JunB promotes Th17 cell identity and restrains alternative CD4+ T-cell programs during inflammation

T helper 17 (Th17) cell plasticity contributes to both immunity and autoimmunity; however, the factors that control lineage flexibility are mostly unknown. Here we show the activator protein-1 (AP-1) factor JunB is an essential regulator of Th17 cell identity. JunB activates expression of Th17 lineage-specifying genes and coordinately represses genes controlling Th1 and regulatory T-cell fate. JunB supports Th17 cell identity by regulating key AP-1 complex constituents. In particular, JunB limits the expression of the subset repressor IRF8, and impedes access of JunD to regulatory regions of alternative effector loci. Although dispensable for homeostatic Th17 cell development, JunB is required for induction and maintenance of Th17 effector responses in the inflammatory contexts of both acute infection and chronic autoimmunity in mice. Through regulatory network analysis, we show that JunB is a core regulator of global transcriptional programs that promote Th17 cell identity and restrict alternative CD4+ T-cell potential.

F unctional plasticity in immune cells enhances the adaptability of responses targeting pathogens, but can also be detrimental to the host. Upon antigenic stimulation, CD4 + T cells adopt one of two opposing fates: a helper T (Th) cell specialized in supporting the clearance of infections, or a regulatory T (Treg) cell that functions to attenuate immune responses. Cytokines and other microenvironmental ligands present during T-cell activation direct diverse effector Th and Treg cell differentiation programs via the induction of function-specifying transcription factors (TF). The resulting subsets include Treg cells defined by Foxp3 expression, Th1 cells defined by T-bet (Tbx21) and IFNγ production, Th2 cells defined by GATA3 and IL-4 secretion, and Th17 cells defined by RORγt and IL-17 production. The identification of CD4 + T cells that co-express TFs and cytokines associated with alternative subsets highlights the vast flexibility inherent to CD4 + T-cell diversification 1 .
Among this diversity, Th17 cells stand out as particularly flexible in acquiring features of multiple other CD4 + T-cell subclasses under altered cytokine conditions 2,3 . At steady state, Th17 cells secreting IL-17 and IL-22 occupy mucosal barriers such as the intestinal lamina propria, where they regulate tissue homeostasis by promoting epithelial barrier function 4 . During infection, Th17 cells provide critical support for immunity against extracellular bacteria and fungi, and can readily adopt Th1-like characteristics (e.g., T-bet and IFNγ expression) in such inflammatory settings 5,6 . Although advantageous during infection, Th17 cell effector flexibility is also associated with deleterious Th17 cell function in autoimmunity. Indeed, pathogenic IFNγ + IL-17A + cells have been identified in numerous disease settings, including multiple sclerosis (MS) and inflammatory bowel disease (IBD) 7,8 , and exacerbate inflammatory disease in mouse models of these autoimmune conditions [9][10][11] . Moreover, genetic fate mapping tools in mice have provided convincing evidence for the context-dependent in vivo conversion of Th17 cells into T follicular helper cells in intestinal Peyer's patches 12 , IFNγ + Th1-like cells in an autoimmune model of MS 11 , and T regulatory type 1 cells (Tr1) during resolution of inflammation 13 . In light of the pervasive role of plasticity in CD4 + T-cell function and dysfunction, an understanding of the underlying mechanisms is of critical importance.
Th17 cell identity is governed by a network of transcriptional regulators that provide dynamic programming downstream of environmental inputs 14,15 . While the central regulator RORγt is essential for the expression of a limited set of key subset effectors 14,16 , additional TFs are required to specify the full Th17 cell program. In particular, a core of obligatory initiator TFs including STAT3 17 , IRF4 18 , and BATF 19 integrate cytokine (IL-6/IL-21/IL-23) and T-cell receptor (TCR) signals to coordinately activate Th17 cell differentiation 14 . Beyond this, less is appreciated about the molecular mechanisms that sustain subset identity and that facilitate functional plasticity. Early studies suggested the flexibility of Th17 cells is facilitated by a permissive, bivalent histone epigenetic status at polarizing TF loci-such as Tbx21 and Gata3-that may allow for subset reversibility and reprogramming 20 . Although epigenetic modulators that control effector status in Th17 cells have been identified 15,21 , the TF regulators that globally program the capacity of CD4 + T cells to dynamically control their functional identity in response to changing contexts are mostly undefined.
TFs of the activation protein-1 (AP-1) family are compelling candidates for mediating CD4 + T-cell identity switches. AP-1 factors are basic leucine-zipper TFs comprised of homodimers or heterodimers of Jun with Fos, or ATF protein classes. These TFs are dynamically regulated by extracellular signals and can either activate or repress transcription. AP-1 consensus motifs predominate in global analyses of Th17 cell cis regulatory regions 14,22 , which coincides with important functional roles for AP-1 TFs in Th17 cell differentiation. In particular, BATF and its cooperative binding partner IRF4 14,23,24 , are essential pioneer factors that establish chromatin accessibility at Th17 regulatory regions downstream of TCR signals. This activity pre-patterns the enhancer landscape for further subset-selective gene regulation 14 . Accordingly, BATF collaborates in high-order regulatory complexes with other Th17 specifying TFs, including STAT3, linking TCR and cytokine signals to epigenetic changes 14,22 . The identification of Fosl2 as a broad repressor of Th effector genes in Th17 cells adds another layer of complexity in the Th17 AP-1 network 14 . Fosl2 restricts Th1 and Treg cell potential, yet also antagonizes important Th17 program genes (e.g. Il17a/f, Ccl20, Ccr6) 14 , suggesting balancing contributions by other AP-1 factors in the regulation of Th17 cell plasticity. In this regard, JunB is a dominant AP-1 dimerization partner for both BATF and Fosl2 in Th17 cells 19,24 , with an established role in CD4 + T-cell differentiation. In Th2 cells, JunB is critical for Il4 transcription, while also restricting inappropriate Ifng expression 25 , intimating a function for JunB in physiological Th17 cell effector conversions. Indeed, JunB may be poised to sense shifts in environmental context as its protein levels are subject to dynamic control via posttranslational modification in CD4 + T cells 26,27 . The contribution of JunB to Th17 cell differentiation and its regulation of effector identity within the growing Th17 cell TF network has not been evaluated.
Here, we identify JunB as a critical regulator of Th17 cell identity. Deletion of Junb in Th17 cell differentiation results in a marked reduction in IL-17A-producing cells and an aberrant emergence of Th1-like and iTreg-like cells. Although dispensable for homeostatic Th17 cells, JunB is essential for induction of these cells in inflammatory settings. Specifically, in the absence of JunB, in vivo inflammation induced by infection with Candida albicans or a model antigen in the context of experimental autoimmune encephalomyelitis (EAE) results in impaired Th17 cell responses with an upregulation of a Th1 cell phenotype. Global analysis of JunB-dependent gene expression and genomic occupancy reveals that JunB controls Th17 cell stability through direct activation of important Th17 effector genes in concert with direct repression of subset-defining regulators of the Th1 and Treg cell lineages (e.g. Tbx21, Foxp3, and Ifng). Beyond these select genes, JunB functions as a core regulatory node in global programs that promote Th17 cell differentiation while constraining alternative effector and regulatory CD4 + T-cell potentials.

