Th9 cells, new players in adaptive immunity

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Th9 cells, new players in adaptive immunity Edgar Schmitt, Matthias Klein, and Tobias Bopp Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstraße 1, Building 708, 55131 Mainz, Germany

Upon antigen-specific stimulation, naı¨ve CD4+ T cells have the potential to differentiate into various T helper (Th) cell subsets. Earlier models of Th cell differentiation focused on IFN-g-producing Th1 cells and IL-4-secreting Th2 cells. The discovery of additional CD4+ Th cell subsets has extended our understanding of Th cell differentiation beyond this dichotomy. Among these is the recently described Th9 cell subset, which preferentially produces interleukin (IL)-9. Here, we review the latest developments in Th9 cell development and differentiation, focusing on contributing environmental signals, and discuss potential physiological and pathophysiological functions of these cells. We describe the challenges inherent to unambiguously defining roles for Th9 cells using the available experimental animal models, and suggest new experimental models to address these concerns. Distinct Th cell subsets control adaptive immune responses Adaptive immune responses are orchestrated by different Th cell subsets, mainly through secretion of lineage-specific cytokines. Initially, the Th1/Th2 concept defined two distinct Th cell subsets based on the secretion of interferon [(IFN)-g, Th1] and IL-4 (Th2) [1–3]. However, experimental data indicated that this model, based on two crossregulatory Th cell subtypes, was insufficient to explain multiple aspects of the initiation, regulation, and finetuning of diverse immune responses. In the past 10 years, three additional Th cell subtypes have been discovered and designated as Th17, Th9, and Th22 cells, according to the signature cytokine secreted by each subset after activation (Th-17: IL-17, Th9: IL-9, and Th22: IL-22) [4–7]. Two decades before the description of Th9 cells, it was reported that IL-9 production by CD4+ T cells depends on IL-2, is promoted by IL-4 and transforming growth factor (TGF)-b), and further enhanced by IL-1, while IFN-g represents a potent inhibitor of IL-9 expression [8,9]. These Th cells were defined as Th9 cells by Veldhoen et al. [7] and Dardalhon et al. [5], and subsequently, other Corresponding author: Bopp, T. ([email protected]). Keywords: T helper cell; interleukin-9; Th9; cytokine; lymphocyte differentiation; transcription factor; plasticity. 1471-4906/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2013.10.004

groups demonstrated that additional stimuli [IL-25, Jagged2, programmed cell death ligand (PD-L)2, cyclooxygenase (COX)-2, 1,25-dihydroxyvitamin D3, calcitonin gene-related peptide (CGRP), tumor necrosis factor receptor superfamily member 4 (TNFRSF4 or Ox40), and thymic stromal lymphopoietin (TSLP)] contribute to Th9 cell development [10–17]. In vitro culture of CD4+ T cells under Th9 cell-inducing conditions, namely in the presence of TGF-b and IL-4, was shown to result in a heterogeneous Th cell population capable of producing not only IL-9 after secondary stimulation, but also other cytokines, specifically IL-10, IL-17, IL-21, and IL-22 [18]. It is apparent that a complex network of signalling pathways orchestrates the development of Th9 cells, and this presents a challenge in terms of unambiguously defining functions for Th9 cells, as we discuss in more depth. Nevertheless, the recent description of human Th9 cells in various disease contexts emphasises the importance of unravelling the functions of this Th cell subtype in both health and disease [19–22]. Here, we review recent studies that have provided important insights into our understanding of Th9 cell development and function. Multiple sources of IL-9 A plethora of studies published within the past two decades demonstrated that IL-9 promotes the development of allergic and autoimmune diseases (reviewed by Stassen et al. [23]). Furthermore, it was shown that IL-9 is essential for the expulsion of certain intestinal nematodes (reviewed by Klementowicz et al. [24,25]). Many of these studies were conducted using multiple mouse models – IL-9-transgenic-, IL-9-deficient-, and IL-9-receptor-deficient mice – but occasionally led to contradictory results (reviewed by Stassen et al. [23]). One study showed that the induction of experimental autoimmune encephalomyelitis (EAE) was delayed and that the severity was reduced in IL-9-receptor-deficient mice, whereas another study reported opposite results. This discrepancy could be explained by a context-dependent regulation of the regulatory T cell/Th17 cell ratio in EAE by IL-9 as proposed by Elyaman and colleagues [12]. Similarly, although lung-specific expression of IL-9 was shown to result in severe asthma symptoms, IL-9-deficient mice developed full-blown disease, thus challenging an essential role for IL-9 in asthma. IL-9-induced asthma symptoms are in part mediated by IL-13, which can also be secreted by Th2 cells. Although this may partially explain the contradictory results reported, further studies will be required Trends in Immunology xx (2013) 1–8

