Identity crisis of Th17 cells: Many forms, many functions, many questions

Seminars in Immunology 25 (2013) 263–272

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Seminars in Immunology journal homepage: www.elsevier.com/locate/ysmim

Review

Identity crisis of Th17 cells: Many forms, many functions, many questions Mark S. Sundrud ∗ , Catherine Trivigno Department of Cancer Biology, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA

a r t i c l e Keywords: Th17 cells Plasticity IL-17 RORC ROR␥t CCR6 CD161

i n f o

a b s t r a c t Th17 cells are a subset of CD4+ effector T cells characterized by expression of the IL-17-family cytokines, IL-17A and IL-17F. Since their discovery nearly a decade ago, Th17 cells have been implicated in the regulation of dozens of immune-mediated inflammatory diseases and cancer. However, attempts to clarify the development and function of Th17 cells in human health and disease have generated as many questions as answers. On one hand, cytokine expression in Th17 cells appears to be remarkably dynamic and is subject to extensive regulation (both positive and negative) in tissue microenvironments. On the other hand, accumulating evidence suggests that the human Th17 subset is a heterogeneous population composed of several distinct pro- and anti-inflammatory subsets. Clearly, Th17 cells as originally conceived no longer neatly fit the long-standing paradigm of stable and irrepressible effector T cell function. Here we review current concepts surrounding human Th17 cells, with an emphasis on their plasticity, heterogeneity, and their many, tissue-specific functions. In spite of the challenges ahead, a comprehensive understanding of Th17 cells and their relationship to human disease is key to ongoing efforts to develop safer and more selective anti-inflammatory medicines. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Inflammation is the fundamental process of immune activation. Despite its “killer” reputation, inflammation is required for immune memory and vaccine responses, protection from infectious microorganisms, and even tissue maintenance and repair in the absence of pathogenic infection [1,2]. Of course, inflammation is also a common underlying pathophysiology observed in autoimmunity, cardiovascular disease, cancer, metabolic syndromes such as diabetes, and fibrosis [3–7]. A key determinant of whether or not inflammation turns pathologic is its ability to be resolved; in autoimmunity, for example, pathologic tissue damage is mediated by chronic, unresolved inflammation resulting from loss of immune tolerance to host tissues and T and B cell autoreactivity. On the other hand, it is also clear that not all inflammatory responses are the same. Qualitatively distinct inflammatory responses are coordinated at the level of antigen presenting cells (APCs) and carried out by CD4+ T helper (Th) cells, which differentiate from multipotent

Abbreviations: JAK, janus kinase; STAT, signal transducer activator of transcription; mTOR, mammalian target of rapamycin; GCN2, general controlled non-derepressible 2. ∗ Corresponding author at: Department of Cancer Biology, The Scripps Research Institute, 130 Scripps Way, #2C2, Jupiter, FL 33458, USA. Tel.: +1 561 228 2328; fax: +1 561 228 2331. E-mail address: [email protected] (M.S. Sundrud). 1044-5323/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.smim.2013.10.021

naïve precursor cells into a variety of phenotypically and functionally distinct effector subsets [8,9]. Cytokine production (i.e., effector function) by Th cell subsets, in turn, orchestrates various aspects of innate and adaptive immune function. Th cell differentiation is an instructive process that starts with the presentation of peptide antigens by APCs to T cells via MHC class II molecules. Cognate antigen recognition by naïve Th cell clones induces an elaborate network of activation signals through the T cell antigen receptor (TCR) complex, which combine with positive or negative co-stimulatory signals provided by major T cell co-stimulatory receptors (CD28, CTLA-4) and their ligands expressed on APCs (B7.1, B7.2) [10]. Whereas these signals initiate T cell activation and can influence effector T cell differentiation – depending on the strength and duration of TCR and co-stimulatory signals [11–13] – additional environmental cues, in the way of local cytokines, are required to specify T cell fate decisions. Cytokines bind to multimeric receptors expressed on Th cells, and regulate T cell differentiation via activation of JAK/STAT signaling pathways. STAT proteins reside in the cytoplasm of resting cells, and undergo rapid dimerization and nuclear translocation following their recruitment to activated (i.e., ligand-bound) cytokine receptors and phosphorylation by receptor-associated JAKs. Once in the nucleus, STAT proteins induce the expression of lineage-specifying transcription factors and coordinate downstream transcriptional programs that underlie Th cell differentiation. There are 7 mammalian STAT proteins (STAT1-4, STAT5a, STAT5b, STAT6), and all but STAT2 play essential roles in Th cell differentiation. A number