Results
JunB regulates Th17 cell identity. To determine the expression profile of JunB in CD4 + T-cell subsets, we evaluated in vitro differentiated CD4 + T cells at 24, 48, and 72 h for levels of JunB. At each time point, the relative mean fluorescence intensity of JunB was on average 3-fold higher when naive CD4 + T cells were cultured under Th17 cell polarizing conditions (IL-6 and TGF-β), as compared to induced Treg (iTreg,TGFβ and IL-2), Th1 (IL-12), or Th2 (IL-4) cell polarizing conditions, or to non-polarizing Th0 conditions (TCR stimulation alone) (Fig. 1a). After 5 days, JunB remained most highly expressed in Th17 cells ( Supplementary Fig. 1a). However, Junb transcript levels were not as differential, consistent with posttranslational mechanisms that regulate JunB protein turnover in CD4 + T cells 26,27 ( Supplementary Fig. 1b). The selective early induction and sustained elevated levels of JunB in Th17 cells suggested that JunB plays an important role during Th17 cell differentiation.
To test this hypothesis, we bred mice with a conditional Junb allele 28 to the CD4-Cre pan T-cell deleter strain 29 . First, we compared αβ T-cell subsets in naive Junb +/+ CD4-Cre (WT CD4cre ) and Junb fl/fl CD4-Cre (KO CD4cre ) mice. Total thymocyte numbers and selection, based on TCRβ and CD69 expression, were unaffected in KO CD4cre mice, and mature CD4 + TCRβ + and CD8α + TCRβ + thymocytes developed in the expected frequencies ( Supplementary Fig. 1c). In the spleen and mesenteric lymph nodes (LN), total cellularity, as well as the frequencies and numbers of CD4 + TCRβ + and CD8α + TCRβ + cells were similar ( Fig. 1b and Supplementary Fig. 1d). Importantly, the frequency of naive CD4 + TCRβ + CD44 lo CD62L + cells was also unaltered in the periphery of KO CD4cre mice ( Fig. 1b and Supplementary  Fig. 1d   such that Th17 cells polarized with IL-6, IL-1β, and IL-23 effectively induce disease compared to non-pathogenic Th17 cells polarized with IL-6 and TGFβ 30,31 . Differentiation of naive CD4 + T cells purified from WT CD4cre and KO CD4cre mice with IL-6 and TGFβ revealed a striking dysregulation in cytokine and TF expression for mutant cells. JunB-deficient cultures displayed a 2-fold to 4-fold decrease in the proportion of IL-17A-expressing cells vs. WT CD4cre cultures, as well as robust populations of aberrant Foxp3 + iTreg-like and T-bet + IFNγ + Th1-like cells (Fig. 1c). IFNγ and IL-17A expression remained nearly mutally exclusive ( Supplementary Fig. 1f). Importantly, the reduction in IL-17A + cells is not due to a defect in culture proliferation, or a selective impairment of IL-17A + cell proliferation or survival in the absence of JunB ( Fig. 1d and Supplementary Fig. 1e). A similar dysregulation occurred using 'pathogenic' Th17 cell-promoting conditions ( Supplementary Fig. 1g), although, KO CD4cre cultures generated fewer Foxp3 + cells, suggesting that efficient release of iTreg-like potential requires TGFβ-mediated signals. Accordingly, we also observed less atypical Foxp3 expression in KO CD4cre naive CD4 + T cells cultured without exogenous TGFβ in Th0 and Th1 conditions relative to 'non-pathogenic' KO CD4cre Th17 conditions (Fig. 1c). In addition, the upregulated Th1-like T-bet + IFNγ + profile of KO CD4cre Th17 cells also occurred in JunB-deficient Th0, iTreg, and Th1 cultures, suggesting a general role for JunB in limiting Th1 cell differentiation (Fig. 1c). Indeed, similar to studies using Junb-hypomorphic mice 25 , KO CD4cre Th2 cell cultures produced reduced levels of IL-4 and increased IFNγ, compared to WT CD4cre by day 5 (Supplementary Fig. 1h). Taken together, JunB has a nonredundant role in promoting Th17 cell identity and restraining alternative Th1 and Treg potential during Th17 cell induction.
Th17 cells require continuous expression of JunB. To interrogate the role of JunB in the maintenance of Th17 cell effector identity we deleted Junb in differentiated Th17 cells and simultaneously employed a fate mapping strategy to track Th17 to Th1 or Treg cell conversion events. Specifically, conditional Junb mutant mice were bred with mice expressing Cre from the Il17a locus (Il17a Cre/+ ) 11 , as well as with a reporter strain encoding a floxed-stop Zsgreen conditional allele in the ubiquitous Rosa26 locus 32 . Consequently, ZsGreen expression permanently marked cells with both a history of Il17a expression and Junb gene deletion in Junb fl/fl cells (Fig. 2a). Th17 cell plasticity under defined, altered conditions can be modeled in vitro 2, 3 . We differentiated naive CD4 + T cells from Junb +/+ Il17a Cre/+ Rosa26 ZsGreen/+ (WT Il17aCre R26 ZS ) and Junb fl/fl Il17a Cre/+ Rosa26 ZsGreen/+ (KO Il17aCre R26 ZS ) mice under Th17 conditions for 4 days to generate Il17a ZsGreen + fate-mapped cells (Il17a ZsGreen-FM+ ). Purified Il17a ZsGreen-FM+ cells were transferred back to Th17 conditions to evaluate the role of JunB in regulating Th17 cell stability, or alternatively, to altered cytokine conditions to establish maximal Th1 and iTreg lineage conversion potential. Compared to wild type, JunB-deficient Il17a ZsGreen-FM+ cells preserved in Th17-promoting conditions, displayed a marked reduction in IL-17A and IL-17F expression ( Fig. 2b and Supplementary Fig. 2c). Moreover, a prominent fraction of JunB-deficient Il17a ZsGreen-FM+ cells underwent effector conversion to Treg-or Th1-like phenotypes (Fig. 2c). The extent of Treg re-direction was similar when Il17a ZsGreen-FM+ cells were transferred to a potent iTreg-polarizing condition ( Supplementary Fig. 2a), indicating that efficient transdifferentiation occurs when JunB expression is extinguished in Th17 cells. Conversely, the level of T-bet in JunB-deficient Il17a ZsGreen-FM+ Th17 cells was higher for cells transferred to Th1 vs. Th17 cell conditions ( Supplementary Fig. 2b), suggesting the full acquisition of Th1 status upon conversion is facilitated by altered cytokine inputs. Therefore, continuous JunB expression is required to both maintain Th17 cell effector identity and constrain plasticity towards alternative CD4 + T-cell fates in cells that have initated the Th17 cell program.