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Review to understand fully the contribution of IL-9 to autoimmune and allergic diseases. Th2 cells were initially described as the main source of IL-9. However, it has been recently shown that IL-9 and IL4 are rarely produced by the same T cell, indicating that Th9 cells represent a discrete Th cell subset [7,19]. The physiological and pathophysiological role of Th9 cell-produced IL-9, however, remains unclear. Studies using in vitro differentiated Th17 cells and peripherally induced T regulatory (iTreg) cells reported that these cells were also capable of producing IL-9 (reviewed by Noelle et al. [26]). This scenario is further complicated by results showing that in a papain-provoked model of airway inflammation, innate lymphoid cells (ILCs) but not IL-9-producing CD4+ T cells represent the main source of IL-9 [27]. The authors used an Il9 fate reporter mouse model and discussed that a combination of IL-2 and IL-33 induced a transient production of IL-9 by ILCs. However, in a recent publication, Licona-Limo´n et al. generated IL-9-deficient and IL-9fluorescent reporter mice to visualize the real-time expression of the Il9 gene [79]. Infection of these mice with the parasitic worm Nippostrongylus brasiliensis demonstrated that next to ILCs, Th9 cells represent the major source of IL-9 in this mouse model. Using an adoptive transfer model the authors elegantly demonstrated that only Th9 cells are essential for rapid expulsion of these worms. Hence, depending on the experimental mouse model, IL-9 can be produced by a variety of cells, and Th9 cells represent an important source of this cytokine in vivo. It is likely that in vivo different cell types are capable of producing IL-9, and further studies with new experimental animal models will be required to define the contribution of IL-9 produced by Th9 cells in health and disease. For the purposes of this review, we mainly focus on studies that convincingly demonstrate IL-9 production by Th9 cells. Th9 cells in health and disease The two studies that originally described in vitro differentiation of Th9 cells in the presence of IL-4 and TGF-b provided only limited data concerning the in vivo relevance of these cells. Veldhoen et al. used mice with an impaired TGF-b responsiveness based on a dominant-negative expression of the TGF-b receptor type II under control of the CD4 promoter and used such mice in an experimental Trichuris muris model known to be dependent on IL-9 for the expulsion of this parasitic worm [7]. Reduced TGF-b signalling resulted in a high parasite burden in the absence of an intestinal mastocytosis, suggesting that impaired Th9 development affected the resolution of parasitic infection. However, the presence of IL-9-producing Th9 cells in the intestine and their frequency was not assessed. Using newly generated IL-9 fluorescent reporter mice, Licona-Limo´n et al. convincingly demonstrated the decisive function of Th9 cell-derived IL-9 for the rapid expulsion of the parasitic worm N. brasiliensis [79]. Dardalhon et al. showed that in vitro generated Th9 cells produce IL-10 in addition to IL-9, and hence, CD4+ T cells from IL-10 reporter mice were used to sort Th9 cells [5]. These cells were found to aggravate the colitis-inducing potency of naı¨ve CD4+ T cells when cotransferred into T cell-deficient mice, but the requirement for IL-9 was not 2

