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. Author manuscript; available in PMC: 2015 Mar 20.
Published in final edited form as: Immunity. 2014 Feb 13;40(3):355–366. doi: 10.1016/j.immuni.2014.01.002

The transcription factors T-bet and Runx are required for the ontogeny of pathogenic interferon-γ-producing T helper 17 cells

Yan Wang 1, Jernej Godec 2, Khadija Ben-Aissa 1, Kairong Cui 3, Keji Zhao 3, Alexandra B Pucsek 4, Yun Kyung Lee 5, Casey T Weaver 5, Ryoji Yagi 6, Vanja Lazarevic 1,7
PMCID: PMC3965587  NIHMSID: NIHMS560522  PMID: 24530058

SUMMARY

T helper 17 (Th17) cells can give rise to interleukin-17A (IL-17A) and interferon (IFN)-γ-double producing cells that are implicated in development of autoimmune diseases. However, the molecular mechanisms that govern generation of IFN-γ-producing Th17 cells are unclear. We found that co-expression of the Th1- and Th17-cell master transcription factors, T-bet and retinoid-related orphan receptor gamma-t (RORγt), respectively, did not generate Th cells with robust IL-17 and IFN-γ expression. Instead, development of IFN-γ-producing Th17 cells required T-bet and Runx1 or Runx3. IL-12-stimulated Th17 cells upregulated Runx1, which bound to the Ifng locus in a T-bet dependent manner. Reciprocally, T-bet or Runx1 deficiency, or inhibition of Runx transcriptional activity, impaired the development of IFN-γ-producing Th17 cells, during experimental autoimmune encephalomyelitis, which correlated with substantially ameliorated disease course. Thus, our studies identify a critical role for T-bet and Runx transcription factors in the generation of pathogenic IFN-γ-producing Th17 cells.

INTRODUCTION

CD4+ T cells are a multipotent population of cells, which are able to differentiate into several different T helper (Th) subsets depending on the transcription factors that they express and cytokine signals that they receive during activation by innate immune cells. Development of Th1 cells is regulated by the Th1-specific transcription factor T-bet (Szabo et al., 2000) while the transcription factors retinoid-related orphan receptor gamma-t (RORγt) and RORα specify the Th17 lineage (Ivanov et al., 2006; Yang et al., 2008). Th1 cells produce interferon-γ (IFN-γ) and are best equipped to clear intracellular bacteria and viruses, while Th17 cells produce interleukin (IL-17A), IL-17F, IL-21 and IL-22 to protect mucosal surfaces against extracellular bacteria and fungi (Kaufmann, 1993; Khader et al., 2009). However, deregulated Th responses can increase susceptibility to autoimmunity. In particular, overactive Th1 and Th17 responses have been linked strongly to the development of autoimmune diseases (Domingues et al., 2010; Jager et al., 2009; Kroenke et al., 2008; Luger et al., 2008; Siffrin et al., 2010; Stromnes et al., 2008; Waldburger et al., 1996). Adoptive transfers of in vitro differentiated Th1 and Th17 cells with TCR specificities for self-antigens have demonstrated that each subset is able to induce disease in animal models of autoimmunity, although disease severity was highest if both Th subsets were present (Domingues et al., 2010). Th17 cells show a high degree of developmental flexibility, and when exposed to IL-12 or IL-23, can rapidly acquire effector functions that are normally associated with Th1 responses such as IFN-γ production (Hirota et al., 2011; Lee et al., 2009; Mukasa et al., 2010; Muranski et al., 2011). The developmental flexibility of Th17 cells and their shift to a Th1-like phenotype has been linked to the pathogenicity of Th17 cells in colitis (Ahern et al., 2010; Lee et al., 2009), Crohn’s disease (Annunziato et al., 2007), arthritis (Nistala et al., 2010), diabetes (Bending et al., 2009), experimental autoimmune encephalomyelitis (EAE) (Hirota et al., 2011) and multiple sclerosis (Kebir et al., 2009). However, the transcription factor network that promotes the genetic plasticity of Th17 cells has not been fully defined.

The current study was undertaken to identify which transcription factors are required for the development of Th1-like Th17 cells. We report that co-expression of Th17- and Th1-cell master transcription factors, RORγt and T-bet, respectively, was not sufficient to generate Th cells with strong dual Th17 and Th1 features. Instead, the development of Th cells that co-produce IL-17A and IFN-γ was dependent on T-bet, Runx1 and Runx3 transcription factors. Chromatin immunoprecipitation (ChIP) analysis revealed that Runx1 bound to the Ifng promoter and conserved regulatory elements within the Ifng locus in a T-bet dependent manner, resulting in the acquisition of IFN-γ production. Indeed, ectopic expression of Runx1 or Runx3 was sufficient to promote the development of IFN-γ-producing Th17 cells in the absence of IL-12 or STAT4. Reciprocally, Runx1 deficiency or inhibition of Runx transcriptional activity prevented conversion of Th17 cells into Th1-like cells either in response to IL-12 or during EAE. Thus, this study identifies a critical role of Runx transcription factors in the generation of Th1-like Th17 cells.

RESULTS

T-bet and RORγt co-expression is not sufficient for the generation of Th1-like Th17 cells