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of outstanding reviews are available elsewhere that detail JAK/STAT signaling pathways and their many functions during Th cell differentiation [14,15]. Th cell differentiation was long considered a binary process, leading to either IFN␥-producing Th1 cells or Th2 cells that secrete IL-4, IL-5 and IL-13. Whereas Th1 cells develop in an IL-12/STAT4and IFN␥/STAT1-dependent manner that requires activation of the “master” Th1 transcription factor T-bet (TBX21), Th2 differentiation requires IL-4/STAT6 and the Th2-specifying transcription factor, GATA-3 [16–19]. Th1 cells, by virtue of IFN␥ production, activate CD8+ cytotoxic T lymphocytes, NK cells, and phagocytes to regulate immunity against intracellular bacterial pathogens and viruses, whereas Th2 cells and their effector cytokines provide help to B cells, regulate class-switch antibody recombination, and recruit eosinophils for immune responses against many extracellular bacteria and large parasitic worms (i.e., helminthes) (reviewed in [20]). For nearly 20 years, the Th1/Th2 paradigm was thought to explain all aspects of immunity and inflammation. However, starting with the discovery of Th17 cells in the mid-2000s, the repertoire of effector T cell lineages has expanded dramatically, and now includes Th17 cells, induced T regulatory (iTreg) cells, T follicular helper (Tfh) cells, as well as Th22 and Th9 cells that express IL-22 and IL-9, respectively [9,21,22]. With the exception of Th17 cells, the regulation and in vivo functions of these new T cell subsets are only beginning to be unraveled. The concept of a distinct, pro-inflammatory, IL-17-producing effector T cell subset began with work from Sedgwick and colleagues [23], who showed that IL-23 and not IL-12 is the critical driver of autoimmunity in mice. IL-23 is an IL-12-related heterodimeric pro-inflammatory cytokine comprised of IL-12 p40 and the unique IL-23 p19 subunit [24,25]. Whereas mice lacking only IL-23 (p19−/− ) were completely protected from experimental autoimmune encephalomyelitis (EAE), animals specifically lacking IL-12 (p35−/− ) remained susceptible to disease, despite a defect in Th1 cell development [23]. IL-23 was found to induce IL-17Aexpressing effector T cells, and these cells were responsible for IL-23-mediated EAE [26,27]. However, because IL-23 did not induce IL-17A expression in naïve T cells in vitro, the notion of Th17 cells remained enigmatic. Shortly thereafter, pioneering work from the Littman, Stockinger, and Kuchroo, labs described the in vitro differentiation of IL-17-expressing Th17 cells, using combinations of IL-6, IL-21, and TGF␤ (i.e., TGF␤1) [28–32]. Because TGF␤ had previously only been considered an anti-inflammatory cytokine that regulated, among other things, development of FOXP3-expressing iTreg cells [33], Weaver et al., thus coined Th17 cells, “an effector lineage with regulatory ties” [34]. Whereas TGF␤ alone induces FOXP3, the combination of TGF␤ and IL-6 induces Th17 cell development via STAT3-dependent induction of the Th17-specific orphan nuclear receptor, ROR␥t (RORC in humans) [31,35,36]. In turn, STAT3 and ROR␥t synergize with other, general transcription factors, including the AP-1 family member BATF, IRF4, NF␬B, and NFAT to regulate transcription and chromatin remodeling at the IL17A/IL17F locus [37]. In addition to IL17A/F, Th17 cells express a cast of pro-inflammatory cytokines, such as IL-22, TNF␣ and GM-CSF and, importantly, they selectively express the IL-23 receptor (IL-23R) [38]. In this way, IL-23 regulates the growth and inflammatory function of Th17 cells following their initial differentiation (discussed below). Indeed, many more molecules and pathways have since been defined that both enhance and inhibit the development of Th17 cells, and those are reviewed elsewhere [38,39]. Here, we focus on the regulation of Th17 cell effector function and plasticity post-initial differentiation, particularly as it relates to inflammation in human autoimmunity and cancer. Unlike inbred mice housed in barrier facilities, humans have a large, heterogeneous, and highly variable (person-to-person) endogenous effector/memory T cell compartment. Thus, the

immune system as seen in human peripheral blood affords a unique view into the natural diversity of T cell effector lineages and provides important and complimentary information to that derived from artificial in vitro T cell culture systems and experimental animal models of autoimmunity. Among the most important insights coming from the study of endogenous human effector/memory T cells is that effector T cell lineages co-express unique lymphocyte homing receptors together with lineage-defining cytokines and lineage-specifying transcription factors [40–44]. For example, IFN␥-producing Th1 cells are highly enriched within human memory cells that express the chemokine receptor CXCR3 [40,43]. Th2 cells not only express IL-4, IL-5, and IL-13; they also express the chemokine receptor CCR4 and the chemotactic receptor of prostaglandin D2, CRTH2 [40,43,45]. Th17 cells uniformly express the inflammatory chemokine receptor CCR6 [46,47], though these cells are further heterogenous and can express additional chemokine receptors in combination (see below). Co-regulation of effector cytokines and lymphoid homing receptors confers tissue-specific T cell effector function. For example, tissues produce a variety of chemokines, both at steady-state and upon infection, stress, or damage. Chemokine receptors thus serve to direct T cell traffic into organs and tissues where their effector cytokines are needed for immune responses against specific microbes or for tissue maintenance and repair (reviewed in [48]). Like most aspects of the immune system, chemokines are also a double-edged sword that can contribute to chronic and autoimmune inflammation. The molecular link between effector cytokines and lymphoid-homing receptors comes by virtue of lineage-specific transcription factors (e.g., T-bet, GATA3, ROR␥t), which activate chemokine receptor gene expression in a subset-specific manner; forced expression of T-bet, GATA-3, ROR␥t in cultured human naïve T cells is sufficient to induce CXCR3, CCR4/CRTH2, and CCR6 expression in Th1, Th2, and Th17 cells, respectively [49–51]. 2. Phenotypes of human Th17 cells All human peripheral blood memory (CD45RO+ ) T cells that express IL-17A following ex vivo stimulation are CCR6+ [46,47]. However, as noted above, CCR6+ human memory T cells are also highly heterogeneous in their expression of other chemokine receptors and cytokines. Two major subsets of endogenous human Th17 cells have been described; CCR6+ CCR4+ cells, which produce IL-17A but not IFN␥; and CCR6+ CXCR3+ cells that can express IL-17A, either alone or together with IFN␥ [46,47,52,53]. Both of these subsets can also express the killer cell lectin-like receptor B1 (KLRB1; a.k.a. CD161) and IL-1R1 [54–56]. The CCR6+ CCR4+ Th17 cell phenotype is shared with Th22 cells, which express IL-22 but not IL-17A [52,53,57]. However, Th22 cells are discriminated from CCR6+ CCR4+ Th17 cells on the basis of CCR10 expression, where CCR6+ CCR4+ CCR10+ Th22 cells also express cutaneous lymphocyte antigen (CLA) and are enriched in the skin [52,53,57]. IL-22 is important for the maintenance of epithelial barrier function in the skin and gut at steady-state [58–60]. As noted above, these distinct chemokine receptor expression profiles of human Th17 subsets (e.g., CCR4 versus CXCR3) provides them with access to distinct sets of tissues. 2.1. Instability of IL-17A The conventional view of effector T cells, based largely on Th1 and Th2 cells, posits that cytokine production by effector T cells is stable following differentiation; stable expression of effector cytokines involves chromatin remodeling, where activating posttranslational modifications (e.g., H3K4tri-me ) are placed on histone