JunB actively promotes Th17 cell identity. To uncover JunB targets that stabilize effector identity, we performed RNA-Sequencing (RNA-Seq) and differential expression analysis of WT CD4cre vs. KO CD4cre Th17 cell cultures. Pathway analysis of the top differential genes identified those associated with Th cell differentiation as most significantly enriched (Supplementary Fig. 3a). Several Th-related genes are highlighted in the volcano plot displaying fold change in JunB-dependent transcripts (Fig. 3a). As KO CD4cre cultures exhibit effector cell heterogeneity, we confirmed JunB regulation via global JunB occupancy determined by chromatin immunoprecipitation sequencing (ChIP-Seq). Of note, we identified Th17 cell effector genes-including Il21, Il23r, and Il17a-as direct JunB targets, being significantly reduced in gene expression in KO CD4cre Th17 cell cultures (Fig. 3a), and displaying significant JunB locus-proximal occupancy ( Fig. 3b and Supplmentary Fig. 3b). At these loci, JunB binding is associated with that of p300, a histone acetyltransferase for which occupancy is predictive of tissue-specfic enhancer activity 33 . Importantly, unlike other Th17 cell regulators, BATF 19 , IRF4 18 , and STAT3 17 , JunB is not required for Rorc (RORγt) expression (Fig. 3a). Thus, JunB directly activates key components of the Th17 cell program, independent of effects on Rorc.
We further explored JunB regulation of Il17a. Motif analysis of three JunB-occupied regions within the Il17a locus identified AP-1 consensus sites in each (Supplementary Table 1), implicating these conserved noncoding sequences (CNS) as putative JunB-dependent enhancers (Fig. 3b). Genomic regions corresponding to CN S+0.9,+6, and +10 were cloned upstream of a minimal promoter driving a luciferase reporter, and assayed for activity in both WT CD4cre and KO CD4cre in vitro differentiated Th17 cells. Only CNS+10, located 10 kb downstream of the Il17a transcription start site (TSS), displayed robust enhancer function, with an 8-fold increase in activity over that of the minimal promoter (Fig. 3c). Although CNS+10 activity was markedly less than that of CNS-5, an in vivo validated 5' Il17a locus enhancer 34 , Fig. 1 JunB promotes Th17 cell identity and represses Th1 and iTreg cell programs. a Flow cytometry of JunB expression in sort-purified Junb +/+ CD4cre (WT CD4cre ) naive CD4 + T cells (CD4 + CD25 − CD44 lo CD62L + ) or WT CD4cre naive CD4 + T cells cultured under Th0, Th17, iTreg, Th1, or Th2 conditions, for the indicated times. Junb fl/fl CD4cre (KO CD4cre ) naive CD4 + T cells were cultured and analyzed in concert as a negative control for staining. Error bars represent SEM of two independent experiments. Histograms depict levels of JunB in Th17 (blue), iTreg (red), and Th1 (green) cells, 48 h post polarization. b Flow cytometry of splenocytes from WT CD4cre and KO CD4cre mice, stained for TCRβ, CD4, and CD8α. Total splenocyte counts, frequencies of CD4 + TCRβ + and CD8 + TCRβ + splenocytes, and total numbers of TCRβ + cells are shown. c Flow cytometry of WT CD4cre and KO CD4cre sort-purified naive CD4 + T cells cultured under Th0, Th17, iTreg, Th1, or Th2 conditions for 72 h followed by restimulation. Data are representative of ten independent experiments which are compiled into bar graphs. Error bars depict SEM. d Flow cytometric analysis of CFSE dilution in restimulated 72 h Th17 cell cultures relative to IL-17A expression (left), and specifically in IL-17A-expressing cells (right). The frequency of cells in each generation is indicated in the line graph. Error bars represent SEM of two independent experiments with at least five mice per group. ****p<0.0001 (unpaired two-tailed Student's t-test) its activity was uniquely JunB-dependent, reduced by 2-fold in JunB-deficient Th17 cells (Fig. 3c). Notably, when test regions were assayed in cis with CNS-5, CNS+10 exclusively synergized with CNS-5 in a JunB-dependent manner (Fig. 3d), suggesting a mechanism for JunB-promoted maintainance of Il17a. The identification of CNS+10 as a novel JunB-dependent enhancer supports the view that JunB directly activates Th17 cell identity.
Among JunB-dependent genes, IRF8 is a compelling candidate mechanistic node for JunB-mediated Th17 cell stability. IRF8 is a negative regulator of Th17 cell differentiation 35 , and can replace IRF4 in BATF-JunB AP-1 complexes in Th17 cells 24 . Irf8 RNA and protein levels were significantly elevated in KO CD4cre Th17 cells and in KOIl17a ZsGreen-FM+ cells (Fig. 3e, f and Supplementary Fig. 3c). Additionally, JunB binds upstream of the Irf8 locus (Fig. 3g), suggesting that direct Irf8 suppression contributes to stabilization of the Th17 cell program.
To assess whether IRF8 upregulation accounts for the decreased differentiation of JunB-deficient Th17 cells, we counteracted Irf8 derepression by conditionally deleting one allele of Irf8 on a KO CD4cre background (generating Irf8 f/+ Junb f/f CD4-Cre mice), thereby restoring Irf8 RNA and protein to near wild-type levels ( Supplementary Fig. 3d and Fig. 3f). This coincided with a 50% rescue in IL-17A production (Fig. 3h); and a significant increase in the expression of lineage-relevant IRF8 repression targets (Il23r, Il21, Ccr6, and Rorc) in Irf8 f/ + KO CD4cre relative to KO CD4cre Th17 cells ( Supplementary  Fig. 3e). Although heterozygous deletion of Irf8 partially restored the Th17 cell program in the absence of Junb, it did not reverse the aberrant elevation of T-bet, IFNγ, and Foxp3 in KO CD4cre Th17 cells (Supplementary Fig. 3f). Therefore, the release of alternative CD4 + T-cell programs upon loss of JunB is independent of IRF8 upregulation. Taken together, JunB promotes Th17 cell identity by both limiting the expression of IRF8 and directly activating effector loci.
JunB directly represses alternative programs in Th17 cells. The lineage dysregulation observed for JunB-deficient Th17 cells was also apparent by global differential gene expression analysis (Fig. 3a). In particular, transcripts for subset-defining regulators of Treg and Th1 differentiation, including Foxp3, Tbx21, and Ifng, were significantly upregulated in KO CD4cre Th17 cells ( Fig. 3a and Supplementary Fig. 4a). This coincided with significant gene-proximal JunB ChIP occupancy (Fig. 4a), identifying these lineage-defining loci as likely direct JunB repression targets. Gene set enrichment analysis was used to characterize the dysregulated transcriptome of KO CD4cre relative to WT CD4cre Th17 cells (Fig. 4b). In the absence of JunB, downregulated targets were significantly enriched in Th17 cell signature genes, whereas upregulated genes were significantly enriched for those associated with Treg and Th1 cell signatures, indicating global shifts in effector programming consistent with the adoption of alternative CD4 + T-cell states.