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assessed, by, for example, neutralizing IL-9 in vivo. An important caveat to interpretation of these results is that IL-10 can also be produced by CD4+FoxP3+ regulatory T cells, type 1 regulatory T cells (Tr1 cells), Th1, Th2, and Th17 cells, excluding its use as a functional marker for Th9 cells [28]. Studies in multiple mouse models indicate a role for Th9 cells in allergic pathologies. In an ovalbumin (OVA)-induced model of airway inflammation, neutralisation of IL-9 strongly reduced allergic symptoms, and a similar outcome was observed when mice with a T cell specific deficiency of the ETS-domain transcription factor PU.1 (encoded by the Sfpi1 gene) were used in this model of experimental asthma. PU.1 is required for Th9 cell development and IL-9 production, therefore, it was concluded that Th9 cell-derived IL-9 causes airway inflammation and expansion of inflammatory cells [20]. In support of this notion, transfer of OVA-specific Th9 cells into T cell-deficient mice led to strong asthmatic symptoms after challenge with OVA. These symptoms could be prevented by treatment with neutralising IL-9 antibodies, indicating that the transferred Th9 cells exhibited a stable IL-9-producing phenotype [19]. Exposure of mice to house dust mite results in the rapid accumulation of PU.1-positive, CD4+ T cells in the lung that produce IL-9 in the absence of IL-13, IL-10, and IFN-g [29]. Recently, the in vivo development of Th9 cells was studied in a chronic model of airway hyper-reactivity (AHR) induced by intranasal Aspergillus fumigatus lysates. Intracellular staining of CD4+CD44+ T cells isolated from the lungs of these mice identified IL-9producing Th9 cells that did not coexpress IL-4, IL-10, and IL-13 [13]. Neutralizing IL-9 reduced AHR symptoms, supporting a role for Th9 cell-derived IL-9 in mediating these pathologies. The function of Th9 cells has also been studied in the EAE mouse model for brain inflammation. Transfer of MOG35-55specific Th cells differentiated in vitro under Th9-promoting conditions led to the development of EAE. Upon recovery from the central nervous system (CNS) of these mice, the transferred cells were capable of producing IFN-g and IL-17, in addition to the cytokines that they had produced prior to transfer (IL-4, IL-10, and IL-9) [18]. Thus, it was not clear which of these cytokines, or which combination of cytokines, induced the EAE symptoms. However, the transfer of in vitro differentiated Th1 and Th17 cells resulted in different pathological phenotypes in this mouse model, suggesting a unique contribution of Th9 cells in EAE pathology. Mechanistically, IL-9 was shown to induce the expression of chemokine CC ligand (CCL)20 in astrocytes, thereby attracting Th17 cells into the CNS in the course of EAE [30]. Furthermore, in IFN-g-deficient mice, MOG35-55-induced EAE was severely exacerbated and could be ameliorated by blocking IL-9 [31]. These findings strongly argue for a disease-aggravating function of Th9 cell-derived IL-9 in EAE, especially in view of the fact that IFN-g impairs the development of Th9 cells [9]. Recent studies in mouse models of cancer suggest a protective function for IL-9. Purwar et al. showed that Th9 cells inhibited the growth of subcutaneous melanoma, and neutralisation of IL-9 led to accelerated tumour growth. Accordingly, administration of IL-9 inhibited tumour growth in a mast cell-dependent manner [32].

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Lu et al. demonstrated that Th9 cell-derived IL-9 inhibited tumour progression in a pulmonary melanoma model [33]. As in Purwar et al., neutralisation of IL-9 led to increased tumour growth. These authors concluded that the antitumour effect of IL-9 was chiefly based on a strong CD8+ cytotoxic T cell (CTL) response and a CCL20/chemokine CC receptor (CCR)6-mediated recruitment of dendritic cells (DCs) and cytotoxic CD8+ T cells into the tumour tissue. Human Th9 cells can be generated in vitro under conditions similar to those used for generating murine Th9 cells, and can be isolated ex vivo. Allergic patients have been shown to have increased numbers of Th9 cells among peripheral blood mononuclear cells (PBMCs) [29]. Similarly, the frequency of Th9 cells detected in tuberculous pleural effusion increases during the course of Mycobacterium tuberculosis infection [34]. By contrast, the frequency and the level of IL-9 production by Th9 cells were both reduced in melanoma biopsies, as compared to T cells isolated from skin of healthy donors, suggesting that an impaired function of Th9 cells favours tumour progression [32]. In summary, results from murine disease models and from human patients indicate that Th9 cells can play a protective (tumour) as well as a pathogenic (allergy, colitis, and EAE) role in different diseases (Figure 1). These findings emphasise the importance of defining the mechanisms underlying Th9 development and function, in both a

physiological and pathophysiological context. Increased understanding of these mechanisms could serve as a basis for improved therapeutic concepts in the future. Signals shaping the transcriptional network of Th9 cell development and function Deficiencies in the Il2 gene or components of the IL-4- or TGF-b-dependent signalling cascades impair the ability of CD4+ T cells to develop into Th9 cells, demonstrating the importance of transcriptional activation downstream of the IL-2 receptor (IL-2R), TGF-b receptors and the IL-4 receptor (IL-4R) [5,7,9,12,35]. It is clear that cytokinesignalling pathways do not function in isolation, but are instead parts of complex interacting networks. The contribution of these cytokine receptor signals to the developmental program that drives Th9 cell development is under extensive study. Below, we review signalling pathways leading to robust Il9 gene expression and the development of Th9 cells (Figure 2). T cell receptor (TCR)-derived signalling Antigen-specific activation of Th cells is an indispensable physiological prerequisite for IL-9 production by CD4+ T cells. TCR-mediated translocation of nuclear factor of activated T cells (NFAT) to the nucleus contributes to Il9 gene expression [17,36,37]. Among other transcription factors, TCR signalling facilitates the expression of the IFN regulatory factor (IRF)4 [38,39]. IRF4 is central to the