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tails to increase transcription factor occupancy at regulatory DNA elements and repressive chromatin modifications (e.g., H3K27tri-me , H3K9tri-me ) are removed (reviewed in [61,62]). Th17 cells have challenged this notion of T cell stability in recent years. In both mouse and human T cells, IL-17A expression induced during in vitro Th17 polarization is labile, and is readily silenced following subsequent restimulation and expansion in vitro. Although the Il17a/Il17f locus in in vitro-polarized Th17 cells and ex vivo-isolated IL-17A+ Th17 cells in mice is remodeled into a predominantly active state, as seen by high H3K4tri-me and low H3K27tri-me , Tbx21 (T-bet) is maintain in a “bivalent” state in Th17 cells, displaying both H3K4tri-me and H3K27tri-me modifications [63–65]. Th17 cells thus readily express T-bet upon secondary activation, which is further enhanced by IL-12, and this results in rapid acquisition of a Th1 phenotype in vitro [63–65]. Consistent with these data, in vitro-polarized Th17 cells transferred into lymphopenic recipients downregulate IL-17A expression, ultimately adopting an IFN␥+ Th1 phenotype [66,67]. More recent experiments using Th17 “fate-mapping reporter” mice have shown that a large portion of Th17 cells that have expressed IL-17A or IL-17F at some point during development, give rise to IL-17A/F− progeny cells [68,69]. In experimental autoimmune encephalomyelitis (EAE), for example, inflamed CNS tissue is infiltrated by a large number of “ex-Th17” cells that phenotypically resemble Th1 cells [69], whereas Th17 cells in the absence of overt inflammation home to Peyer’s patches in the gut and develop into Tfh cells that express IL-21 but not IL-17A that are ultimately important for B cell IgA responses [70]. Contrary to the notion that IL-17A expression is inherently transient in Th17 cells, Levings and colleagues presented evidence that the IL17A/IL17F locus is epigenetically stable in endogenous human CCR6+ CCR4+ CD161+ Th17 memory cells. Even when human Th17 memory cells are exposed to Th1-polarizing cytokines and induce expression of IFN␥, this does not coincide with epigenetic repression of the IL17A/IL17F locus [71]. These results argue that Th17 cells remain capable of expressing IL-17A/F, even in microenvironments that do not favor active/acute expression. In support of this notion, we have shown that Th17 cytokine expression (i.e., IL-17A, IL-17F, IL-22) can be rapidly activated in CCR6+ , but not CCR6− , human memory T cells simply by stimulation with homeostatic cytokines such as IL-2, IL-7, IL-15 [72]. Boniface et al. similarly showed that IL-17A− IFN␥+ effector T cells generated during in vitro Th17 polarization retain capacity to express IL-17A following subsequent expansion and restimulation [73]. Cytokine-driven activation of Th17 cytokine expression involves common gamma (␥c ) chain signaling through PI-3K and AKT, and downstream inhibition of the transcription factors FOXO1 and KLF4; ectopic expression of FOXO1 and KLF4 inhibits Th17 cytokine expression by CCR6+ memory T cells, whereas shRNA-mediated silencing of these factors leads to increased IL-17A, IL-17F, and IL-22 expression [72]. It is unclear whether these transcription factors directly bind and regulate transcription at the IL17A/IL17F locus, or if this regulation is indirect. More recent data from the mouse indicate that Foxo1 also represses Il23r expression in Th17 cells; activation of the Sgk1 kinase via increased sodium chloride exposure blocks Foxo1 transcriptional repression resulting in increased Il23r expression and pathogenic Th17 cell function in vivo [74]. Cytokine-dependent activation of Th17 cytokine expression in human CCR6+ memory T cells does not result from selective outgrowth of stable IL-17A-producing cells, but rather involves cell-intrinsic activation of IL-17A expression in “poised” Th17 cells, which express CCR6 and RORC but lack IL17A expression ex vivo [72]. Collectively, these data illustrate that Th17 cytokine expression is subject to extensive regulation in Th17 cells post-lineage commitment. Further, these findings suggest that plasticity in the Th17 system can work both ways, to enhance or inhibit IL-17A/IL-17F/IL-22 expression, depending on cues in the local microenvironment.