JunB occupancy defines novel silencer elements. Cell identity is governed by the interaction between environment-sensing TFs, chromatin context, and the DNA elements that regulate lineage-relevant loci. The cis elements that restrict alternative potentials in Th17 cells are undefined. To address this, putative JunB-dependent silencer elements were identified as JunB-occupied CNS proximal to lineage-defining repressed loci (Irf8, Foxp3, Tbx21, and Ifng), which also contained AP-1 a JunB % of max   Table 1). Candidate regions were cloned upstream of the JunB-independent Il17a CNS-5 enhancer in a minimal promoter-driven luciferase reporter construct, and assayed in Th17 differentiation cultures for the ability to diminish enhancer activity. Three of seven regions displayed silencer activity, including Ifng CNS+18, Foxp3 CNS+13 (3'UTR-localized), and Irf8 CNS-34. Each significantly attenuated the activity of Il17a CNS-5 (Fig. 4c), and did not display enhancer activity ( Supplementary Fig. 4b). Notably, when these 3 silencer CNSs were assayed in KO CD4cre Th17 cells, Il17a CNS-5 activity was significantly restored and, in the case of Irf8 CNS-34, enhanced ( Fig. 4c), indicating that their repressive potential is JunB-dependent. Interestingly, none of the Tbx21 CNS demonstrated silencing activity ( Supplementary Fig. 4c), potentially reflecting the presence of distal cis elements 36 . Together, these data implicate Ifng CNS+18, Foxp3 CNS+13, and Irf8 CNS-34 as novel JunB-dependent silencer elements, which coordinate to protect Th17 cell status, and through regulation of Ifng and Foxp3, are potential restriction elements that actively repress alternative Th1/Treg cell fates.

Test element minP Luciferase
Test element AP1-IRF composite motifs with BATF and IRF4 23 , essential pioneer factors for Th17 cell specification 14 . JunB also dimerizes with Fosl2, a negative regulator of effector potential in Th17 cells 14,19,24 . Consistent with this diversity, ChIP-Seq revealed a high degree of colocalization of JunB with known partners at Th17 cell (Il17a), and alternative Th subset (Ifng) loci ( Fig. 5a and Supplementary Fig. 5a) 23 . To examine global patterns of TF binding in Th17 cells, we merged overlapping binding peaks for AP-1 TFs, IRF4, RORγt, and p300 into putative cis regulatory modules (pCRM), and generated a clustered heatmap of TF distribution at pCRMs. JunB showed a striking, near-exclusive binding overlap with BATF, Fosl2, and IRF4, indicating global occupancy of common regulatory regions (Fig. 5b, clusters 1, 2, and 3).
In light of the strong regulatory interaction between AP-1 TFs, we characterized how the loss of JunB influences the chromatin occupancy of JunB binding partners in Th17 cells. Importantly, Batf, Irf4, and Fosl2 transcript levels were not reduced in KO CD4cre relative to WT CD4cre Th17 cell polarization cultures by 48 h (Fig. 5c). ChIP for BATF, IRF4, and Fosl2 from KO CD4cre cultures demonstrated comparable occupancy to WT CD4cre at select JunB-bound regions in the Il17a, Irf8, Tbx21, Ifng, and Foxp3 loci (Fig. 5d). A notable reduction was observed for IRF4 at the Il17a locus, potentially reflecting replacement by elevated IRF8 24 (Fig. 3f), which can bind to and regulate Il17a both directly and indirectly 35 . Therefore, the DNA binding pattern of Th17 cell-relevant AP-1 complex TFs is largely independent of JunB, unlike the collaborative binding of BATF with IRF4, which is greatly reduced in cells lacking the respective partner 14,23,24 .
The incomplete block in Th17 cell differentiation in the absence of JunB, compared to either BATF or IRF4, implies that other Jun family members play a compensatory role 23,24 . JunD is an excellent candidate in this regard. Whereas cJun transcripts were dramatically reduced in KO CD4cre relative to WT CD4cre cultures by 48 h (Fig. 5c), Jund expression was elevated in Junb-deficient cells specifically under Th17-promoting conditions ( Fig. 5c and Supplementary Fig. 5b). Moreover, 68% of significant genome-wide JunD binding peaks, vs. only 24% of cJun peaks, overlapped with those of JunB (Supplementary Fig. 5c and d). Like JunB, JunD can bind cooperatively to DNA with BATF and IRF4 23 . Accordingly, the global binding pattern of JunD in Th17 cells highly overlapped with that of BATF, and IRF4 as reported 23 , and also with that of Fosl2 and JunB (Fig. 5a, b, cluster 1, 5).
The overlap in JunD and JunB occupancy suggests an antagonistic relationship. In addition to shared pCRMs (Fig. 5b, cluster 1), JunB binding in regions devoid of JunD implies that JunD is excluded in some contexts (cluster 2,3). Consistent with TF antagonism, in the absence of JunB, we observed a striking elevation in the binding of JunD at many AP-1 consensus sites typically bound by JunB in lineage-defining regulatory loci (Fig. 5d). Notably, global ChIP-Seq revealed elevated JunD binding in JunB-deficient Th17 cell cultures at numerous sites throughout the genome (Fig. 5e). Among these, there are regions that acquire novel JunD binding when JunB is eliminated ( Supplementary Fig. 5d) indicative of factor antagonism, either through competition for DNA binding (Fig. 5f, JunB + cluster 4), or for co-factors (Fig. 5f, JunB-devoid cluster 6). Importantly, JunD binding is not globally elevated, as an abundance of JunB-bound pCRMs displayed significantly diminished JunD occupancy in the absence of JunB (Fig. 5e), potentially reflecting binding of JunB-JunD heterodimers. Furthermore, unlike JunB, Batf, or Fosl2, JunD localizes near-exclusively to p300-occupied pCRMs (Fig. 5b, clusters 1,5), implying a dominant activating role in Th17 cells. Taken together, JunD can effectively replace JunB as an AP-1 dimerization partner at select genomic regions that include key effector loci, suggesting a potential mechanism for repression to activation switches during lineage conversion events mediated by loss of JunB.
Global coordination of JunB with Th17 specification factors. The interplay between AP-1 proteins in Th17 cells is complex as JunB can homodimerize, or form heterodimers with partners such as BATF or Fosl2. To examine global trends in the regulation of shared targets by individual AP-1 factors in Th17 cells, we compared the JunB-dependent differential expression profile with that of BATF and Fosl2 (Fig. 6a, red dots). Notably, the majority of TF-dependent genes (90%) were similarly affected (i.e. both up-or downregulated) in JunB-and BATF-deficient Th17 cells, in contrast to a comparison of JunB-and Fosl2-dependent genes, where a relatively large proportion (32%) demonstrate discordant regulation (Fig. 6a). Highly coordinate regulation of gene expression by JunB and BATF is consistent with the predominant formation of a heterodimer pair, vs. potentially greater JunB-independent diversity in AP-1 or co-factor associations for Fosl2.