preTh IL-2, TGF-β IL-4, IL-1

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Figure 1. Physiological and pathophysiological functions of T helper (Th)9 cell-derived interleukin (IL)-9. The development of Th9 cells from naive precursor Th cells (preTh) depends on IL-2, is induced by transforming growth factor (TGF)-b in combination with IL-4, and enhanced by IL-1 [5,7–9,47]. Th9 cell-derived IL-9 was shown to favour allergic asthma, especially upon induction of IL-13 and eotaxin [13]. Experimental autoimmune encephalomyelitis (EAE) was found to be induced by MOG35-55-specific Th9 cells through IL-9-mediated recruitment of autoimmune Th17 cells [30,31]. A beneficial function was demonstrated for Th9 cell-derived IL-9, which inhibits the growth of melanoma cells in the presence of mast cells and/or via the recruitment of CD8+ T cells and dendritic cells (DCs) [32,33]. However, the survival of IL-9-receptor-positive EL4 thymoma cells was not impaired but rather supported [32]. A protective effect of an IL-9-induced mastocytosis against intestinal nematodes was demonstrated a decade ago and could be indirectly confirmed for Th9 cell-derived IL-9 by using mice with an impaired Th9 cell development that could not expel the parasitic worms [7].

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TGF-βRs NOTCH1/2 SMAD

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Figure 2. Transcriptional regulation of T helper (Th)9 cell development and function. The development of Th9 cells from naı¨ve CD4 T cells strictly depends on T cell receptor (TCR)-derived signals and interleukin (IL)-2 and is synergistically enhanced by a combination of transforming growth factor (TGF)-b, IL-4 [5,7,9], and IL-1 [8,47]. Thus, signals derived from the T cell receptor (TCR), IL-2 receptor (IL-2R), TGF-b receptors (TGF-bRs) and IL-4 receptor (IL-4R) also induce expression of the transcription factors interferon (IFN)-regulatory factor (IRF)4 and PU.1 in a nuclear factor of activated T cells (NFAT)- (TCR), nuclear factor (NF)-kB- (TCR), signal transducer and activator of transcription (STAT)5- (IL-2R), SMAD- (TGF-bRs), and STAT6-dependent (IL-4R) manner. Essentially, these transcription factors contribute to chromatin modifications at the Il9 locus and the initiation of Il9 expression [19,20,35]. In addition, downstream signals of the TCR leading to the expression of BATF–JUN family protein complexes may contribute to Il9 expression in a cooperative manner with IRF4 [42,78]. IFN-g strongly inhibits Il9 expression via STAT1 [9,31] whereas IL-1R1-, Notch1/2- or thymic stromal lymphopoietin receptor (TSLPR)-mediated signals further enhance IL-9 production by Th9 cells [8,10,12]. Abbreviations: BATF, basic leucine zipper transcription factor, ATFlike; GATA-3, GATA-binding protein 3; NICD, Notch intracellular domain, RBP-Jk, recombination signal binding protein for immunglobulin kJ region.

development and function of murine as well as human Th9 cells [19]. Detailed analyses, using chromatin immunoprecipitation and Il9 reporter gene studies revealed a direct binding to and transactivating activity of IRF4 on the Il9 promoter in developing murine Th9 cells. Furthermore, genetic ablation of the Irf4 gene strongly reduced Il9 expression and Th9 cell development. As a result, Irf4-deficient mice did not develop IL-9-dependent immunopathologies of asthma. IL-4R signalling was shown to facilitate Irf4 expression, as discussed further below [35]. Notably, IRF4 is also involved in the development of Th2 and Th17 cells [40,41]. IRF4 thus seems to regulate the development of different Th cell subsets in a concerted interaction with additional, subtype-specific transcription factors. Accordingly, it was recently demonstrated that IRF4 cooperates with activator protein (AP)1 complexes, binding to so-called AP1–IRF composite elements in conjunction with BATFJUN (basic leucine zipper transcription factor, ATF-likeJUN) family protein complexes. This cooperative binding was shown to promote initial chromatin accessibility in 4