Th17 cells demonstrate a high degree of developmental flexibility, often acquiring qualities of Th1 cells through mechanisms that are not yet fully understood. Exposure of Th17 cells to IL-12 or IL-23 results in the up-regulation of the Th1 lineage-specifying transcription factor T-bet and their deviation into Th1-like cells (Hirota et al., 2011; Lee et al., 2009). Published studies have described CD4+ Th cells that simultaneously produce IL-17A and IFN-γ and stably co-express RORγt and T-bet (IL-17A+IFN-γ+ or Th1-like Th17 cells) (Annunziato et al., 2007; Boniface et al., 2010; Cohen et al., 2011; Kebir et al., 2009). Such observations led to a hypothesis that co-expression of RORγt and T-bet results in a hybrid gene expression profile, which would create CD4+ T cell phenotypes with features of Th17 and Th1 cells (Oestreich and Weinmann, 2012). To directly test this hypothesis, we transduced CD4+ T cells with retroviruses expressing RORγt and T-bet under Th17-polarizing conditions, and asked whether the co-expression of master regulators of Th17 and Th1 differentiation programs was sufficient for the generation of IL-17A+IFN-γ+ cells. Consistent with previously published results (Lazarevic et al., 2011; Zhang et al., 2008), retroviral transduction of CD4+ T cells with RORγt alone promoted the differentiation of Th17 cells, while T-bet expression effectively blocked Th17 lineage commitment. Simultaneous expression of RORγt and T-bet did not result in the generation of IL-17A+IFN-γ+ cells (Figure 1). Similarly, T-bet overexpression in established Th17 cells did not induce a IL-17A+IFN-γ+ state (data not shown). The inability of RORγt and T-bet co-expression to generate Th17 cells with Th1-like features suggests that co-expression of master transcription factors in CD4+ T cells is not sufficient to explain the flexibility of Th17 cells.

Figure 1. T-bet and RORγt co-expression is not sufficient for the generation of Th1-like Th17 cells.

Figure 1

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Flow cytometry analysis of CD4+ T cells transduced with various combinations of retroviruses expressing GFP, Thy1.1, T-bet-Thy1.1 or RORγt-GFP (above plots). Cells were stimulated with PMA and ionomycin for 4 hours before intracellular cytokine staining for IL-17A and IFN-γ. Numbers in quadrants indicate percent IL-17A-producing and IFN-γ-producing cells within GFP+Thy1.1+ gate. Data are representative of 2 independent experiments.

Runx1 and Runx3 drive Th1-like Th17 cell differentiation

To identify which transcription factor(s) could promote the development of Th1-like Th17 cells, we took advantage of the fact that exposure of committed Th17 cells to IL-12 results in a rapid induction of IFN-γ and deviation to a Th1-like phenotype (Lee et al., 2009; Mukasa et al., 2010). As expected, reactivation of committed Th17 cells in the presence of IL-12 resulted in their conversion to IL-17A+IFN-γ+ and IFN-γ+ cells (Figure S1). IL-12 treatment substantially upregulated Ifng expression by committed Th17 cells, although mRNA and protein expression was lower than that observed in Th1 cells. Under these conditions, Th17 and IL-12-treated Th17 cells expressed similar amounts of Il17a transcripts (Figure S1). Hence, we generated a heterogeneous population of IFN-γ-producing cells from polarized Th17 cells without completely extinguishing their IL-17A expression.

We next sought to determine which transcription factors were upregulated in Th17 cells after IL-12 treatment that could account for the generation of Th1-like Th17 cells. To ensure that we started with a homogeneous population of IL-17A-producing Th17 cells, we used previously described Il17fThy1.1/Thy1.1 reporter mice in which the Thy1 gene was introduced into the endogenous Il17f gene (Lee et al., 2009). The IL-17F Thy1.1 reporter is expressed early in Th17 development, thus marking with high accuracy almost all IL-17A-producing cells, while IFN-γ-producing Th1 cells did not express the reporter (Figure 2A). Naïve CD4+ T cells were isolated from Il17fThy1.1/Thy1.1 mice and activated under Th17 polarizing conditions. On the sixth day of culture, sorted Thy1.1+ and Thy1.1− cells (Figure 2B, >99% purity) were reactivated in the presence of IL-12 for 48 h. In agreement with previous results (Lee et al., 2009), sorted IL-17-producing Th17 cells gave rise to IL-17A+IFN-γ+ and IFN-γ+ cells while Thy1.1− cells gave rise predominantly to IFN-γ+ cells when reactivated in the presence of IL-12 (Figure 2B). The conversion of IL-12-stimulated Th17 cells into IL-17A+IFN-γ+ and IFN-γ+ cells was accompanied by increased expression of mRNA encoding the transcription factors Tbx21, Eomes, Runx1 and Runx3 (Figure 2C), all of which have been previously linked to regulation of the Ifng gene. To investigate whether these transcription factors can replace IL-12 in shifting the phenotype of Th17 cells to Th1-like cells, we transduced CD4+ T cells with retroviruses expressing T-bet, Eomes, Runx1 or Runx3 under Th17 polarizing conditions. Consistent with published reports (Ichiyama et al., 2011; Lazarevic et al., 2011), overexpression of T-bet or Eomes constrained Th17 cell differentiation. However, neither T-bet nor Eomes induced development of IL-17A+IFN-γ+ cells (Figures 3A and 3B). Ectopic expression of Runx1 or Runx3 promoted differentiation not only of IL-17A+ cells, but also a significant proportion of IL-17A+IFN-γ+ and IFN-γ+ cells (Figures 3A and 3B). Thus, Runx1 and Runx3 are able to replace IL-12 in promoting the generation of Th1-like Th17 cells. Mechanistically, overexpression of Runx1 or Runx3 in Th17 cells resulted in the induction of Stat4 and Tbx21, known downstream factors of IL-12 signaling (Figure 3C). Runx1 was a stronger activator of Tbx21 and Stat4 genes than Runx3, resulting in higher amounts of Ifng mRNA and a higher frequency of IFN-γ-producing cells (Figure 3). STAT4 and T-bet epigenetically silence the Rorc locus in Th17 cells (Mukasa et al., 2010). Both Runx1 and Runx3 maintained high Rorc expression despite elevated expression of Tbx21 and Stat4. These results demonstrate that high expression of RORγt, T-bet and especially Runx1 (or Runx3) are needed to stabilize the IL-17A+IFN-γ+ mixed phenotype (Figure 3C).

Figure 2. Conversion of Th17 cells into IL-17A+IFN-γ+ and IFN-γ+ cells after IL-12 treatment is accompanied by induction of Tbx21, Eomes, Runx1 and Runx3 expression.