To evaluate the regulatory collaboration between JunB and Th17 cell identity factors (BATF, Fosl2, IRF4 and STAT3), we defined direct, functional targets for each TF as those that are significantly differentially expressed in TF knockout vs. wild-type RNA-seq; and for which one or more significant TF ChIP-seq peaks localize within 5 kb of the gene. The high-confidence network of TF-target interactions are visualized in Cytoscape ( Supplementary Fig. 6a). JunB target genes display a high degree of co-regulation by other Th17 cell specifying TFs, with 83% regulated by at least one additional TF. Notably, AP-1 TF subnetworks reveal a striking, near-absolute activation-repression concordance by JunB and BATF, with a lesser degree of coordinate regulation by JunB and Fosl2 (Fig. 6b), consistent with the comparisons of RNA differential expression  (Fig. 6b, c). Conversely, in instances where JunB and Fosl2 show opposing regulation, differential gene expression upon Th17 differentiation is most consistent with JunB activity, implicating JunB as a more influential activator and repressor (Fig. 6c). Does JunB globally silence alternative CD4 + T-cell programs? Direct repression targets of JunB are significantly enriched for genes that are differentially expressed in Th1 vs. Th17 polarization cultures (Th1 signature in orange; Supplementary  Fig. 6b and Fig. 6d), and similarly in iTreg vs. Th17 differentiation ( Supplementary Fig. 6c), supporting a role for JunB in broad silencing of Th1-and iTreg-associated loci. This strong correlation is mirrored for repression targets of BATF, IRF4, and STAT3, but not of RORγt (Fig. 6d,  Supplementary Fig. 6c). Consistent with a more general role in limiting effector plasticity, Fosl2 represses genes that are upregulated in Th1, iTreg, and Th17 signatures. Focusing our analysis on functional gene classes (e.g., cytokines, receptors, and TFs) reveals a high level of coordinated regulation of global Th17 (colored blue), Th1, and iTreg cell signature genes by JunB, BATF, IRF4, STAT3, and Fosl2 (Fig. 6e, and Supplementary Fig. 6d). The concerted negative regulation is highlighted for key Th1 and Treg loci (Ifng, Tbx21, Il18r1,Tnfsf4, Nrp1, and Foxp3) among others that are upregulated upon Th1 and iTreg differentiation. JunB is also highly integrated into the global Th17 cell activation program coordinated by initiator TFs BATF, IRF4 and STAT3 14 . Indeed, the Th17 cell signature is strongly enriched among direct JunB activation targets (Fig. 6d), and JunB co-activates many of these signature genes (Il17a, Il23r, Ccl20, and Rora) in conjunction with Th17 pioneer TFs (Fig. 6e).
In addition to influencing 'master' regulator loci, JunB directly contributes more broadly to global regulatory programs that promote Th17 cell identity and restrict alternative Th1 and Treg programs.
Steady-state Th17 cells are unaltered in the absence of JunB.
Given the dysregulation of JunB-deficient polarization cultures, we examined whether Th17 cell differentiation was also altered in vivo. Notably, there was no difference in the proportion of IL-17A-or IFNγ-expressing steady-state CD4 + T cells localized to the spleen, mesenteric LNs, or small intestine lamina propria (SILP) between WT CD4cre and KO CD4cre mice (Fig. 7a)   frequency of SILP CD4 + T cells expressing RORγt or T-bet was also unaltered in JunB mutants (Fig. 7b, c). However, there was a modest reduction in the frequency and number of Foxp3 + Treg cells in KO CD4cre mice in every tissue evaluated ( Fig. 7d and Supplementary Fig. 7a). The decrease in Treg cells in vivo is similar to the defect observed when IL-2 is omitted from mutant iTreg cultures in vitro ( Supplementary Fig. 7b). This is in contrast to the unperturbed status of IL-2 supplemented cultures (Fig. 1c), suggesting a potential non cell-intrinsic role for JunB in Treg cell development or maintenance. Accordingly, deletion of Junb exclusively in Foxp3 + cells resulted is similar frequencies of Tregs compared to control mice in secondary lymphoid organs ( Supplementary Fig. 7c). Overall, steady-state CD4 + Th cell subsets, including homeostatic Th17 cells, were similar in WT CD4cre and KO CD4cre mice.
JunB promotes Th17 cell subset stability in vivo. To evaluate the role of JunB in restricting alternative CD4 + T-cell programs in an inflammatory setting where Th17 cell plasticity occurs physiologically, we used the EAE mouse model of MS, which is dependent upon the activity of CD4 + Th cells 37 . In EAE, IFNγ + encephalitogenic T cells originate nearly entirely from Th17 cells that have undergone subset conversion 11 . Notably, JunB levels decrease as Il17a ZsGreen-FM+ CD4 + T cells transition from IL-17A + to IL-17A + IFNγ + or to IFNγ + status post EAE induction. Consistent with in vitro differentiation cultures (Fig. 1a), JunB is more highly expressed by IL-17A-vs. IFNγ-producing CD4 + T cells (Fig. 8a). These trends indicate that JunB protein levels are modulated during physiological Th17 cell plasticity, suggesting that JunB is part of the mechanism that regulates effector conversions. Thus, EAE is an ideal model to evaluate the role of JunB in Th17 cell lineage stability. Following induction of EAE, WT CD4cre mice progressed to near-complete paralysis, whereas KO CD4cre mice were absolutely protected from disease (Fig. 8b). Analysis of cerebellum and spinal columns at peak disease in WT CD4cre mice, uncovered a dramatic reduction in encephalitogenic CD4 + CD45 + cells infiltrating the central nervous system (CNS) of KO CD4cre mice, precluding cellular phenotyping (Fig. 8c). This likely accounted for the lack of symptoms, and revealed an unexpected requirement for JunB for pathogenic T-cell CNS infiltration. Accordingly, genes involved in migration are enriched in the altered transcriptome of JunB-deficient Th17 cells (Supplementary Fig.3a). To assess early CD4 + T-cell responses, draining LNs were examined at 6 days post induction of EAE, where there is no significant difference in baseline effector profiles in naive KO CD4cre mice (Fig. 8d). This revealed a striking dysregulation in CD4 + T-cell cytokine production in KO CD4cre mice similar to that of JunB-deficient Th17 cell cultures (Fig. 8d). In particular, KO CD4cre mice had fewer IL-17A-producing CD4 + T cells compared to WT CD4cre mice, with numbers similar to naive levels (Fig. 8d). Importantly, mutant CD4 + T cells were not refractory to activation; rather, KO CD4cre LNs showed a striking predominance of IFNγ-and GM-CSF-producing CD4 + T cells relative to WT CD4cre EAE mice (Fig. 8d). Enhanced GM-CSF was also a feature of KO CD4cre Th17 cell cultures in vitro ( Supplementary Fig. 8a). Moreover, taking into account that Treg cells are reduced in steady-state KO CD4cre mice, we observed a selective twofold increase in the frequency of LN Foxp3 + CD4 + T cells relative to naive mice at day 6 of EAE, implying that enhanced Treg induction also occurs in vivo in the absence of JunB (Fig. 8e and Supplementary Fig. 8b). Notably, while JunB is essential for inflammatory Th17 cell induction and expression of important effector genes, JunB does not globally regulate the previously defined pathogenic Th17 cell profile 30 ( Supplementary Fig. 8c). Thus, in an inflammatory setting, JunB is essential for the induction of appropriate Th17 cell responses; for restricting alternative CD4 + Th and Treg programs; and for enabling pathogenic CD4 + T-cell CNS migration during EAE.