CD4+ T cells and to contribute to the transcriptional program leading to Th17 cell differentiation [42,43]. IL-4R-derived signalling Triggering of the IL-4Ra results in the activation of Janus kinase (JAK)1 and JAK3, recruitment and phosphorylation of signal transducer and activator of transcription (STAT)6 and the subsequent expression of genes that regulate Th2 cell development [44,45]. Notably, Stat6-deficient CD4+ T cells are limited in their ability to produce IL-9 upon stimulation under Th9 cell-skewing conditions [5,7,35]. Upon activation, phosphorylated STAT6 facilitates the transcription of Gata3 and Irf4; two transcription factors essential for the differentiation of both Th2 cells and Th9 cells [5,19,40,46]. However, constitutive expression of IRF4 and/or GATA-binding protein 3 (GATA-3) upon retroviral transduction into Stat6-deficient CD4+ T cells did not rescue IL-9 production, indicating the involvement of additional factors dependent on STAT6 activity in the regulation of Il9 expression [35].

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Review Adding to this complexity, two studies have shown that IL-4R-mediated signalling might be of negligible importance for Il9 expression under certain circumstances [9,47]. Strong evidence for IL-4-independent IL-9 production was provided by results showing robust induction of IL-9 upon TCR-mediated stimulation of Il4-deficient CD4+ T cells in the presence of TGF-b and a neutralising IFN-g antibody [9]. IFN-g is a potent inhibitor of Th9 cell development and function [9,31], thus, its neutralisation, or counterregulation by IL-4, as discussed below, might be essential for Th9 development. Recently, Dong and colleagues provided evidence for a pathway initiated by IL-4 that attenuates Th2 and Th9 cell development via negative feedback [48]. IL-4R signalling was shown to result in the expression of the cytokineinduced SH-2 protein (CIS), a member of the suppressor of cytokine signalling (SOCS) family of proteins, leading to the inhibition of STAT5 and STAT6 phosphorylation. Conditional genetic ablation of Cis in T cells resulted in an enhanced expression of IL-9 and IRF4, and greater susceptibility to development of experimental allergic asthma, demonstrating the physiological relevance of this regulatory circuit. In addition, several groups have demonstrated that Gata3 transcription can occur in a STAT6-dependent as well as STAT6-independent fashion [44,45,49]. Consistent with this, two recent reports suggested that Notchdependent signalling can regulate transcription of Gata3 in the absence of STAT6 [50,51]. Continuative research demonstrated that the Delta family of Notch ligands induces Th1 cell development, whereas Jagged promotes Th2 cell development in an IL-4- and STAT6-independent manner. Likewise, Elyaman et al. recently demonstrated that Notch1- and Notch2-deficient Th9 cells show decreased IL-9 production and that Jagged2 but not Deltalike 1 is able to induce IL-9 production in the presence of TGF-b alone [12]. Exogenous IL-4 compensates for Notch deficiency, therefore, it can be assumed that above described Notch-dependent expression of Gata3 is involved in the underlying molecular mechanism. Thus, the requirement for IL-4R signalling may not be absolute due to redundancy with Notch-mediated induction of GATA-3 early during Th9 differentiation. TGF-b-derived signalling TGF-b initiates expression of forkhead box (Fox)p3 in naı¨ve CD4+ T cells and is a key cytokine in the extrathymic development of iTreg cells [52]. Upon ligand binding, the TGF-b receptor complex phosphorylates specific members of the SMAD family of proteins. SMAD phosphorylation leads to their translocation to the nucleus and to the transcription of SMAD-dependent genes [53]. TGF-b-activated phosphoSMAD3 was shown to bind to the Il9 locus in a cooperative manner with the Notch intracellular domain (NICD) and RBP-Jk (recombination signal binding protein for immunglobulin kappa J region) [12]. CD4+ T cells expressing a dominant-negative TGF-b receptor II fail to develop into Th9 cells [7]. Detailed analyses revealed that TGF-b is required for Th9 development and function most probably by preventing expression of the transcription factor T-bet, and by inducing the expression of PU.1 [20,54]. Like IRF4,