Figure 2

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(A) Naïve CD4+ T cells were sorted from Il17fThy1.1/Thy1.1 mice and cultured under Th1- or Th17-polarizing conditions for 5 days. Recovered cells were stimulated with PMA and ionomycin for 4 hours and stained for cell surface Thy1.1 and CD4 and intracellular IL-17A and IFN-γ. Numbers in quadrants indicate percent Thy1.1 positive, IL-17A-producing and IFN-γ-producing cells within CD4 positive gate. Data are representative of 2 independent experiments.

(B) Naïve CD4+ T cells were sorted from Il17fThy1.1/Thy1.1 mice and cultured under Th17-polarizing conditions for 5 days. Recovered cells were stained with anti-Thy1.1 antibody and sorted into Thy1.1+ and Thy1.1− fractions (>99% purity). Thy1.1+ and Thy1.1− cells were washed and reactivated on anti-CD3 and CD28 antibody-coated plates in the presence of IL-12 alone (5 ng/ml). Cells were cultured for 3 days and stained for cell surface CD4 and intracellular IL-17A and IFN-γ following PMA and ionomycin stimulation for 4 hours. Numbers in quadrants represent the percentage of IL-17A-producing and IFN-γ-producing cells within CD4+ gate. Data are representative of 2 independent experiments.

(C) Quantitative RT-PCR analysis of Tbx21, Eomes, Runx1 and Runx3 mRNA isolated from Thy1.1+ cells that were reactivated under Th17-polarizing conditions or in the presence of IL-12 alone. Data are representative of 2 independent experiments (mean ± SEM of replicate wells). See also Figure S1 and Table S1.

Figure 3. Runx1 and Runx3 promote differentiation of Th1-like Th17 cells.

Figure 3

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(A) Naïve CD4+ T cells were sorted from WT mice, activated and transduced with retroviruses expressing GFP, T-bet-GFP, Eomesodermin-GFP, Runx1-GFP or Runx3-GFP (above plots) under Th17-polarizing conditions. Cells were stimulated with PMA and ionomycin for 4 hours and stained for intracellular IL-17A and IFN-γ. Numbers in quadrants represent the percentage of IL-17A-producing and IFN-γ-producing cells within GFP+ gate. The data are representative of 5 independent experiments.

(B) The bars represent mean ± SEM of cytokine producing cells within GFP+ cells from 5 independent experiments (as described in A).

(C) Quantitative RT-PCR analysis of Ifng, Il17a, Tbx21, Stat4 and Rorc mRNA isolated from WT Th17 cells transduced with retroviruses expressing GFP, Runx1-GFP or Runx3-GFP. The data are representative of 3 independent experiments (mean ± SEM of replicate wells).

(D–G) Cytokine production by WT and Tbx21−/− Th17 cells (D) and WT and Stat4−/− Th17 cells (F) transduced with retroviruses expressing GFP, Runx1-GFP or Runx3-GFP (above plots). Numbers in quadrants indicate percent IL-17A-producing and IFN-γ-producing cells within GFP+ gate. Quantitative RT-PCR analysis of Ifng mRNA and intracellular staining of IFN-γ protein in Tbx21−/− and WT Th17 cells (E) and WT and Stat4−/− Th17 cells (G) transduced with Runx1- or Runx3-expressing retrovirus. MFI, mean fluorescence intensity. The data are representative of 2 – 3 independent experiments. See also Figure S2.

T-bet and Runx3 interact and functionally cooperate to induce maximal IFN-γ production in Th1 cells (Djuretic et al., 2007), while we have shown previously that T-bet and Runx1 interact in primary CD4+ T cells (Lazarevic et al., 2011). Based on these findings, we sought to determine whether the formation of T-bet and Runx1 or T-bet and Runx3 transcriptional complexes was required for Runx-mediated conversion of Th17 cells into Th1-like Th17 cells. To address this question, we expressed Runx1 or Runx3 in WT or T-bet deficient (Tbx21−/−) Th17 cells. Both Runx1 and Runx3 promoted differentiation of Th1-like Th17 cells in both the presence and absence of T-bet, as similar percentages of IL-17A+, IL-17A+IFN-γ+ and IFN-γ+ cells were detected in Tbx21−/− and WT cultures (Figures 3D). However, Tbx21−/− Th17 cells transduced with Runx1- or Runx3-expressing retroviruses had diminished Ifng transcription and IFN-γ protein compared to WT controls (Figures 3E), indicating that Runx transcription factors were dependent on endogenous T-bet expression for maximal induction of IFN-γ production in Th17 cells. In fact, co-expression of T-bet and Runx1 or T-bet and Runx3 in Th17 cells resulted in the highest frequency of IFN-γ-producing cells and higher amounts of Ifng mRNA and IFN-γ protein than when either transcription factor was overexpressed alone (Figure S2). Similar amounts of Ifng mRNA and IFN-γ protein were produced by Stat4−/− and WT Th17 cells after transduction with Runx1 and Runx3-expressing retroviruses, demonstrating that STAT4 was not required for Runx-mediated generation of Th1-like Th17 cells (Figure 3F and 3G). Based on these results, we conclude that expression of either Runx1 or Runx3 is sufficient to promote the differentiation of Th cells with Th1 and Th17 characteristics in the absence of IL-12 or STAT4; however, maximal Ifng transcription and IFN-γ protein expression by Th17 cells requires both T-bet and Runx transcription factors.

Conversion of committed Th17 to Th1-like cells requires T-bet and Runx

To assess whether T-bet and Runx transcription factors were essential for IL-12-mediated conversion of Th17 cells into IFN-γ-producing cells, we examined the effects of T-bet and Runx deficiency on the ability of polarized Th17 cells to convert into Th1-like cells after exposure to IL-12. Because Runx1 is required for the survival of CD4+ T cells, in part by regulating IL-7Rα expression, and Runx1-deficient mice have a substantial reduction in peripheral CD4+ T cells (Egawa et al., 2007), we introduced the pro-survival Bcl2 transgene into Cd4-cre Runx1fl/fl and Runx1fl/fl mice. We found that Runx1-deficient CD4+ T cells differentiated poorly into Th17 cells (Figure 4A), which is consistent with a previously reported role of Runx1 in the differentiation of Th17 cells (Zhang et al., 2008). Isolated deletion of T-bet, Runx1 or Runx3 in CD4+ T cells markedly diminished the Th17 to Th1 conversion after IL-12 treatment, as evidenced by a decreased percentage of IL-17A+IFN-γ+ and IFN-γ+ cells and reduced Ifng mRNA expression (Figures 4A and 4B). Based on these results, we conclude that T-bet, Runx1 and Runx3 are all critical for IL-12-induced conversion of differentiated Th17 cells into IFN-γ-producing cells.