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2.2. Stable features of the Th17 lineage Although Th17 cells are classically defined by expression of IL17A following acute stimulation, these cells express a variety of other molecules that distinguish them from other effector T cell lineages. A number of Th17-associated molecules are stably expressed in effector/memory T cells even if IL-17A expression itself is downregulated. As noted above, CCR6 expression broadly defines human memory T cells that have the capacity to express IL-17A, even if they do not express IL-17A ex vivo and even if they have a conventional Th1 cytokine phenotype (IL-17A− IFN␥+ ). The capacity of CCR6+ IL-17A− cells to rapidly activate Th17 cytokine expression is associated with stable and high-level RORC expression, is independent of their ex vivo cytokine profile (e.g., IFN␥− , IFN␥+ ), and is present in both CD161+ and CD161− CCR6+ subsets [72]. In addition to CCR6 and RORC, ex vivo CCR6+ memory T cells (and in vitro-polarized Th17 cells) express CCL20 independent of IL-17A [72,73]. CCL20 is a Th17-associated chemokine that mediates Th17 tissue homing via its cognate receptor, CCR6. It is unclear if CCL20 is also expressed by CCR6− CD161+ IFN␥+ “non-classic” (a.k.a. Th17derived) Th1 cells found in inflamed tissue. Th17-derived Th1 cells are thought to be the human counterpart of murine Th1 cells that develop in vivo from in vitro-generated Th17 cells. Unlike “classic” Th1 cells that develop directly from naïve T cells, Th1 cells resulting from Th17 cell maturation or differentiation in inflamed tissues are thought to possess marked pro-inflammatory functions. Th17-derived Th1 cells are discriminated from classic Th1 cells by expression of CD161, as noted above, and these cells also retain expression of IL23R, IL1R1, and IL17RE [75–78]. Both classic Th1 cells and Th17-derived Th1 cells lack surface CCR6 expression and display low levels of RORC mRNA relative to Th17 and Th17/Th1 cells [75–78]. 2.3. The Th17–Th1 “phenotypic drift” Evidence from both human and mouse model systems suggest a model wherein Th17 cells, at least as defined by IL-17A expression, are not an end stage of effector/memory T cell differentiation. Rather, as noted above, a substantial proportion of human Th17 cells acquire Th1-associated features, including expression of IFN␥, CXCR3, and T-bet. At least 3 distinctive effector/memory T cell subsets have been suggested to represent various stages of a Th17–Th1 “phenotypic drift” [75,79]. First are CCR6+ IL-17A+ cells that lack Th1-associated gene expression by-and-large. A second, hybrid Th17/Th1 subset has been described in both healthy donor peripheral blood and clinically inflamed tissue; these cells are defined by surface co-expression of CCR6 and CXCR3, and can produce IL-17A alone, IFN␥ alone, or IL-17A and IFN␥ together upon stimulation. Th17-derived Th1 cells, discussed above, are thought to represent the final stage of the Th17-Th1 differentiation pathway; molecules shared by all three subsets include CD161, IL-23R, and IL-1R1 [75–78]. The notion that Th17 cells are precursors that differentiate into Th1-like effector progeny in a progressive, linear fashion in inflamed tissues is suggested by ratio of these subsets found in autoimmune target organs; Th17-derived Th1 cells (as well as classic Th1 cells) constitute the majority of tissue-infiltrating CD4+ T cells in the joints of RA patients [76,77], affected gut of Crohn’s disease (CD) patients [55,80], and cerebrospinal fluid of multiple sclerosis (MS) patients [81]. Th17/Th1 cells are generally present in these tissues in lower numbers than Th17-derived Th1 cells, though Th17/Th1 cells are strongly enriched within inflamed tissues relative to the peripheral blood. IL-17A+ IFN␥− Th17 cells, by contrast, are generally observed in RA, CD, and MS patient tissues at similar levels to Th17/Th1 cells, though Th17 cell enrichment in inflamed tissue versus peripheral blood

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Fig. 1. Differentiation versus selection underlying Th17 plasticity in chronically inflamed tissues. (A) Local, progressive, and linear differentiation of IL-17A+ IFN␥− Th17 “precursor” cells into non-classic (NC) Th1 cells in inflamed tissue. Distinct Th17 and Th1 subsets extravasate from blood into tissue. Inflammatory cytokines (IL-12, IL-23, IL-1␤, TNF␣) from local monocytes, macrophages, and dendritic cells promote gradual acquisition Th1-associated gene expression by Th17 cells. During their differentiation, Th17 cells first pass through an intermediate Th17/Th1 stage, and progress to the terminal NC Th1 stage, which is marked by loss canonical Th17 cell molecules, most notably IL-17A, ROR␥t, and CCR6. However, NC Th1 cells remain distinguishable from “classic” Th1 cells (Th1 – light blue cells) based on residual expression of CD161, IL-1R1, and IL-23R expression. (B) The selection model involves tissue infiltration of distinct and stable Th17, Th17/Th1, non-classic (NC) Th1, and classic Th1 subsets from the blood. Th17/Th1 and NC Th1 cells preferentially home to inflamed tissue versus Th17 cells, due to co-expression of CCR6 and CXCR3. Chronic inflammation and constitutive expression of inflammatory cytokines (IL-12, IL-23, IL-1␤, TNF␣) by local monocytes, macrophages, and dendritic cells drive further expansion and survival of Th17/Th1, NC Th1, and classic Th1 cells, whereas Th17 cells fail to expand or undergo apoptosis. In both models, chronic inflammation manifests as reduced numbers of IL-17A+ Th17 cells and increased levels of Th17/Th1, NC Th1, and classic Th1 cells in tissues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