To assess the role of JunB in regulating lineage conversion events during inflammation, we induced EAE in WT Il17aCre R26 ZS and KO Il17aCre R26 ZS mice and tracked fate-mapped Il17a ZsGreen-FM+ cells in vivo. Similar to KO CD4cre mice, Junb deletion in Il17a-expressing cells resulted in a complete rescue from symptoms ( Supplementary Fig. 8d). At peak disease, 50% of CD4 + CNS infiltrating cells in WT Il17aCre R26 ZS mice were Il17a ZsGreen-FM+ and enriched for encephalitogenic IL-17A + IFNγ + CD4 + T cells, as reported 11 (Supplementary Fig. 8e). In contrast, although CD4 + T cells were identified in the CNS in KO Il17aCre R26 ZS mice, these were selectively devoid of the Il17a ZsGreen-FM+ fraction that marks JunB-deficient cells. Unexpectedly, this specific depletion was mirrored in the draining LN early post-induction of EAE in KO Il17aCre R26 ZS vs. WT Il17a-Cre R26 ZS mice ( Supplementary Fig. 8f), and this coincided with a dramatic elevation in the frequency of AnnexinV + apoptotic cells ( Supplementary Fig. 8g). While the loss of JunB-deficient Il17a ZsGreen-FM+ T cells precluded analysis of fate conversion events, it revealed a distinct requirement for sustained JunB expression for survival of differentiated Th17 cells. Importantly, an unaltered frequency of AnnexinV + cells among homeostatic SILP Th17 cells suggests that the role of JunB in Th17 cell survival is restricted to an inflammatory setting ( Supplementary Fig. 8h).
In addition to the chronic inflammatory context of EAE, we employed an acute cutaneous infection model with Candida albicans to assess the role of JunB in an inflammatory setting where Th17 cells do not typically exhibit plasticity 11 . IL-17Aproducing Th17 cells are important in the defense against C. albicans 38, 39 . On day 5 post infection, similar frequencies of CD4 + cells were observed in the skin explants from WT CD4cre and KO CD4cre mice, both significantly enhanced relative to mocktreated controls (Fig. 8f). As expected, WT CD4cre mice produced a balanced IL-17A/IFNγ cytokine response to C. albicans 11 . However, comparable to our findings in the EAE model, infected KO CD4cre mice failed to induce a Th17 cell response, instead  having a proportion of IL-17A + cells ressembling uninfected mice (Fig. 8f). Moreover, T-cell loss of Junb skewed the CD4 + effector profile toward a predominantly Th1 phenotype, characterized by a striking enhancement in IFNγ and GM-CSF production. This dysregulation reflects an inflammation-dependent role for JunB in effector cell generation as steady-state cytokine-producing skin CD4 + cells are unaltered in KO CD4cre mice (Fig. 8f). However, as in other tissues, there is a reduction in Foxp3 + Treg cells in the skin of naive KO CD4cre mice that persists post infection ( Supplementary Figs. 8i and 7d), despite a similar relative expansion in WT CD4cre and KO CD4cre mice (Fig. 8g). The lack of increased enhancement of Treg cells in mutants relative to controls may reflect the non-supportive environment for Treg cells in KO CD4cre mice ( Fig. 7d and Supplementary Fig. 7), which is perhaps selectively exacerbated in an acute inflammatory setting of C. albicans infection that is inherently non-permissive to plasticity. Taken together, the in vivo findings identify an absolute and selective requirement for JunB in the induction and maintenance of Th17 effector cells during inflammation, in the context of both chronic autoimmunity and acute infection. Coordinately, JunB serves an essential role in constraining Th1 and Treg features, suggesting that the modulation of JunB in an environment-dependent manner can permit controlled adoption of alternative effector programs.

Discussion
Previous studies have identified the pioneer factors and transcriptional regulators required to implement the Th17 cell program downstream of TCR and cytokine signals, however, the mechanisms underlying the inherent plasticity of the Th17 cell subset are less characterized 14,[17][18][19] . Here, we define JunB as a novel, nonredundant regulator of global programs that support Th17 cell identity and that restrain alternative Th1 and Treg cell fates in inflammatory contexts.
Th17 cells are functionally heterogeneous. Those produced at steady-state promote intestinal barrier homeostasis and defense 4 , whereas those elicited by IL-23 exposure are pathogenic, express IFNγ and GM-CSF, and exacerbate inflammatory disease 10,11,40,41 . Although all subsets are RORγt-dependent 16 , the TFs that distinguish the development of homeostatic vs. inflammatory Th17 cells are less defined. In this regard, JunB is a selective, obligatory regulator of inflammatory Th17 cell induction, but dispensable at steady-state. In contrast to Blimp-1 and RBPJ, which confer IL-23R-driven pathogenicity to Th17 cells 42,43 , JunB restrains pathogenic IFNγ and GM-CSF production, and supports the underlying Th17 cell program. However, the selective role of JunB during inflammation does not discriminate pathogenic vs. non-pathogenic Th17 cell differentiation. Rather, JunB is necessary in the context of IL-6. JunB is essential for Th17 cell induction in the IL-6-dependent inflammatory contexts of EAE and C. albicans infection 44,45 , but not for homeostatic IL-6-independent intestinal Th17 cell development 46 . In addition, JunB expression is upregulated by TCR activation in vitro, suggesting that the priming environment may drive unique inflammatory vs. homeostatic Th17 programs, whereby JunB differentially integrates into AP-1 complexes that program the regulatory landscape 14 .
JunB is required at distinct stages of inflammatory Th17 cell differentiation. It is necessary during initiation for robust induction of the Th17 cell effector program, and restraint of Th1 and Treg cell potentials. Thereafter, sustained JunB expression is essential to maintain identity and limit plasticity in differentiated Th17 cells. Examination of lineage conversion events in vivo were precluded by apoptosis following JunB withdrawal from Il17a-expressing cells. Therefore, once established, continued JunB activity supports Th17 cell viability in inflammatory environments. Notably, no CD4 + T-cell subset completely lacks JunB expression. Thus, whereas JunB deletion causes apoptosis, reduced levels of JunB, as observed in EAE for non-Th17 or ex-Th17 converted subsets, appear to be compatible with lineage conversion events. A similar modulation of JunB may contribute to Th17 cell transdifferentiation into pathogenic IFNγ + ex-Th17 cells during colitis, or into regulatory cells during resolution of inflammation 6,13 . Lastly, JunB is obligatory for the infiltration of inflammatory CD4 + T cells into the CNS during EAE, but not the skin in C. albicans infection, impling that JunB regulates a factor that mediates CNS-specific entry. Taken together, JunB plays multiple essential roles in CD4 + T cells.