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PU.1 promotes Th9 development [20]. In mice, PU.1 is encoded within the Sfpi1 locus and T cells deficient in Sfpi1 show diminished IL-9 production upon TCR stimulation in the presence of TGF-b and IL-4. Consequently, Sfpi1-deficient mice show reduced allergic airway inflammation. Ectopic expression of PU.1 not only represses Th2-associated cytokines, possibly by interfering with GATA-3 function [55], but also enhances IL-9 production in Th9 cells. Another function of PU.1 in the regulation of Il9 gene expression was suggested recently. Here, the authors demonstrated that Sfpi1-deficient T cells stimulated under Th9 skewing conditions showed decreased overall histone H3 acetylation at the Il9 locus [54]. Detailed analysis revealed that PU.1 can recruit the histone acetyltransferase (HAT) proteins Gcn5 [Kat2a, K(lysine) acetyltransferase 2A] and PCAF [Kat2b, K(lysine) acetyltransferase 2B], resulting in an open chromatin formation at the Il9 locus, thereby facilitating the binding of further transcription factors like IRF4 and enabling transcription of the Il9 gene. IL-2R-derived signalling Binding of IL-2 to the high-affinity receptor consisting of three subunits, IL-2Ra (CD25), IL-2Rb (CD122), and the common g-chain (CD132) promotes proliferation and differentiation of T cells [56]. IL-2 transmits signals to the nucleus in part via activation of STAT5, a feature that is shared with the cytokine TSLP [57]. In fact, TSLP was shown to promote IL-9 production by T cells and TSLP fostered allergic airway inflammation in an IL-9-dependent manner [10]. In contrast to TSLP, IL-2 is indispensable for IL-9 production by Th9 cells because it has been demonstrated that CD4+ T cells from IL-2-deficient mice cannot produce IL-9 in the absence of exogenous IL-2 [9]. This IL-2-dependency was recently further supported by analyses showing direct binding of STAT5 to the Il9 locus [48]. Conversely, IL-2 signalling via STAT5 constrains Th17 cell differentiation [58,59]. Thus, it is conceivable that early in the development of Th9 cells, IL-2-mediated STAT5 activation inhibits the development of Th17 cells while simultaneously promoting differentiation towards the Th9 cell phenotype. In parallel to STAT5 activation, IL-2 also activates the phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) pathways [60], leading to, among other activities, the triggering of the nuclear factor (NF)-kB pathway and the activation of downstream genetic programmes. A broad range of cytokines and some costimulatory molecules can induce translocation of NF-kB to the nucleus; IL-1a, IL-1b, IL-25, IL-33, and Ox40 have been shown to signal to the nucleus in a NF-kB-dependent manner and to enhance IL-9 production of Th9 cells [8,11,17,47,61,62]. However, IL-1b was shown additionally to enhance Th17 development, excluding a unique role for NF-kB in the transcriptional regulation of the Il9 gene in Th9 cells [63–65]. Co-stimulatory signals There is emerging evidence that strong co-stimulatory signals are important for efficient Th9 cell development and high IL-9 production. In addition to TCR-mediated signal transduction, co-stimulation via CD28 is required 5