Figure 4. Runx1, Runx3 and T-bet regulate the conversion of Th17 to Th1-like cells in response to IL-12 signaling.

Figure 4

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(A) Naïve CD4+ T cells from Cd4-cre Tbx21fl/fl, Cd4-cre Runx1fl/fl (on Bcl2 transgenic background), or Cd4-cre Runx3fl/fl mice and their respective controls were cultured under Th17-polarizing conditions for 5 days. Recovered cells were washed, and one half of the recovered cells was reactivated on anti-CD3 and CD28 antibody-coated plates under Th17-polarizing conditions while the second half of the cells was reactivated in the presence of IL-12 alone (5 ng/ml) for 3 days. Numbers in quadrants indicate percent IL-17A-producing and IFN-γ-producing cells within CD4+ gate. Data are representative of 3 independent experiments.

(B) Quantitative RT-PCR analysis of Ifng mRNA from cultures as described in A. Data are representative of 3 independent experiments (mean ± SEM of replicate wells). See also Figure S3.

Recently, several studies have shown the importance of IL-23 and IL-1β in the ontogeny of pathogenic Th1-like Th17 cells (Chung et al., 2009; Ghoreschi et al., 2010; Hirota et al., 2011; Lee et al., 2009). To determine whether Runx1 and Runx3 expression was also induced upon IL-23 or IL-1β treatment, we reactivated established Th17 cells in the presence of Th17-skewing cytokines, IL-12 alone, IL-23 alone or IL-1β alone. Similar to IL-12, IL-23 and IL-1β enhanced Runx1 and Runx3 expression in Th17 cells (Figure S3). These results suggest that the upregulation of Runx1 and Runx3 may be a central component in the generation of Th1-like Th17 cells in response to multiple cytokines.

Runx1 binds to the Ifng locus in a T-bet dependent manner

Our experiments showed that conversion of Th17 cells into Th1-like Th17 cells required expression of three transcription factors: T-bet, Runx1 and Runx3. In addition, the ability of Runx1 and Runx3 to induce maximal IFN-γ production by Th1-like Th17 cells was dependent on endogenous T-bet expression. These observations prompted us to investigate the relationship between T-bet and Runx transcription factors in the IL-12-stimulated transition of Th17 cells into IFN-γ-producing cells. T-bet is the key transcriptional activator of the Ifng gene, and the ability of Runx3 to enhance T-bet-mediated activation of the Ifng gene in Th1 cells is documented (Djuretic et al., 2007). Hence, one possibility is that the Runx transcription factors act as co-activators to enhance T-bet mediated transcriptional activity at the Ifng locus in Th17 cells. However, because Runx1 and Runx3 can induce IFN-γ expression in T-bet-deficient Th17 cells (Figures 3D and 3E), Runx transcription factors might be directly involved in regulating Ifng gene expression in Th17 cells.

The Ifng promoter and the nine conserved non-coding sequences (CNS) located at −54 kb, −34 kb, −22 kb, −6 kb, +17–19 kb, +30 kb, +40 kb, +46 kb and +54 kb from the Ifng start site have regulatory functions that ensure proper IFN-γ expression in Th1 cells (Balasubramani et al., 2010; Hatton et al., 2006; Shnyreva et al., 2004). The Ifng promoter and −54 kb, −34 kb, −6 kb CNS are epigenetically silenced in Th17 cells (Mukasa et al., 2010). However, these regulatory elements acquire permissive epigenetic modifications in a T-bet- and STAT4-dependent manner after IL-12 treatment of Th17 cells (Mukasa et al., 2010). To determine whether Runx1 binds to the Ifng promoter and the regulatory elements in IL-12-treated Th17 cells, we performed ChIP analysis of Runx1 precipitated from nuclear lysates that had been isolated from Th17 or IL-12-treated Th17 cells. We focused our analysis on the Ifng promoter and the −54 kb, −34 kb, −6 kb regulatory elements, because their change from repressive to permissive state in response to IL-12 treatment is dependent on T-bet, and on the −22 kb and +17–19 kb CNSs, which are partially accessible in Th17 cells (Mukasa et al., 2010). We found marked enrichment of Runx1 binding to the Ifng promoter and −54 kb, −34 kb, −6 kb CNSs within the Ifng locus only in Th17 cells treated with IL-12 (Figure 5A). We also detected a modest binding of Runx1 to the partially accessible −22 kb and +17–19 kb CNSs in Th17 cells, which was further enriched by IL-12 treatment (Figure 5A). No significant Runx1 binding was observed at the Ifng promoter and −54 kb, −34 kb, −6 kb CNSs in WT Th17 cells, in IL-12-treated Runx1-deficient Th17 cells, or in the control ChIP with immunoglobulin G (IgG) (Figure 5A). These data establish that Runx1 operates directly at the Ifng locus in IL-12-treated Th17 cells to promote IFN-γ expression by transitioned Th17 cells.

Figure 5. Runx1 binds to the Ifng locus in IL-12-treated Th17 cells in a T-bet dependent manner.

Figure 5

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(A–B) ChIP analysis of Runx1 binding in Th17 cells that were reactivated under Th17-polarizing conditions or in the presence of IL-12 alone (as described in 4A).

(A) Runx1 specific binding to Tcrb enhancer, Actb gene, and Ifng promoter and conserved non-coding sequences (CNS) within the Ifng locus in Th17 and IL-12-treated Th17 cells. Tcrb enhancer was used as a positive control. Actb promoter, ChIP with IgG control antibody and ChIP with Runx1-specific antibody on Runx1-deficient cells (Cd4-cre Runx1fl/fl) were used as negative controls. Results are presented as percent of input DNA. Data are representative of 2 independent experiments (mean ± SEM of replicate wells).

