.
    OpenUrlAbstract/FREE Full Text
  18. Li, S., A. L. Symonds, B. Zhu, M. Liu, M. V. Raymond, T. Miao, and P. Wang. 2011. Early growth response gene-2 (Egr-2) regulates the development of B and T cells. PLoS One 6: e18498.
    1. Lawson V. J.,
    2. K. Weston,
    3. D. Maurice
    . 2010. Early growth response 2 regulates the survival of thymocytes during positive selection. Eur. J. Immunol. 40: 232–241.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Lauritsen J. P.,
    2. S. Kurella,
    3. S. Y. Lee,
    4. J. M. Lefebvre,
    5. M. Rhodes,
    6. J. Alberola-Ila,
    7. D. L. Wiest
    . 2008. Egr2 is required for Bcl-2 induction during positive selection. J. Immunol. 181: 7778–7785.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Lazarevic V.,
    2. A. J. Zullo,
    3. M. N. Schweitzer,
    4. T. L. Staton,
    5. E. M. Gallo,
    6. G. R. Crabtree,
    7. L. H. Glimcher
    . 2009. The gene encoding early growth response 2, a target of the transcription factor NFAT, is required for the development and maturation of natural killer T cells. Nat. Immunol. 10: 306–313.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Seiler M. P.,
    2. R. Mathew,
    3. M. K. Liszewski,
    4. C. Spooner,
    5. K. Barr,
    6. F. Meng,
    7. H. Singh,
    8. A. Bendelac
    . 2012. Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling. Nat. Immunol. 13: 264–271.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Mittelstadt P. R.,
    2. J. D. Ashwell
    . 1999. Role of Egr-2 in up-regulation of Fas ligand in normal T cells and aberrant double-negative lpr and gld T cells. J. Biol. Chem. 274: 3222–3227.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Rengarajan J.,
    2. P. R. Mittelstadt,
    3. H. W. Mages,
    4. A. J. Gerth,
    5. R. A. Kroczek,
    6. J. D. Ashwell,
    7. L. H. Glimcher
    . 2000. Sequential involvement of NFAT and Egr transcription factors in FasL regulation. Immunity 12: 293–300.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Okamura T.,
    2. K. Fujio,
    3. M. Shibuya,
    4. S. Sumitomo,
    5. H. Shoda,
    6. S. Sakaguchi,
    7. K. Yamamoto
    . 2009. CD4+CD25−LAG3+ regulatory T cells controlled by the transcription factor Egr-2. Proc. Natl. Acad. Sci. USA 106: 13974–13979.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Zhu B.,
    2. A. L. Symonds,
    3. J. E. Martin,
    4. D. Kioussis,
    5. D. C. Wraith,
    6. S. Li,
    7. P. Wang
    . 2008. Early growth response gene 2 (Egr-2) controls the self-tolerance of T cells and prevents the development of lupuslike autoimmune disease. J. Exp. Med. 205: 2295–2307.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Polman C. H.,
    2. S. C. Reingold,
    3. G. Edan,
    4. M. Filippi,
    5. H. P. Hartung,
    6. L. Kappos,
    7. F. D. Lublin,
    8. L. M. Metz,
    9. H. F. McFarland,
    10. P. W. O’Connor,
    11. et al
    . 2005. Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria”. Ann. Neurol. 58: 840–846.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Miao T.,
    2. D. Wu,
    3. Y. Zhang,
    4. X. Bo,
    5. M. C. Subang,
    6. P. Wang,
    7. P. M. Richardson
    . 2006. Suppressor of cytokine signaling-3 suppresses the ability of activated signal transducer and activator of transcription-3 to stimulate neurite growth in rat primary sensory neurons. J. Neurosci. 26: 9512–9519.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Kaye J.
    2000. Regulation of T cell development in the thymus. Immunol. Res. 21: 71–81.
    OpenUrlCrossRefPubMed
  30. ↵
    Li, S.,T. Miao, M. Sebastian, P. Bhullar, E. Ghaffari, M. Liu, A. L. Symonds, and P. Wang. 2012. The transcription factors Egr2 and Egr3 are essential for the control of inflammation and antigen-induced proliferation of B and T cells. Immunity 37: 685–696.
  31. ↵
    1. Tzartos J. S.,
    2. M. A. Friese,
    3. M. J. Craner,
    4. J. Palace,
    5. J. Newcombe,
    6. M. M. Esiri,
    7. L. Fugger
    . 2008. Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am. J. Pathol. 172: 146–155.
    OpenUrlCrossRefPubMed
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