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(Compl)Ex-Th17–Treg cell inter-relationship

Codependent development and Th17-to-FoxP3+ T cell inter-conversion account for the enigmatic coexistence of IL17-producing and FoxP3+ cells in tumor-associated inflammation. In addition to Treg cells, exTh17–FoxP3+ cells present a novel subpopulation of FoxP3+ cells. Yin-yang of IL17+ and FoxP3+ cells presents an important principle for improved approaches in cancer immunotherapy.


Introduction
While Th17 and regulatory T (T reg ) cells functions per se are important to maintain the homeostasis, Th17/T reg -governed sterile (para-)inflammation (a response defined by Medzhitov 1 ) is a corollary of immune responses against self-/neo-antigens and Th17-T reg imbalance has a crucial role in the induction and maintenance of sterile chronic inflammation, 2 associated with cancer. The development of Th17 and T reg cells demonstrate common characteristics. Herein, we discuss the newly identified immunological mechanisms involved in the induction of Th17 cells 3 and the development of converted ex-Th17 IL17 ¡ FoxP3 C T cells 4 in sterile inflammation. As a novel subpopulation of forkhead box P3 (FoxP3 C ) cells, ex-Th17-FoxP3 C cells present a paradigm shift with important consequences for tumor initiation, progression and novel therapeutic approaches targeting mutual Th17-T reg pathways of tumor immune surveillance.

Developmental Co-dependence of Th17-T reg Cells
Differentiation of Th17 cells proceeds through a developmental pathway partially shared with the anti-inflammatory FoxP3 C T reg population. This developmental mechanism can present itself in the perplexing co-existence of both populations. Marks et al. demonstrated that the environment may directly regulate the balance between the development of natural T reg and Th17 subsets from a common precursor and implied that T reg enrichment may be a necessary component preceding natural Th17 development. 5 By T reg cell depletion prior to in vivo Th17 priming, resulting in a reduced frequency of antigen-specific IL17 producers and reduced inflammatory skin responses, Chen et al. demonstrated that FoxP3 C T reg cells promote Th17 cell development in vivo, and identified IL2 (single l) signaling to play an important role. 6 Similarly, Pandiyan et al. observed enhanced IL2dependent induction of Th17 cell differentiation of na€ ıve CD4 C cells in the presence of T reg cells in vivo. 7