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Review for PDK1 (pyruvate dehydrogenase kinase, isoenzyme 1)mediated activation of NF-kB and robust T cell activation [66]. Given the above outlined important role of NF-kB for Il9 gene expression, it is tempting to speculate that CD28derived signals enhance IL-9 production by Th9 cells. Indeed, crosslinking of CD28 results in strongly enhanced IL-9 production by Th9 cells [67]. Like CD28, triggering of Ox40 activates the NF-kB pathway, and Xiao et al. have recently demonstrated that Ox40-derived signalling favours the development of Th9 cells while restricting iTreg and Th17 cell development [17]. Importantly, costimulation via Ox40 selectively induces IL-9 production (and not the production of the Th2 cytokines IL-4 and IL-5), and results in the recruitment of TRAF6 (TNF receptor associated factor 6) and an enrichment of p52-RelB at the Il9 locus, revealing a role for the noncanonical NF-kB pathway to strengthen Il9 gene expression. It should be noted that contrary to Ox40, TCR-induced Il9 gene expression requires NF-kB activated by the canonical NF-kB pathway in cooperation with NFATc2 [37]. Jash et al. suggested that NFATc2 (also known as NFAT1 or NFATp) enables binding of a member of the NF-kB family of transcription factors p65 (RelA) to and transactivation of the Il9 locus by epigenetic regulation. Supporting this, siRNA-mediated ablation of p65 as well as Nfatc2 deficiency results in reduced IL-9 production by CD4+ T cells [37]. Another co-stimulus for IL-9 production that acts via translocation of NFATc2 is induced by CGRP [16]. Mechanistically, CGRP inhibits glycogen synthase kinase (GSK)3b; an enzyme known to phosphorylate NFATs and to promote their nuclear export [68]. In addition, CGRP enhances IL-9 production in a cAMP/protein kinase (PK)A-dependent manner and results in enhanced expression of Sfpi1 (PU.1) and Gata3. Next to this Th9-promoting effect, cAMP was shown also to have an effect of inhibitory potency on Th9 cell development. In this context, Li et al. showed that mice deficient in the prostaglandin synthase COX-2 show strongly enhanced Th9 cell numbers and higher IL-9 concentration in an OVA-induced allergic inflammation model [14]. Further analysis demonstrated that the prostaglandins PGD2 and PGE2, which elevate intracellular levels of cAMP, inhibit Il17rb expression, likely in a PKA-dependent manner. Notably, IL-17RB serves as the receptor for IL-25, a cytokine known to enhance IL-9 production [11]. Previously, IL-25 was also shown to promote Th2 responses [69]. In this context, Boonstra et al. recently demonstrated that 1a,25-dihydroxyvitamin D3 promotes the expression of Gata3 and enhances IL-4 production in CD4+ T cells, thus favouring Th2 cell development [70]. In addition, 1a,25-dihydroxyvitamin D3 was shown to inhibit NFATc2/AP1 complex formation and IL-2 production [71], and recently an inhibitory effect of 1a,25-dihydroxyvitamin D3 on IL-9 production by Th9 cells was demonstrated [15]. Thus, the regulatory role of 1a,25-dihydroxyvitamin D3 in IL-9 production needs to be further defined, and will likely be context specific. Recently, the co-inhibitory receptor PD-L2 was shown to regulate Th9 cell development negatively in vivo. Using an Aspergillus fumigatus-induced model of chronic AHR, Kerzerho et al. demonstrated that blocking of PD-L2 by a monoclonal antibody or genetic ablation of the gene 6

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encoding for PD-L2 results in increased numbers of IL-9-producing Th9 cells, whereas the number of IL-4producing Th2 cells was unaffected [13]. This increase in Th9 cells was associated with higher levels of IL-1a and TGF-b; cytokines known to promote IL-9 production by CD4+ T cells [8] or to be required for Th9 cell development [5,7,9,47]. Given the multiplicity of cytokines and co-stimulatory signals modulating Th9 cell differentiation, it is clear that the underlying transcriptional programme is the result of well-balanced and concerted interactions wherein signals downstream of the TCR, the high affinity IL-2R, and the TGF-b receptors play central roles that are further modulated by co-stimulatory receptors and by IL-4 and IL-1. As a consequence, this sophisticated developmental programme can be expected to favour a rather fragile stability of Th9 cell development prone to reprogramming towards other Th cell subtypes. Th9 cell paradigm: stability and plasticity The stability of the Th9 cell subset in vivo remains matter of debate. The publications that support relatively high plasticity in Th9 cells are mainly based on adoptive transfer of in vitro differentiated Th9 cells. In this regard, adoptive transfer of Th9 cells specific for MOG35-55 induced EAE symptoms concomitant with the ability to secrete IFN-g and IL-17 [18]. Another report demonstrated that re-stimulation of Th9 cells in vivo by exposure to their cognate antigen results in diminished IL-9 production [72]. In contrast, various studies have demonstrated that neutralisation of IL-9 abrogated the effects evoked by adoptively transferred Th9 cells, suggesting stability of the IL9-producing phenotype in distinct disease models and organs [19,32,33]. In these studies, the entirety of the in vitro differentiated Th cell population was adoptively transferred without certainty regarding the purity of the IL-9-producing Th9 cells. Furthermore, several cytokines including IFN-g, IL23, and IL-27 can dampen IL-9 production in vivo, and thereby diminish Th9 cell frequency [9,18,31,32,73]. Nevertheless, depending on multiple environmental signals including the local cytokine milieu present in particular disease models in vivo adoptively transferred Th9 cells