(B) Runx1 specific binding to the Ifng promoter and conserved non-coding sequences (CNS) within the Ifng locus in WT and T-bet-deficient Th17 and IL-12-treated Th17. Data are representative of 2 independent experiments (mean ± SEM of replicate wells).

To determine whether Runx1 binding to the Ifng promoter and conserved regulatory elements was dependent on T-bet-mediated epigenetic changes after IL-12 treatment, we performed Runx1-specific ChIP on nuclear lysates from T-bet-deficient and WT Th17 and IL-12-treated Th17 cells. We found that binding of Runx1 to the Ifng promoter and to the −54 kb, −34 kb, −6 kb CNSs in IL-12-treated Th17 cells was strictly dependent on T-bet, whereas binding of Runx1 to the −22 kb and +17–19 kb CNSs was mostly T-bet independent (Figure 5B). These results demonstrate that Runx1 binding to the Ifng locus is dependent on T-bet at specific sites in the Ifng locus which are normally silenced in Th17 cells, and which require T-bet to become accessible for transcription after IL-12 treatment.

T-bet and Runx control Th17 to Th1 conversion in passive EAE model

To determine whether T-bet was required for the conversion of Th17 cells into Th1-like Th17 cells during an inflammatory response in vivo, we examined the fate of polarized, T-bet-deficient Th17 cells in the adoptive transfer model of EAE. 2D2 WT and 2D2 Tbx21−/− CD4+ T cells were cultured under Th17 conditions in the presence of IL-23, reactivated on anti-CD3 and anti-CD28-coated plates and transferred into recipient mice. Seventeen days after transfer, 2D2 WT and 2D2 Tbx21−/− CD4+ T cells were isolated from the CNS of recipient mice and assayed for cytokine expression. The majority of transferred 2D2 WT CD4+ Th17 cells converted into IL-17A+IFN-γ+ and IFN-γ+ cells, suggesting that the shift in cytokine production occurred in the recipient mice during EAE (Figure 6A). The conversion of IL-17A-producing Th17 cells into IFN-γ-producing cells did not occur in the absence of T-bet. Adoptively transferred 2D2 Tbx21−/− Th17 cells were not pathogenic and failed to induce EAE in recipient mice, which correlated with significantly reduced frequencies of IL-17A+IFN-γ+ and IFN-γ+ cells compared to WT controls (Figure 6A). Thus, the in vivo conversion of Th17 cells into IL-17A+IFN-γ+ and IFN-γ+ cells and their pathogenic potential is dependent on T-bet expression.

Figure 6. Runx1 transcription factors and T-bet are required for conversion of Th17 cells into Th1-like cells in the adoptive transfer EAE model.

Figure 6

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(A) Flow cytometry analysis of 2D2 WT and 2D2 Tbx21−/− Th17 cells before and 17 d after adoptive transfer into C57BL6 recipients. Bar graphs represent percent of cytokine producing cells within 2D2 TCR transgenic positive CD4+ T cell population isolated from the central nervous system (CNS) of the recipient mice. Graphs represent combined data of 2 independent experiments with 8 mice per experimental group (mean ± SEM; P-values as determined by unpaired Student’s t-test). Mean clinical score of mice that received 2D2 WT or 2D2 Tbx21−/− Th17 cells is shown.

(B) Flow cytometry analysis of 2D2 WT (on Bcl2 transgenic background) Th17 cells transduced with empty vector (EV) or Runx dominant negative (Runx DN) expressing retrovirus, before and 17 d after adoptive transfer into T cell-deficient (Rag2−/−Il2rg−/−) recipients. Retrovirally transduced CD4+ Th17 cells (GFP+) were sorted prior to injection into recipient mice. Bar graphs represent percent of cytokine producing cells within 2D2 TCR transgenic positive CD4+ T cell population isolated from the CNS of the recipient mice. Graphs represent combined data of 2 independent experiments with 6 – 8 mice per experimental group (mean ± SEM; P-values as determined by unpaired Student’s t-test). Mean clinical score of mice that received 2D2 WT Th17 cells transduced with empty vector (EV) or Runx dominant negative (Runx DN) expressing retrovirus is shown. See also Figure S4.

To further evaluate the role of Runx transcription factors in the functional plasticity of Th17 cells, we specifically disrupted the DNA binding activity of Runx transcription factors using a retrovirus overexpressing the Runt domain with dominant negative activity. Using this experimental system, we effectively blocked transcriptional activity of both Runx1 and Runx3 in Th cells. As Runx1 is required for the survival and functional fitness of CD4+ T cells (Egawa et al., 2007), we introduced the pro-survival Bcl2 transgene into 2D2 WT mice (2D2 Bcl2+). CD4+ T cells isolated from 2D2 Bcl2+ mice were transduced with either empty retrovirus (EV) or retrovirus expressing the Runt domain (Runx DN). Overexpression of Runx DN resulted in a reduced frequency of IL-17A-producing 2D2 Th17 cells prior to transfer, indicating that the Runt domain interfered with the activity of Runx1 as a positive regulator of Th17 differentiation (Figure 6B). Seventeen days post-transfer, a significant percentage of IL-17A+IFN-γ+ and IFN-γ+ cells was detected in the CNS of mice that received 2D2 Bcl2+ Th17 cells transduced with the empty vector. In contrast, inhibition of Runx transcriptional activity resulted in a significant reduction in the frequency of IL-17A+ and IL-17A+IFN-γ+ cells. Consistent with this result, adoptively transferred 2D2 Bcl2+ Th17 cells expressing Runx DN induced mild EAE in recipient mice (Figure 6B). As the Runt domain may have effects unrelated to endogenous Runx protein functions, we deleted Runx1 from MOG35–55-reactive CD4+ Th17 cells using Cre-expressing retrovirus. CD4+ T cells were isolated from Runx1fl/fl mice immunized with MOG35–55 and CFA and re-activated in vitro with MOG35–55 peptide and IL-23 to expand the population of MOG35–55-reactive Th17 cells. Activated CD4+ T cells were transduced with Cre-expressing retrovirus to induce Runx1 gene deletion or empty vector control. Both Runx1-expressing and Runx1-deficient cells (as marked by GFP expression) CD4+ T cells were sorted and injected into the same host (Rag2−/−Ilr2g−/−) at a 1:1 ratio. Sixteen days later, the cells were isolated from the CNS of recipient mice and IL-17A and IFN-γ production by transferred CD4+ T cells was analyzed by intracellular cytokine staining. CD4+ T cells lacking functional Runx1 (GFP+) expanded less efficiently in a competitive setting compared to Runx1-expressing CD4+ T cells, confirming previously published data (Egawa et al., 2007). Nevertheless, we observed that the frequency of IL-17A+IFN-γ+ cells was substantially reduced in GFP+ Cre-expressing Runx1fl/fl cells compared to Runx1fl/fl control cells (Figure S4). Collectively, these results demonstrate a requirement for both T-bet and Runx transcription factors in the generation of pathogenic IL-17A+IFN-γ+ cells.