is not on par with that of Th17/Th1 cells. These findings also suggest that Th17–Th1 conversion may occur locally in autoimmune target tissues. Because availability of normal human tissue is limiting, it is unclear whether all, or even most, Th17 cells at some point end up in non-lymphoid tissues as Th1-like cells, or if this conversion is a unique feature linked to chronic inflammation/autoimmunity. As discussed above and below, Th17 to Th17/Th1 conversion is readily induced in vitro by Th17 cell stimulation with IL-12, IL-23, IL-1␤, and TNF␣ [55,76–78,80]. Further differentiation of Th17/Th1 cells into CCR6− RORC− Th17-derived Th1 cells has not yet been demonstrated in vitro. An alternative/complimentary explanation exists for the altered distribution of Th17, Th17/Th1, and Th17-derived Th1 cells observed in the blood and inflamed tissues of autoimmune patients; this posits that Th17, Th17/Th1, and Th17-derived Th1 cells are distinct, parallel, and stable lineages of effector/memory T cells, and that each subset persists at different proportions in blood versus inflamed tissue. Enrichment for Th17/Th1 and Th17-derived Th1 cells in inflamed tissues over Th17 cells, for example, could involve preferential tissue homing to the gut, joints and CNS due to CXCR3 expression. In addition, Th17/Th1 and Th17-derived Th1 cells may also possess unique, cell-intrinsic survival mechanisms vis-à-vis Th17 cells that allows these subsets to preferentially survive within the hostile environment of chronically inflamed tissues [82,83]. Supporting such a “selection model” is the fact that all IL-17+ IFN␥− Th17

cells are prevalent within the highly differentiated/mature CCR710 TEM compartment of healthy donor peripheral blood [84]. Further, enrichment of Th17/Th1 and Th17-derived Th1 cells over Th17 cells appears to be tissue-specific. Whereas Th17/Th1 and Th17-derived Th1 cell enrichment is well-established in inflamed gut and synovial tissue [55,76,77,80], numerous reports in both human patients and autoimmune mouse models indicate that IL17A-expressing Th17 cells are elevated in chronically inflamed skin (psoriasis) [85,86] and lung (asthma) [87–90]. Ultimately, it is likely that both selection and cell-intrinsic signaling mechanisms contribute to the apparent plasticity of Th17 cells during chronic inflammation (Fig. 1). More recent data suggest that Th17 cell plasticity may not be limited solely to acquisition of Th1 effector functions. For example, small subsets of CCR6+ and CD161+ cells expressing both IL-17A and IL-4 (“Th17/Th2” cells) have been reported in the circulating blood of asthma patients and in inflamed lungs of allergic mice [91,92]. In addition, it is well-established that Th17 cells, particularly in the gut, can acquire stable FOXP3 expression and acquire potent immunoregulatory functions [93]. Finally, and as noted above, endogenous Th17 cells from Th17 “fate-mapping reporter” mice can seed Tfh cells in Peyer’s patches [70]. Indeed, it will be key in the coming years to determine the extent to which Th17 cells represent a broader, dynamic state of effector/memory T cell development that is specialized in acquiring additional lineage- and tissue-specific effector functions.

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3. Functions of human Th17 cells

3.2. Th17 plasticity in cancer immunosurveillance

The functions of Th17 cells are as poorly understood as they are important. However, after nearly a decade of research, several general principals have begun to emerge. In this section we discuss the consequences of Th17 cell plasticity in autoimmunity and cancer.

As in autoimmune target tissues, plasticity of Th17 cells within tumor microenvironments can influence the course of malignant disease. Whereas solid tumors are highly immunogenic and initiate chronic inflammatory responses, prevailing immunologic conditions within the tumor microenvironment tend to be immunoregulatory in nature and favor tumor growth [108]. Tumor-induced immune suppression is achieved by recruitment of FOXP3+ Treg cells, myeloid-derived suppressor cells (MDSC), and anti-inflammatory non-classical (i.e., M2) macrophages [109–111]. These cells restrain inflammatory CD4+ Th cell activation and CD8+ cytolytic T lymphocyte (CTL) responses against tumors, and their numbers at tumor sites are inversely correlated with disease prognosis. In contrast, infiltration of CTL and natural killer (NK) cells is mostly associated with improved prognosis in a variety of cancers [112–114]. Because CTL and NK cells are mobilized and activated by IFN␥, tumor infiltration of Th1 and Th1-like Th subsets also decreases tumor burden in animal model experiments [115]. Two clinical approaches to boost immune-mediated tumor killing have shown promise in trials of patients with advanced solid tumors. Cancer immunotherapy targets Treg-mediated immune suppression and inhibitory co-stimulation by APCs [116]; two modalities, anti-CTLA-4 and anti-PD-L1 antibodies, have provided key proof-of-concept data in human cancer patients [117,118], and anti-CTLA-4 has now been approved for the treatment of advanced melanoma. Similarly, adoptive cell therapy (ACT) is the process by which autologous CTLs are expanded in vitro and infused back into cancer patients (reviewed in [119]). ACT has been shown to induce durable remission in a subset of melanoma patients with advanced metastatic disease [120,121], though antigen specificity, local immune suppression, and limited cell engraftment remain as hurdles in ACT [122]. Tumor-infiltrating Th17 cells in tumor-bearing mice and human cancer patients come in several varieties. IL-17A+ FOXP3+ “regulatory” Th17 cells are commonly found in association with gastric tumors and these cells promote tumor development [123,124]. Conventional (IL-17A+ FOXP3− IFN␥− ) Th17 cells seem to play tissue-specific roles in cancer, either enhancing or impairing antitumor immunity [125]. In some cancers, IL-17A acts directly on the tumor to activate oncogenic signaling pathways and induce tumor growth, increase angiogenesis, and even promote drug-resistance [126]. In addition, IL-17A fosters tumor development in lung cancer via recruitment of macrophages that undergo local differentiation into M2 macrophages [127]. In contrast to these Th17 subsets that favor tumor growth, the same Th17/Th1 hybrid effector T cells that are highly pathogenic in the context of autoimmunity mediate protective anti-tumor immunity. Kryczek et al. reported no differences in the level of circulating Th17 cells between ovarian cancer patients and healthy donors but found higher proportions of Th17 cells within the tumor microenvironment [128]. Many of these tumor-infiltrating Th17 cells were Th17/Th1 cells, they expressed IL-17A, IFNy, and TNF␣, and their numbers at the tumor site were predictive of improved patient outcome [128]. Whereas IL-17A alone can favor tumor growth, IL17A and IFNy cooperatively induce CXCL9 and CXCL10 expression in tumor-associated macrophages (TAMs), which in turn recruit NK cells and CTLs [128]. Although it is not clear whether development and tumor infiltration by Th17/Th1 cells is associated with improved prognosis in all cancers, similar correlations have been reported in many; including lung cancer [129], gastric adenocarcinoma [130], and prostate cancer [131]. The mechanisms regulating Th17 cell plasticity in various tumor microenvironments are not well understood, though Th17/Th1 cell development, at least within cervical tumors, is influenced by IL-1␤ and IL-23 [128].