AP-1 complexes are fundamental to Th17 cell identity. Like JunB, Fosl2 modulates plasticity in Th17 cells 14 . Analysis of the collaboration between JunB and Fosl2 revealed both concordant and discordant regulation of shared target genes, highlighting shared and unique roles in Th17 cells. Indeed, both TFs restrain Th1 and Treg potential, however, whereas JunB predominantly activates the Th17 program, Fosl2 represses many Th17-relevant genes 14 . Conversely, the highly coincident gene regulation by JunB and BATF identifies BATF as the dominant regulatory partner for JunB in Th17 cells. Further, JunB is integrated into a broad collaboration with pioneer TFs, BATF and IRF4 14 . This encompasses both coordinate activation of Th17 lineage genes and repression of alternative Th1/Treg programs. The latter implies an unappreciated role for BATF and IRF4 in limiting alternative CD4 + T-cell potentials, which is otherwise masked by their absolute requirement during Th17 cell differentiation 18,19 . Thus, it is tempting to speculate that BATF and IRF4 establish a general CD4 + T-cell enhancer  landscape downstream of the TCR that is poised to respond to inductive cytokine signals, and exploited by various AP-1 complexes to facilitate effector switches. Alterations in AP-1 complex composition coincide with CD4 + T-cell effector flexibility. In the absence of JunB, the binding of BATF, IRF4, and Fosl2 are largely unaltered at effector loci, whereas JunD binding is significantly elevated, suggesting that JunD replaces JunB in AP-1 complexes and alters regulatory dynamics. Accordingly, this coincides with a repression-to-activation switch at Th1-and Treg-promoting loci, IFNγ IL-17A   suggesting antagonistic roles for JunB and JunD, consistent with their regulation of Il4 expression in Th2 cells 47 . Moreover, TFs such as BACH2, the global repressor of CD4 + T-cell effector programs 48 , may collaborate with JunB in restricting alternative fates. Indeed, in CD8 + T cells, BACH2 impedes access of JunD to enhancers of terminal differentiation effector loci and prevents their aberrant activation 49 . A similar mechanism may regulate JunD function in Th17 cells, where extensive colocalization with p300 implies a dominant activating role for JunD. However, at Th17-relevant loci (e.g., Il17a), which are downregulated in JunB deficiency, JunD activity may be countered by the concomitant derepression of the Th17 cell negative regulator, IRF8 35 . The capacity of IRF8 to replace IRF4 in AP-1 complexes suggests a mechanism for the downregulation of Il17a and other Th17 cell-defining loci repressed by IRF8. Altogether, this supports the model that dynamic changes in AP-1 complex and co-regulator composition coordinates the loss of Th17 cell effector function with the acquisition of alternative fates during lineage conversion.
Silencers are conserved elements that permit contextdependent expression of target loci. Capitalizing on the role of JunB as a plasticity regulator, we identified novel silencers at Ifng, Foxp3, and Irf8 where negative regulation by JunB is central to maintenance of Th17 cell effector status. It is tempting to speculate that the coordinated loss of JunB activity at a network of such silencers facilitates transdifferentiation by simultaneously reversing the restriction of alternative potentials (e.g., via Foxp3, and Ifng) and releasing antagonists of Th17 cell identity (e.g., Irf8). Although there are examples of silencers that enforce long-term lineage identity (e.g., Cd4 50 , and Zbtb7b 51, 52 ), those elements that underlie plasticity by permitting flexible changes in expression between CD4 + T-cell states are undefined. An interesting candidate is Ifng CNS-18, implicated as an enhancer in Th1 cells 53 , and here, as a potent silencer in Th17 cells. Such dual-activity elements have been described 54,55 . Encompassing activating and repressive functions in single cis elements proposes a mechanism for efficient toggling between effector states in changing environments.
The mechanisms governing Th17 cell plasticity in immunity and autoimmunity are unclear. Here, we identify JunB as an obligatory regulator of Th17 cell stability that promotes the development of inflammatory Th17 cells and restricts flexibility towards alternative effector and regulatory programs. Targetted modulation of JunB to attenuate conversion to pathogenic Th17 cell subtypes, or rather, to promote transdifferentiation to antiinflammatory regulatory cells presents possible avenues for therapeutic intervention in disease.

Methods
Mice. Mice encoding a conditional Junb floxed allele were obtained from Erwin Wagner (CNIO, Spain), and backcrossed at least five generations onto the C57BL/6 background. Junb fl/fl mice were bred with CD4cre 29 and Il17a Cre deleter strains 29,11 , allowing for pan T-cell deletion or Th17 cell deletion, respectively (Jackson Laboratory). To enable tracking of Il17a Cre activity, mice encoding a loxP-STOP-loxP-ZsGreen conditional allele in the ubiquitous Rosa26 locus 32 (Jackson Laboratory), were bred to Junb fl/fl mice in order to obtain Junb fl/fl Il17a Cre+/− R26 ZS +/− mice. Junb fl/fl CD4cre mice were also mated with mice harboring a conditional Irf8 floxed allele, to generate Junb fl/fl Irf8 fl/+ CD4cre mice (Jackson Laboratory). For deletion in Treg cells, Junb fl/fl mice were bred with Foxp3 YFP-Cre mice (Jackson Laboratory). C57Bl/6 wild-type mice were obtained from Taconic Biosciences. Experimental mice were between 7 and 12 weeks of age, with no preference to gender. Sample size was chosen using Mead's equation to ensure adequate power. No blinding or randomization was used. Mice were maintained in specific-pathogen-free conditions at Duke University and all experiments were performed with permission from and in accordance with the guidelines of the Duke Institutional Animal Care and Use Committee.