Th17, Th1-like Th17 and Th1 cells are readily detected in the CNS of WT mice after active immunization with MOG35–55 and CFA. To determine whether Th1-like Th17 cells are generated in the absence of T-bet, Runx1 or Runx3 during active EAE, we immunized mice with Cd4-cre-specific deletions of T-bet, Runx1 or Runx3 and their respective controls with MOG35–55 and CFA, and analyzed cytokine production by CNS-infiltrating CD4+ T cells at the peak of disease. Consistent with the negative regulatory role of T-bet in the differentiation of Th17 cells (Lazarevic et al., 2011; Mukasa et al., 2010), we observed a higher frequency of IL-17A+ cells in the absence of T-bet. T-bet was required for development of IL-17A+IFN-γ+ and IFN-γ+ cells (Figure 7A). In contrast, development of IL-17A+ and IL-17A+IFN-γ+ cells was dependent on Runx1 (Figure 7B). There was also a significant reduction in the frequency of IL-17A+IFN-γ+ cells in Runx3-deficient mice (Figure 7C). We detected a reduced proportion of IL-17A+IFN-γ+ cells in mice with a T cell-specific deletion of T-bet, Runx1 or Runx3, suggesting that all three transcription factors regulate the development of IL-17A+IFN-γ+ cells during EAE (Figure 7). Thus, we conclude that IL-17A+IFN-γ+ cells are generated as a result of a concerted action of T-bet and Runx transcription factors. In contrast, the development of IL-17A+ and IFN-γ+ cells is dependent on Runx1 and T-bet, respectively.

Figure 7. T-bet and Runx transcription factors are required for the generation of IL-17A+IFN-γ+ CD4+ T cells during EAE.

Figure 7

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(A–B) Flow cytometry analysis of cytokine producing CD4+ T cells isolated from the CNS of Cd4-cre Tbx21fl/fl, Cd4-cre Runx1fl/fl (on Bcl2 transgenic background) and Cd4-cre Runx3fl/fl mice and their respective controls 17 d after MOG35–55 and CFA immunization. Data depict percent cytokine producing cells within CD4+ gate. Each symbol represents an individual mouse; small horizontal line indicates the mean. Graphs represent pooled data of 2 independent experiments with 10–11 mice per experimental group (A–B) and one of two independent experiments (C). P values were determined by Student’s t-test.

DISCUSSION

In this study, we have identified Runx1 and Runx3 transcription factors as critical regulators of Th1-like Th17 cell differentiation. We showed that ectopic expression of Runx transcription factors in Th17 cells promoted the development of IFN-γ-producing Th17 cells, which expressed high amounts of T-bet, STAT4 and RORγt. Although ectopic expression of Runx1 or Runx3 induced both T-bet and STAT4, only T-bet (and not STAT4) was required for maximal IFN-γ production by Th1-like Th17 cells. T cell-specific deletions of T-bet or Runx transcription factors impeded differentiation of Th1-like Th17 cells during EAE, suggesting that such cells were generated as a result of a concerted action of T-bet and Runx transcription factors. Our mechanistic studies showed that exposure of Th17 cells to IL-12 increased Runx1, Runx3 and Tbx21 expression in Th17 cells, and enhanced binding of Runx1 to the Ifng promoter and to major regulatory elements within the Ifng locus in a T-bet dependent manner. Thus, we have identified a transcription factor module consisting of Runx1, Runx3 and T-bet that supports differentiation of Th1-like Th17cells.

Fate mapping experiments using Il17a-cre Rosa26eYFP reporter mice have revealed that a significant proportion of IFN-γ-producing cells isolated from tissues with an ongoing inflammatory response had expressed IL-17A at some point during their development (Hirota et al., 2011). The acquisition of Th1 effector functions by Th17 cells, including IFN-γ expression, is crucial for effective anti-tumor activity of Th17 cells, as T-bet- or IFN-γ-deficient Th17 cells had profoundly impaired anti-tumor responses (Muranski et al., 2011). However, Th1-like Th17 cells have also been implicated as major drivers of immunopathology in multiple sclerosis (Kebir et al., 2009), rheumatoid arthritis (Nistala et al., 2010), colitis (Ahern et al., 2010; Lee et al., 2009) and diabetes (Bending et al., 2009). The transcription factor networks that regulate the developmental program of Th1-like Th17 cells are poorly understood. Here we showed that the co-expression of T-bet and RORγt was not sufficient to establish a hybrid gene expression profile that would give rise to CD4+ T cells equipped with Th1 and Th17 effector functions. Instead, the differentiation of Th1-like Th17 cells was controlled by T-bet and Runx transcription factors.