T reg Lineage is Stable
Genetic fate mapping and adoptive T cell transfer approaches led to the notion that the sustained expression of lineagespecifying transcription factors under control of inherent signaling pathways is likely a common feature of late T reg and Th17 cell differentiation, 8 required for the maintenance of a given cell identity in the progeny of dividing differentiated cells. 9 Rubtsov et al. have shown that T reg cell lineage is remarkably stable under physiologic conditions and following a variety of challenges, 10 where stable FoxP3 expression in committed T reg cells is likely facilitated by a positive autoregulatory loop 11 and continuous self-renewal of the established T reg cell population combined Keywords: myeloid-derived suppressor cells, regulatory T cells, sterile inflammation, T cell plasticity, Th17 cells Abbreviations: COX, cyclooxygenase; FoxP3, forkhead box P3; HIF-1a, hypoxia inducible factor 1 a; IDO, indoleamine 2,3-dioxygenase; IRF, interferon regulatory factor; MDSC, myeloidderived suppressor cell; MMF, mycophenolate mofetil; MSC, mesenchymal stem cell; mTOR, mammalian target of rapamycin; NO, nitric oxide; NOS, nitric oxide synthase; PGE 2 , prostaglandin E 2 ; PPARg, nuclear receptor proliferator-activated receptor gamma; RA, retinoic acid; RheumA, rheumatoid arthritis; Rorc/ RORgt, retinoid-related orphan receptor gamma t; T reg , regulatory T cell.
with heritable maintenance of FoxP3 expression serves as a major mechanism of maintenance of this lineage. Miyao et al. confirmed that T reg cells constitute a stable cell lineage and identified a minor population of non-regulatory FoxP3 C conventional T cells exhibiting promiscuous and transient FoxP3 expression, which give rise to FoxP3 ¡ cells and selectively accumulate in inflammatory milieus. 12 Plasticity is an Integral Part of FoxP3 C and IL17 C T Cell Biology While committed T reg cells are a stable population, ex-FoxP3 IL17A C cells selectively accumulating in inflammatory milieus reveal the emergence of a plastic and conversion-prone minority within the FoxP3 C population. [13][14][15] IL17 C FoxP3 C pathogenic cells that can arise in conditions of disrupted immune homeostasis, present a new possibility to restore the balance: rather than focusing on the biology of the differentiated populations, the relevant targets of future clinical interventions could well be the mechanisms regulating plastic subsets.
In line with T reg lineage stability, Kryczek et al. reported that only a population of CCR6 C memory FoxP3 ¡ and CCR6 C FoxP3 C T cells are preferential precursors of IL17 C FoxP3 C16 . Similar data by Mercer et al. substantiate the selective differentiation of IL17 C FoxP3 C T cells from lineage-committed naive CCR6 C FoxP3 C precursors. 17 Opposite, Th17 cells can convert to IL17 C FoxP3 C cells and ex-Th17-FoxP3 C cells. Whether IL17 C FoxP3 C cells represent a stable lineage or a transient state remains to be determined.
Bona fide Th17-T reg transcription factors integrate the functional phenotype of both lineages. While FoxP3 determines the suppressive potential, retinoid-related orphan receptor gamma t (RORgt) instructs the inflammatory phenotype. Cells harboring a FoxP3 reporter null allele exhibit some of the characteristics of FoxP3 C T reg cells, but are devoid of suppressor activity and also produce IL17. This phenomenon demonstrates the essential purpose of FoxP3 in T reg cell regulatory function (i.e. in the stable T reg cells and the plastic subsets), but not its suggested requirement in initiating T reg cell lineage commitment. 18 FoxP3-mediated repression of IL17 is likely due to a modulation of transcriptional activity of RORgt through a direct interaction. 19 Ablation of the RORgt gene in FoxP3 C cells stabilizes T reg anti-inflammatory functions, suppresses inflammation, and improves immune surveillance. 14 Further, signal transducer and activator of transcription (Stat) 3 is a transcription factor activated in both, Th17 and T reg cells, and is required for Th17 induction, while it interacts with FoxP3 in T reg cells, limits the expression of soluble mediators of Th17 differentiation, and endows T reg cells with the ability to suppress Th17 responses. 20 Lineage specifying cytokine signaling induces a specific metabolic signature of differentiated T cells (extensively reviewed by Pearce et al. 21 ). Metabolic signals (i.e., nutrient, energy, oxygen availability, and stress level) are integrated by mammalian target of rapamycin (mTOR), hypoxia inducible factor 1 a (HIF1a), and AMPactivated kinase (AMPK) and allow for the fine-tuning of the Th17-T reg cell balance (reviewed by Barbi et al. 22 ). That metabolites can help shape Th cell fate was recently demonstrated by Smith et al., showing that short-chain fatty acids regulate colonic T reg cell homeostasis. 23 With Th17-T reg inter-conversion, Th cells display a high degree of metabolic plasticity and can adapt their metabolism to support proliferation and viability.
Visceral adipose tissue FoxP3 C CD4 C T reg have further revealed an additional association between metabolic regulation and Th17-T reg polarization. Nuclear receptor proliferator-activated receptor gamma (PPARg) is a key negative regulator of human and mouse Th17 differentiation 24 and a crucial molecular orchestrator of visceral adipose tissue T reg cell accumulation, phenotype, and function. 25 A recent report by Carbo et al. demonstrates that PPARg activation promotes a phenotype switch from Th17-to-T reg cells and can thereby alleviate colitis. 26 Th17-to-T reg conversion only occurs in lean fat tissue and allows for insulin sensitivity and homeostasis, while Th17 accumulation in adipose tissue is associated with increased insulin resistance. 27 Factors with Dual T reg -and Th17-Promoting Functions Apart from developmental codependence and Th17-FoxP3 C inter-conversion, an additional level of complexity in discriminating Th17 from T reg lineages is their dependence on the same differentiation factors. Here, the same signal may have either pro-T reg or pro-Th17 driving functions depending on the inflammatory micromilieu. PGE 2 , an essential homeostatic factor and a mediator of chronic inflammation, has been shown to exert pro-Th17 effects and expand Th17 cells through both, enhanced macrophage and DC production of IL23 and IL1b and its direct action on Th17 cells. 28 On the contrary, PGE 2 produced by tumor cells induces the expansion of type-1 T reg cells and induces their endogenous expression of cyclooxygenase (COX2)/ PGE 2 . 29 TGFb also drives both, T reg and Th17 cell differentiation in mice, while its role in human Th17 cell differentiation is controversial. While the induction of FoxP3 upon chronic antigen exposure in vivo requires TGFbR signaling, 31 TGFb as well contributes to mouse, 32-36 although not human 37,38 Th17 cell differentiation. TGFb drives T reg and Th17 cell differentiation through the repression of Gfi-1, a transcriptional repressor that inhibits the differentiation of both iT reg and Th17 cells. 39 It is the IL2, required for TGFbmediated induction of FoxP3 in peripheral T cells in vitro 40 that opposes differentiation of activated CD4 C T cells into Th17 cells. 41 Whereas the latter differentiation pathway is favored when T cell receptor and TGFbR activation in naive CD4 C T cells coincides with IL6R stimulation. 42 Interestingly, recent results indicate that only in the presence of TGFb increased salt (NaCl) conditions in combination with IL1b, IL6, and IL23 drive autoimmune disease by the induction of pathogenic Th17 cells. 30 Inherent iNOS expression in CD4 C T cells limits T reg induction by repressing TGFb 43 . This negative regulation of TGFb in human and mouse CD4 C T cells may explain the conflicting data on iNOS-mediated regulation of Th17 differentiation: whereas the NO/iNOS-cGMP-cGK pathway induces Th17 cell differentiation in naive and memory human CD4 C T cells, 3 murine T cellderived iNOS plays a negative role in the regulation of Th17 cell differentiation. 44