Isolation of cells from tissues. Single cell suspensions of the thymus, spleen, and lymph nodes were obtained by mechanical disruption of the tissues through a 40 μm cell strainer (Falcon). To isolate leuckocytes from the small intestine lamina propria (SILP), the small intestine was harvested, fat removed, Peyer's patches excised, and luminal contents expelled. The intestine was cut into 1-2 cm pieces and shaken in PBS containing 1 mM DTT to remove mucus. The intestinal pieces were then transferred to PBS supplemented with 5 mM EDTA and 10 mM Hepes, and incubated twice at 37°C for 10 min with rotation, to remove the epithelial cell layer. After the second incubation, intestinal pieces were collected and washed with HBSS. Pieces were transferred to HBSS supplemented with collagenase D (1 mg/ml; Roche), DNaseI (0.1 mg/ml; Sigma), and dispase (0.1 U/ml; Worthington), and subsequently, minced with scissors and incubated in the digestion cocktail at 37°C for 25 min with rotation. The intestinal suspension was passed through a 100 μm cell strainer and a discontinuous 40:80 Percoll gradient used to further remove Error bars represent SEM of six mice in each cohort, from three independent experiments. c Flow cytometry of IL-17A, IFNγ, and Foxp3 expression in CD4 + CD45 + cells isolated from the cerebellum at day 18 post induction of EAE and restimulated ex vivo. Plots are representative of all mice analyzed in a. Total cell numbers of CD4 + CD45 + cells in the cerebellum and spinal column of WT CD4cre and KO CD4cre mice, as well as naive control, when WT CD4cre mice were at max score are shown. d Flow cytometry of cytokine production in WT CD4cre and KO CD4cre CD4 + TCRβ + cells from the draining lymph node of mice on day 6 post induction of EAE and restimulated ex vivo. Total cell numbers of CD4 + TCRβ + cells producing IL-17A, IFNγ, or GM-CSF are shown. ns, not significant. e Relative frequency of Foxp3 + CD4 + TCRβ + cells in the draining lymph node of WT CD4cre and KO CD4cre mice at day 6 post-induction of EAE relative to naive mice of the same genotype. f C. albicans. cutaneous infection model in WT CD4cre and KO CD4cre mice. Flow cytometry of cytokine production by ex vivo restimulated CD4 + CD45 + cells harvested from the skin 5 days post infection, as well as naive control. Data are compiled from three independent experiments. g Relative frequencies of Foxp3 + CD4 + CD45 + by ex vivo restimulated WT CD4cre and KO CD4cre cells harvested from the skin 5 days post infection, compared to their respective naive controls. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (unpaired two tailed Student's t-test)  Table 3. Sort-purified naive CD4 + T cells were cultured for 48 h in Th17 cell polarizing conditions (20 ng/ml IL-6 and 0.3 ng/ml TGFβ), and subsequently, harvested for electroporation. In short, 2.5−5 × 10 6 cells were incubated on ice in 500 μl RPMI with 2 μg pCMVRL and 10 μg pGL4 construct, empty or encoding a JunB-bound region. Cells were electroporated using a BioRad GenePulser II at 325 V and 960 μF, and subsequently, placed back on ice for 10 min to recover. Cells were transferred to prewarmed Th17 cell culture medium, and harvested 20 to 24 h later. Luciferase assays were performed using the Dual Luciferase Reporter Assay System (Promega; E1910). For each sample, firefly luciferase measurements were normalized to renilla luciferase readings and data presented as fold change over empty pGL4minP or CNS-5-pGL4minP, as appropriate.
Quantitative real-time PCR. RNA was extracted with Trizol, from sort-purified naive CD4 + T cells or naive CD4 + T cells cultured under Th0, Th17, iTreg, Th1, or Th2 cell conditions. The RNA was reverse transcribed using SSRTIV (Invitrogen; 18091050) according to the manufacturer's protocol. The resultant cDNA served as the template for amplification of genes of interest and housekeeping gene (Actb), by QPCR using iQ SYBR Green Supermix (Bio-Rad Laboratories). Primers used can be found in Supplementary Table 2. Relative expression was calculated using the ΔCT method.
EAE induction. To induce EAE, mice were injected subcutaneously at three locations on day 0 with 200 μg MOG 35-55 peptide (United Biosystems) emulsified in CFA supplemented with 2 mg/ml heat-killed Mycobacterium tuberculosis (VGD, Inc./Voigt Global). On days 0 and 2, mice were also injected intraperitoneally with 200 ng of pertussis toxin (List Biologicals). Mice were assessed daily for symptoms and scores were assigned as follows: 0, no symptoms; 0.5, limp tail tip; 1.0, limp tail; 1.5, quick recovery after inversion; 2.0, cannot recover after inversion; 2.5, one weak/partially paralyzed hind leg; 3.0, two weak/partially paralyzed hind legs; 3.5, complete hind and partial front paralysis; 4.0, complete hind and front paralysis. Mice that reached score 4.0 were deemed moribund and euthanized. Leukocytes from the cerebellum and spinal column of mice with a score of 2.5 to 3.5 were harvested for phenotyping by flow cytometry. Briefly, mice were perfused with PBS containing 5 mM EDTA. The cerebellum was removed and placed in PBS supplemented with 1% FBS and 1 mM Hepes (PFH). The spinal column was expelled with PFH, using a 21 G needle affixed to a 20 mL syringe, and also placed in PFH. Following mincing, samples were digested in PFH containing 10 mg/ml collagenase D for 30 min in a 37°C tissue culture incubator, and EDTA added to a final concentration of 12.5 mM to terminate the digestion. The digests were subsequently aspirated through a 3 ml syringe with 18 G needle and filtered through a 100 μm cell strainer. Cells were washed once with PFH, resuspended in 38% Percoll solution, and pelleted at 2000 r.p.m. for 30 min at RT with no brake. The cell pellet was washed once with PFH and resuspended in complete IMDM, followed by stimulation and flow cytrometic analysis as detailed above.
Cutaneous infection with Candida albicans. C. albicans strain SC5314 was kindly provided by the laboratory of Dr. Joseph Heitman. They were grown in YPD medium at 30°C overnight for yeast morphology, and the next day diluted to OD 600 of 0.6 and cultured until OD 600 reached 1.5 44 . The C. albicans was washed once with PBS, and resuspended to infect each mouse with 2 × 10 8 yeast in 50 μl PBS. An~2 cm 2 region of the ventral skin was plucked and scotch tape used to abraize the region. The 50 μl C. albicans suspension was subsequently applied to the skin. On day 5 post infection, mice were euthanized and leukocytes isolated from the infected skin for phenotyping by flow cytometry. Briefly, the infected skin was excised and minced in complete IMDM. The tissue was digested for 1 h at 37°C in complete IMDM supplemented with Collagenase D (1 mg/ml), dispase (5 U/ ml), and DNase I (0.1 mg/ml). The digests were subsequently filtered through a 100 μm cell strainer and the cells washed once with PBS. The cells were then resuspended in 38% Percoll solution, and pelleted at 2000 r.p.m. for 30 min at RT with no brake. The cell pellet was washed once with PBS and resuspended in complete IMDM, followed by stimulation and flow cytrometic analysis as detailed above.
Statistical analysis. A standard unpaired two tailed Student's t test was used, unless otherwise stated, to determine the statistical probability of the difference observed between two populations of cells in our phenotyping analyses (GraphPad Software): *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Parametric tests were used when variance between groups was similar, as determined by F-tests; however, when variance was significantly different non-parametric tests were performed.
Library preparation and sequencing. RNA was purified from Th cultures for RNA-Seq using the Trizol method and used directly for library preparation. For ChIP-Seq, the same general ChIP protocol (see above) was used with some modifications. Briefly, 4 × 10 7 Th17 cells were cross-linked and the resulting sonicate was subjected to immunoprecipitation using 10 μg of anti-JunB (N-17; sc-46x), anti-JunB (C-11; sc-8051x), or anti-JunD (329; sc-74x). Following reverse-crosslinking, ChIP and input samples were processed to obtain purified DNA as described 56 .
Raw material (RNA or DNA) was provided to the Duke Center for Genomic and Computational Biology for library preparation and sequencing. Sample