Runx1 and Runx3 are ubiquitously expressed in hematopoietic cells, including T cells, in which they play essential roles in T cell development and the acquisition of T cell effector functions (Collins et al., 2009). Runx transcription factors are relatively weak transcriptional regulators on their own; thus, they regulate gene expression in a context-dependent manner through interactions with other transcription factors to activate or repress gene expression (Mikhail et al., 2006). Runx proteins have been shown to form transcriptional complexes with Foxp3, RORγt and T-bet to ensure appropriate Th differentiation and cytokine production (Djuretic et al., 2007; Ono et al., 2007; Zhang et al., 2008). For example, Runx3 interacts with T-bet to promote Th1 differentiation through maximal activation of the Ifng gene and through repression of the Il4 gene (Djuretic et al., 2007). Runx1 promotes Th17 differentiation by enhancing the expression of the Th17-specific transcription factor RORγt and by augmenting RORγt transcriptional activity at the Il17a locus (Zhang et al., 2008). Based on these findings, we postulated that Runx1 and Runx3 transcription factors could potentially support the differentiation program that is characteristic of both Th1 and Th17 cells depending on whether the Runx-interacting partner is a Th1- or Th17-cell master transcription factor. In this scenario, the differentiation of IL-17A+, IFN-γ+ and IL-17A+IFN-γ+ cells, would be determined by the relative molar ratios of T-bet, RORγt and Runx transcription factors in an activated CD4+ T cell. If the concentration of Runx1 is high enough to support formation of both Runx1-T-bet and Runx1-RORγt transcription complexes, then a Th cell that has the ability to simultaneously express IFN-γ and IL-17A will develop. In our study, ectopic expression of Runx1 or Runx3 induced strong Tbx21 expression in Th17 cells. Although T-bet is capable of epigenetically silencing the Rorc locus in Th17 cells (Mukasa et al., 2010), we found that both Runx1 and Runx3 maintained high levels of Rorc expression in Th17 cells. This was necessary for stabilizing the Th1-like Th17 cell state characterized by dual expression of IFN-γ and IL-17A. However, if the amount of Runx1 is limited and the cells are activated in the presence of cytokines that induce RORγt expression, then Runx1-RORγt interactions will promote the differentiation of IL-17A+ cells. Conversely, the reactivation of Th17 cells in the presence of IL-12 enhances not only T-bet expression, but also induces repressive epigenetic changes in the Rorc locus (Mukasa et al., 2010); thus, tipping the balance towards the differentiation of IFN-γ cells. Indeed, when we sorted IL-17-expressing cells marked by Thy1.1 expression and exposed them to IL-12, we observed that they gave rise to IL-17A+IFN-γ+ and IFN-γ+ cells. This shift in cytokine production was accompanied by an increase in Tbx21, Eomes, Runx1 and Runx3 mRNA. All four transcription factors have been linked to the regulation of the Ifng gene; however, only Runx1 and Runx3 promoted the differentiation of IL-17A+IFN-γ+ cells in the absence of IL-12 and independently of STAT4.

Although increased Runx1 or Runx3 expression was sufficient to generate similar percentages of IL-17A+IFN-γ+ and IFN-γ+ cells in the absence of T-bet, the abundance of Ifng transcripts was substantially diminished in T-bet-deficient Th17 cells transduced with Runx1 or Runx3 compared to WT controls. These data suggest that there is a requirement for T-bet and Runx1 co-expression for maximal induction of IFN-γ in Th17 cells. This conclusion is supported by loss-of-function genetic studies which demonstrated that IL-12-mediated reprograming of Th17 cells into IFN-γ-producing cells was severely impaired in the absence of T-bet, Runx1 or Runx3. Previously, it has been shown that treatment of Th17 cells with IL-12 led to the increased binding of STAT4 and T-bet to the Ifng locus, which correlated with the acquisition of permissive epigenetic marks and opening of the Ifng locus (Mukasa et al., 2010). In this study, we provide evidence for Runx1 binding to the Ifng promoter and at regulatory elements within the Ifng locus only in IL-12-treated Th17 cells, indicating that Runx1 may be directly involved in regulating Ifng expression. Runx1 was dependent on T-bet for binding to the Ifng promoter and regulatory elements that are normally silenced in Th17 cells, as these require T-bet for IL-12-induced changes from a repressive to permissive state.

Using the passive EAE model, we provide evidence that T-bet and Runx transcription factors were indeed required for the pathogenicity of adoptively transferred, MOG35–55-specific cells, which correlated with the presence of dual expressing IL-17A+IFN-γ+ cells in the CNS. However, the percentage of IFN-γ+ cells was not affected by Runx1 deficiency in the passive or active EAE models. In passive EAE, approximately 60% of CD4+ T cells in “2D2 Bcl2+ Th17 + Runx DN” culture did not produce IL-17A and IL-17F at the time of transfer. We have shown using IL-17F-Thy1.1 reporter mice that IL-17-non-producing cells (Thy1.1 negative) can give rise to IFN-γ+ cells when reactivated in the presence of IL-12. Similarly, immunization of mice with MOG35–55 and CFA induces high levels of IL-12 and IL-18, which drive the development of Th1 cells during active EAE. Differentiation of Th1 cells is dependent on T-bet and not on Runx1. In fact, we and others have shown that Runx1 is not expressed in Th1 cells (Djuretic et al., 2007; Lazarevic et al., 2011). Thus, the results from passive and active EAE experiments raise the possibility that a substantial portion of IFN-γ+ cells detected in the CNS could be bona fide Th1 cells, whose development is T-bet dependent, while the development of IL-17A+IFN-γ+ (Th1-like Th17) cells is dependent on both T-bet and Runx1.

In conclusion, we found that Runx1 contributes to the pathogenicity and functional plasticity of Th17 cells. In over-expression studies, Runx transcription factors did not require IL]-12 signaling, STAT4 or T-bet to promote the differentiation of IFN-γ-producing Th17 cells. Nevertheless, co-expression of T-bet and Runx transcription factors was needed for maximal production of IFN-γ by Th1-like Th17 cells. This finding was a consequence of enhanced Runx1 binding to the Ifng locus in the presence of T-bet. Collectively, these studies highlight Runx1 as a potential genetic risk factor for the development of autoimmune diseases and beg for detailed analysis of Runx1 target genes in CD4+ T cells. The analysis of ChIP-Seq data sets from other transcription factors, such as RORγt and T-bet, will reveal how T-bet-Runx1 and RORγt-Runx1 transcriptional complexes orchestrate and coordinate T cell effector functions during an inflammatory response, whether in the setting of infectious or autoimmune diseases.