Box 1. Future Needs 1. Identification and characterisation of Th9 cells ex vivo with aid of an IL-9 reporter mouse. 2. Characterisation of chemokine receptors critical for Th9 cell homing and function in different experimental animal models such as asthma, parasitic worm infections, EAE, and melanoma. 3. Characterisation of human Th9 cells in asthma, parasitic worm infections, multiple sclerosis, and melanoma. 4. Analysis of the role of Th9 cell-derived IL-9 for anti-melanoma immune responses, asthma, EAE, and parasitic worm infections. 5. Comparison of the transcriptome of tumour-associated (melanoma) versus lung-associated (asthma) Th9 cells to identify unique transcriptional programmes. 6. Identification of factors driving stability and plasticity of Th9 cells in vivo. 7. Characterisation of transcription factors physically interacting with IRF4 to promote Th9 cell development.

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Review might, just as Th2 cells, be reprogrammed to improve and facilitate adaptive immunity [74]. Therefore, the use of the recently described IL-9 fluorescent reporter mouse [79] that directly visualises the real-time expression of the Il9 gene represents an inevitable prerequisite for future studies using in vitro generated Th9 cells (Box 1). Different from Il9 fate reporter mice described by Wilhelm et al. [27], these mice would enable real-time analyses of murine IL-9-producing Th9 cells in different disease models in vivo and they can serve as a source for the transfer of purified IL-9-producing Th9 cells to analyse their role, plasticity, and flexibility in vivo. Concluding remarks IL-9 was cloned and characterised more than two decades ago as a Th2 cell-derived cytokine. Subsequently, mast cells and eosinophils were also identified as sources of IL-9 and recently a new IL-9-producing Th cell subset – Th9 cells – has been defined. These Th cells are distinct from Th2 cells, and can be differentiated in vitro via TCR stimulation in the presence of TGF-b in combination with IL-4. This newly defined Th cell subset has been the subject of a multiplicity of studies, which have described additional regulators (IL-25, PD-L2, and COX-2) and basic transcriptional control mechanisms of Th9 cell development. The pathophysiological role of Th9 cell-derived IL-9 in allergic and autoimmune diseases has been demonstrated in vivo, as has its protective role in a N. brasiliensis infection model and in a melanoma tumour model. The isolation and characterisation of IL-9-producing murine and human CD4+ T cells directly ex vivo further corroborates that Th9 cells represent a separate Th cell subset with important physiological functions. Nevertheless, we are far from a comprehensive understanding of the transcriptional control of Th9 cell development and the molecular basis of the functional properties of this Th cell subset. The main reason for this is that in vitro generation of Th9 cells depends on a cocktail of cytokines (IL-2, IL-4, and TGF-b) resulting in the development of a heterogeneous population of Th cells. Hence, the use of the IL-9 reporter mouse, which was described recently by Licona-Limo´n et al. [79] will allow tracking and isolation of individual IL-9-producing Th9 cells (Box 1). Such Th9 cells will provide the basis for single cell genomic analyses by next-generation sequencing to identify subsetspecific transcription factors and miRNAs contributing to their development and function. In addition, they will allow profiling of individual cells by a combination of CyTOF-Analyses, a multi-parametric mass cytometry technology based on metal-conjugated antibodies [75] and ‘serial microengraving’ allowing for nondestructive kinetics studies of cytokine secretion by single T cells during sustained stimulation under Th9 cell-skewing conditions [76,77]. In addition to these sophisticated analyses, IL-9 reporter mice could be used in combination with different experimental in vivo disease models – chronic as well as acute – to elucidate the contribution of Th9 cellderived IL-9, and the role of IL-9 produced by other cell types such as mast cells, eosinophils, natural killer (NK)-T cells, and ILCs. These future studies should be further corroborated by studies in T and B cell-deficient (Rag1- or Rag2-deficient) and mast cell-deficient [Kit(W-sh)] mice, in

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which the consequences of IL-9 production by Th9 cells or mast cells could be directly examined by adoptive transfer of wildtype or Il9-deficient T cells and mast cells. Further understanding of Th9 cell development and function obtained through these types of approaches will aid in the design of therapeutic intervention targeted to pathologies that are affected, both positively and negatively, by Th9 cells. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft DFG, grant SFB TR128 (T.B.), grant DFG SCHM 1014/5-1, GRK 1043: International Graduate School of Immunotherapy (project C4; E.S. and T.B.), and the ‘‘Forschungszentrum Immunologie (FZI)’’ of the university medical center (E.S. and T.B.).

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