3.1. Th17 plasticity in autoimmunity That Th17 cell plasticity contributes to autoimmune pathogenesis is suggested by data from human clinical trials as well as experimental models of autoimmunity. In mice, Il17a is largely dispensable for pathogenic Th17 cell function in vivo – for example in experimental autoimmune encephalomyelitis (EAE) and T cell-induced colitis [94–96]. Further, IL-17A expression by groups of Th17 cells polarized in vitro using different cytokine combinations does not discriminate between Th17 subsets that have or lack pathogenic activity in vivo [97,98]. Rather, “pathogenic” Th17 cells that induce autoimmunity in mice are discriminated from “non-pathogenic” Th17 cells by virtue of a unique transcriptional profile, which includes a number of genes also expressed in classic Th1 cells (e.g., Tbx21, Stat4, Gzmb) [97]. There are conflicting reports as to whether or not T-bet, and other canonical Th1-associated molecules are required for Th17-driven autoimmune pathology [99–103]. In human clinical trials, anti-IL-17 monoclonal antibody (␣-IL-17 mAb) therapy shows variable and mostly modest effects in autoimmune patients, suggesting that the function of IL-17A in autoimmune disease is context- or tissue-dependent. For example, whereas ␣-IL-17 mAb treatment shows marked benefit in psoriasis, it shows more modest effects in autoimmune uveitis and rheumatoid arthritis, and it actually exacerbates disease symptoms in patients with Crohn’s disease [104,105]. The efficacy of ␣-IL-17 mAb in psoriasis is consistent with the high levels of IL17A observed in affected skin of patients [85], though IL-17A in inflamed skin is produced by both Th17 cells and skin-resident ␥␦ T cells [106]. In contrast, and as mentioned above, IL-17A+ IFN␥− Th17 cells are a smaller constituent of tissueinfiltrating CD4+ T cells in Crohn’s disease and RA [55,76,77,80]. In these instances, Th17 cells are present alongside larger numbers of Th17/Th1, Th17-derived Th1, and classic Th1 cells; many, if not most IL-17A-producing Th17 cells in these tissues also express IFN␥. The distinct phenotypes of Th17 cells in psoriatic skin versus inflamed gut of Crohn’s disease patients or inflamed joints of RA patients likely involves both differential recruitment of distinct circulating Th17 subsets as well as local conditioning of Th17 cells in the tissue. Related to cytokine-driven Th17 conditioning in tissues, one recent report showed that Th17 cells isolated from healthy donor peripheral blood acquire a Th1 phenotype in vitro upon exposure to inflamed synovial fluid from juvenile idiopathic arthritis (JIA) patients [76]. Further, Taams and colleagues have shown that monocytes recovered from synovial fluid or synovial membranes of RA patients are superior to those from patient-matched peripheral blood in inducing expression of both IL-17A and IFN␥ in T cells [107]. In both instances, IL-1␤ and TNF appear to play important roles in regulating local Th17 plasticity in the inflamed joint. Mechanistically, it is unclear whether such Th17 plasticity is due primarily to: (1) cell-intrinsic activation of Th1 transcription factors and cytokines in Th17 cells, or (2) selective survival/expansion of Th17/Th1 versus Th17 cells. In mice, IL-23 is required for acquisition of Th1-associated gene expression in Th17 cells during neuroinflammation and colitis [27,69,103].