EXPERIMENTAL PROCEDURES

Mice

C57BL/6 (WT) mice were purchased from Charles River Laboratories. Mutant strains were kindly provided by the following researchers: Il17fThy1.1/Thy1.1 reporter (Dr. C. Weaver; University of Alabama at Birmingham) (Lee et al., 2009), Stat4−/− (Dr. John O’Shea, MIIB, NIH), Tbx21fl/fl (Drs. S. Reiner, N. Cereb, S.Y. Yang and L. Boring) (Intlekofer et al., 2008) and Runx3fl/fl (Dr. D. Littman; NYU) (Egawa et al., 2007). 2D2 TCR transgenic, Runx1fl/fl and Cd4-cre mice were purchased from Jackson Laboratories and Bcl2 transgenic mice were obtained from Dr. A. Singer (NCI, NIH). Animal experiments were approved by the NCI Institute Animal Care and Use Committee.

Plasmids

For a detailed list see the Supplemental Experimental Procedures.

Flow cytometry

See the Supplemental Experimental Procedures for details.

Quantitative Real-Time PCR

mRNA was extracted from T cells with Qiazol (Qiagen) and treated with molecular grade DNase I (Roche) to eliminate possible genomic DNA contamination. Reverse transcription was performed using high capacity cDNA reverse transcription kit (Applied Biosystems). For quantitative RT-PCR, we used the Syber Green PCR system (Applied Biosystems). The sequences of primers are listed in the Supplementary Table 1.

Isolation, differentiation and functional analysis of CD4+ T cells

Naïve (CD62LhiCD25−) CD4+ T cell purification from lymph nodes and spleens of mice and culture conditions for Th differentiation are described in the Supplemental Experimental Procedures.

Retroviral transduction

See the Supplemental Experimental Procedures for details.

EAE induction and isolation of CNS cells

Active EAE was induced by subcutaneous injection of mice with 100 μg MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in complete Freund’s adjuvant supplemented with 250 μg Mycobacterium tuberculosis extract H37Ra (Difco). Mice received 180 ng pertussis toxin (List Biological Laboratories) intraperitoneally on days 0 and 2. For passive EAE experiments, 2D2 CD4+ T cells were activated and transduced under the Th17-polarizing condition with either a control retroviral vector or retrovirus overexpressing the Runx dominant negative mutant. On day 2, mIL-23 (10 ng/ml; R&D System) was added to retrovirally transduced 2D2 CD4+ T cells and cells were expanded for additional 3 days. Five days after initial stimulation, cells were sorted for GFP expression. Retrovirally transduced 2D2 T cells (>99% GFP+) were reactivated on anti-CD3/CD28 coated plates (each at 2 μg/ml) for 48 hours and injected into C57BL/6 or Rag2−/−Il2rg−/− recipients (5×106/mouse intravenously). Mice were monitored daily for the development of EAE according to the following criteria: 0, no disease; 1 limp tail; 2, hind limb weakness/partial paralysis; 3, complete hind limb paralysis; 4, front and hind limb paralysis; 5, moribund state.

Chromatin immunoprecipitation assay (ChIP)

Chromatin was prepared from 1.5×107of cells cultured under Th17 and Th17 and IL-12 conditions as described above. Briefly, cells were fixed with 1% (vol/vol) methanol-free formaldehyde for 10 minutes at room temperature and glycine was added to a final concentration of 0.125 M. Cell were washed with cold 1xPBS followed by a 10 minute lysis with 0.5% Triton-X on ice. Chromatin was sonicated to obtain fragments around 500 bp – 1 kb in length using Ultrasonic Processor XL (Heat System). Chromatin was incubated with Protein A beads coupled to anti-Runx1 antibody (Abcam; ab23980) overnight at 4°C. Beads were washed extensively and immunoprecipitated chromatin was treated with Proteinase K overnight at 56°C. DNA was extracted by phenol-chloroform extraction followed by ethanol precipitation in the presence of 0.3 M sodium acetate and 20 μg of glycogen. Extracted DNA was analyzed by real-time PCR using previously published primer sequences spanning the Ifng promoter and Ifng-specific CNS regions (Mukasa et al., 2010). We used the enhancer of the Tcrb gene as a positive control and the Actb promoter as a negative control (Rudra et al., 2009).

Statistics

Statistical significance was calculated by unpaired Student’s t-test using Prism software. All p values < 0.05 are considered significant.

Supplementary Material

01

Acknowledgments

The authors are thankful to Dr. Afred Singer, Dr. Hyun Park and members of the Lazarevic laboratory for critical reading of the manuscript, comments and suggestions. We gratefully acknowledge S. Sharrow, L. Granger and T. Adams for flow cytometry and sorting. We also thank Dr. W. Strober (at NIAID, NIH) for providing us with pMCsIg-EV-GFP, pMCsIg-Runx1-GFP and pMCsIg-Runx1-DN-GFP retroviral plasmids, and Drs. J. O’Shea (at MIIB, NIH), S. Reiner (at Columbia University), and D. Littman (via Dr. R. Bosselut at NCI, NIH) for providing us with Stat4−/−, Tbx21-floxed and Runx3-floxed mice, respectively.

Footnotes

AUTHOR CONTRIBUTIONS:

Y.W. did experiments, analyzed data and contributed to the writing of the manuscript; J.G., K.B.A., K.C., and A.B.P did experiments and analyzed data; R.Y. cloned Eomes-RV and Y.K.L. and C.T.W. generated Il17fThy1.1/Thy1.1 reporter mice; V.L. designed the study, did experiments, analyzed the data and wrote the manuscript.

COMEPETING FINANCIAL INTERESTS:

The authors declare no competing financial interests.

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