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In tumor-bearing mice, in vitro-generated Th17 cells are superior to Th1 cells in controlling tumor burden in vivo [82,83,115]. Here, as with pathogenic Th17 cell function in some experimental models of autoimmunity, potent anti-tumor activity in Th17 cells requires the Th1-associated transcription factor Tbx21 (T-bet), and is associated with acquisition of Th1-like effector function [83]. Interestingly, the transcriptional signature of Th17 cells that control tumor burden in mice shows overlap with that of pathogenic Th17 cells that efficiently induce EAE [83,97]. Another hallmark of Th17 cells in mice and humans is their unique, stem cell-like features, which include self-renewal, the ability to give rise to multiple distinct effector subsets (e.g., Th17 cells, Th17/Th1 cells, Th17-derived Th1 cells), and their expression of early progenitor cell-associated transcription factors, such as Tcf7 and Lef1 [82,83]. Although it is unclear how these “stem-ness” qualities of Th17 cells contribute to pathogenesis in autoimmunity, protective anti-tumor immunity, and overall Th17 cell plasticity, it is reasonable to assume that there is more to Th17 cell function than simply expression of one or two cytokines. Further, if Th17 cells do represent a pluripotent CD4+ memory T cell compartment, these cells are almost certainly important for long-term T cell memory and beneficial vaccine responses. 4. Regulation of human Th17 cells Much has been learned about how Th17 cells develop from naïve precursor cells. In contrast, the molecules and signaling pathways that regulate Th17 cell effector function and plasticity are only beginning to be understood. As noted throughout, cytokines play a major role in the regulation of Th17 cells, both in quiescent and inflamed tissues. However, Th17 cells also appear to be uniquely sensitive (versus other effector T cell lineages) to fluctuations in metabolic parameters. In some instances, cytokines directly activate core metabolic signaling pathways; in other cases, unrelated metabolic inputs, such as insulin, oxygen, and amino acids indirectly influence cytokine signaling to control Th17 cell growth and effector function. Because of their selective regulatory impacts on Th17 cells, these “metabolic checkpoints” hold exciting therapeutic promise in the regulation of local inflammatory Th17 cell function in tissues. 4.1. Cytokines in control of Th17 cell plasticity The IL-12-related pro-inflammatory cytokine, IL-23, is arguably the most important feature underlying pro-inflammatory Th17 cell function. First, genome-wide association (GWA) studies have linked polymorphisms in IL23R to several human autoimmune diseases [132–134]. Second, whereas murine Th17 cells deficient in Il17a or Il17f display little-to-no loss in in vivo inflammatory function, IL-23/IL-23R are absolutely required for pathogenic Th17 function in mice [94,96,97,135]. The IL-23 receptor (IL23R) is not expressed on naïve T cells but is instead induced during Th17 cell development [31,136]. Thus, IL-23 selectively regulates the growth and function of mature Th17 cell subsets. IL-23 is produced by activated dendritic cells (DCs) and macrophages, and is predominantly found in inflamed tissues [23,137]. IL-23 predominantly activates STAT3 [138,139], and drives both Th17 cell proliferation and inflammatory cytokine expression. In both mice and humans, IL-23 activates canonical Th17 cytokines (e.g., IL-17A, IL-17F, IL-22), though a number of studies indicate that IL-23 also drives development of Th17/Th1 cells that express IL-17A and IFN␥ together [55,77,103]. Further, IL-23 promotes expression of the non-canonical Th17 cytokine, GM-CSF, and this has been shown to be important in EAE pathogenesis [140,141]. It is unclear whether IL-23 directly activates IFN␥ expression in Th17 cells or whether apparent enrichment

of Th17/Th1 cells by IL-23 is due to selective expansion/survival within inflamed tissues. At least in mice, Il23r expression is elevated on subsets of Th17 cells that also have Th1 characteristics and display pathogenic functions in vivo, for example in EAE [93,97,98]. IL-1␤ is another major regulator of Th17 cells in inflamed tissues. The impact of IL-1␤ stimulation on Th17 cell growth and inflammatory effector function is often synergistic with IL23. As noted above, Th17, Th17/Th1, and Th17-derived Th1 cells all express IL-1R1 [56,69,78]. Further, IL-1␤ represses antiinflammatory cytokine expression, namely IL-10, in human Th17 cells activated by whole pathogens [142]. Here, IL-1␤ concomitantly inhibits IL-10 and enhances IFN␥ in IL-17A-expressing Th17 cells. As with IL-23, it is again unclear whether IL-1␤ regulates Th17 cell plasticity via subset-selective expansion or through cell-intrinsic regulation of cytokine gene expression. Additional cytokines implicated in the regulation of Th17 cell plasticity in inflamed tissues are IL-12, IL-18, and TNF␣. Although IL-12 and IL18 are generally regarded as Th1-polarizing cytokines, more recent data indicates that both can also impact Th17 cell function. As noted above, Th17 cells maintain expression of a functional IL-12R, which activates T-bet and IFN␥ expression via STAT4 [64,76,78]; the role of IL-18 is less clear, though Th17 cells can express IL-18R, and, at least in mice, IL-18 synergizes with IL-23 to induce IL-17A and IFN␥ in Th17 cells and IL-17A-expressing ␥␦ T cells [143]. More recent evidence in Sjogren syndrome patients suggests that increased IL18 levels in inflamed salivary glands drives expression of IL-17A and IFN␥ [144]. The role of TNF␣ in facilitating Th17 plasticity during chronic inflammation has been shown both in vitro and through ex vivo analysis of autoimmune patient cohorts treated with anti-TNF mAb’s [80,107,145] 4.2. Metabolic regulation of Th17 cell plasticity That Th17 cells are tightly regulated by cell metabolism should be evident by the fact that STAT3 is an essential Th17 transcriptional regulator. In addition to directly binding and trans-activating IL17A/IL17F gene expression, STAT3 is a growth factor that directly regulates the expression of hundreds of genes associated with cell proliferation and cell survival [35]. STAT3 is also an established oncoprotein, particularly in B cell lymphomas, where constitutive STAT3 activation is a common feature of transformed B cells [146]. Further, intriguing studies by Levy and colleagues have shown that STAT3 has non-transcriptional functions in regulating cell metabolism and transformation [147]. Here, STAT3 localizes to the mitochondria of cells independent its DNA-binding domain, and induces a metabolic switch from oxidative phosphorylation to aerobic glycolysis (i.e., Warburg effect). The STAT3-dependent glycolytic program in tumor cells requires hypoxia-inducible factor (HIF1␣) and coincides with repression of mitochondria function, which typically mediates oxidation phosphorylation [148]. Although these metabolic functions of STAT3 in Th17 cells is unclear, it is tempting to speculate that the pro-glycolytic function of STAT3 could serve to protect Th17 subsets from apoptosis in hypoxic tissues during chronic inflammatory responses. One fact that has become clear in Th17 cells is that they are highly glycolytic vis-à-vis other effector and regulatory T cell lineages, and HIF1␣ regulates both Th17 cell development as well as the previously mentioned stem cell-like features of Th17 cells [82,149,150]. Consistent with their pro-glycolytic nature, Th17 cells express high levels of glucose receptor GLUT1, which is induced cooperatively via HIF1␣ and mTOR, whereas treatment of 2-deoxyglucose (2-DG) limits inflammatory pathology in mice with EAE [151]. mTOR is a key nutrient sensing kinase that coordinates cell growth and metabolism in response to growth factors, insulin, and amino acids; mTOR functions via two discreet protein complexes, termed mTOR complex 1 and complex 2 (i.e., mTORC1,

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mTORC2). mTORC1 is activated by PI-3K and AKT, which inhibit the tuberous sclerosis (TSC) 1/2 complex and activate the small GTPase, Rheb. In turn, activated mTORC1 drives protein synthesis in cells via phosphorylation-dependent activation of p70-S6 kinase (S6K1) and ribosomal S6 (rS6) and phosphorylation-dependent inhibition of eIF4E-BP1/2, which are inhibitors of the translation initiation factor eIF4E [152]. The mTORC1 pathway is essential for the development of Th17 cells, as has now been established through studies of a variety of mice conditionally lacking components of the mTORC1 pathway in CD4+ T cells [153–155]. Although the function of mTORC1 in controlling Th17 effector function and plasticity in clinical inflammation has yet to be directly evaluated, several anecdotal lines of evidence suggest it is important. First, IL-1␤ promotes differentiation/expansion of Th17/Th1 cells in chronically inflamed tissues and this cytokine signals, in part, through mTORC1 [156]. Second, the homeostatic cytokines IL-7 and IL-15 activate Th17 cytokine expression and expand Th17/Th1 cells in a manner that is sensitive to treatment with PI-3K and AKT inhibitors [72]. Third, low-dose rapamycin attenuates preestablished autoimmune disease in mice, and reduces Th17 and Th1 cell numbers in autoimmune target organs [157,158]. Collectively, these data call for further investigation into the role of mTOR and the broader mTORC1 pathway in the local regulation of Th17 cell function and plasticity during chronic inflammatory diseases. mTORC1 activity is dominantly regulated by amino acid availability, where local amino acid starvation resulting from amino acid consumption or amino acid degradation blocks mTORC1 activation, even in the presence of insulin and other growth factors [152]. Indeed, amino acid deprivation selectively blocks Th17 cell development, and these effects can be mimicked, not only by rapamycin, but also by small molecule inhibitors of tRNA synthetases, such as the plant natural product derivative and prolyl-tRNA synthetase inhibitor, halofuginone (HF) [159–161]. Halofuginone treatment activates a cellular stress response pathway known as the amino acid starvation response (AAR), which, unlike mTORC1, is directly regulated by the status of tRNA amino-acylation (i.e., charging) [159,161,162]. Here HF-induced uncharged prolyl-tRNAs leads to activation of the protein kinase GCN2, phosphorylation and inhibition of the translation initiation factor, eIF2, and translational upregulation of the stress-induced transcription factor ATF4. HF treatment selectively inhibits Th17 cell development, impairs IL23-induced IL-17A expression in human memory T cells, reduces STAT3 activation, and blocks Th17-associated immune pathology in mice [161]. Although it remains unclear to what extent amino acid availability is regulated and can become limiting in chronically inflamed tissues, consumption by activated immune cells with high protein synthesis requirements could conceivably lead to such a phenomenon. Regardless of the extent to which these and other metabolic fluctuations occur physiologically during chronic inflammation, the STAT3, HIF1␣, mTORC1, and AAR pathways offer attractive therapeutic opportunities to regulate Th17 cell function and plasticity in the context of chronic and autoimmune inflammatory diseases.

5. Concluding remarks Th17 cells play major roles in the development and persistence of inflammatory pathologies. Yet while substantial gains have been made in our understanding of Th17 cell function, many more questions are now evident. As discussed here, Th17 cells come in many forms, display highly dynamic functions, and appear uniquely adept at taking on additional effector functions as dictated by inflammatory tissue microenvironments. In order to gain a truly holistic appreciation for all the various disease- and tissue-specific functions of Th17 cells, we submit that parallel work in both human

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