MDSCs -Central Regulators of Th17-T reg Cell Balance
Myeloid-derived suppressor cells (MDSCs) are a cell type that promote T reg cell expansion and/or drive the differentiation of Th17 cells. 3,4 Most importantly, MDSCs promote IL17 C FoxP3 C CD4 C Th cell induction and can skew IL17 C FoxP3 ¡ CD4 C cells toward IL17 ¡ FoxP3 C CD4 C cells. 45 When immune homeostasis is disturbed in tissue malfunction, inflammation occurs through activating tissue-resident macrophages and/or recruiting additional myeloid cells. These in turn provoke expansion of MDSCs, which are associated with cancer, autoimmunity, and inflammation. MDSCs are thus a highly plastic key element of para-inflammation. The plasticity of MDSCs is displayed by their heterogeneity, and also helps to explain how various pathological settings can have similar biological effects on myeloid cells. PGE 2 has emerged as one key molecule in MDSC biology and appears to be the common denominator of diverse settings in the induction of MDSCs. 4,46 The most prominent cell type to induce MDSCs are cancerous cells. Additionally, bone marrow derived stromal cells, mesenchymal stem cells 4 or adipose cells 47 can also reprogram myeloid cells upon perturbed homeostasis (see Fig. 1). Ericksen et al. have shown that a sole subtle commensal stimulus suffices for sensitization and inflammation of adipose tissue that results in MDSCs mobilization, associated with increased Th17 responses and accelerated preneoplasia. 47 MDSCs inherently express factors and cytokines that instruct the development and plasticity of Th17 or T reg cells. The integration of factors produced under defined circumstances ultimately defines the fate of Th17 or T reg predominance. MDSCs spontaneously produce significant amounts of PGE 2 , IL1b, IL6, TGFb 1 , as well as arginase, indoleamine 2,3-dioxygenase (IDO), and Figure 1. Commensalism of Th17 and T reg cells in sterile inflammation is governed by MDSCs. In the absence of any inflammation, retinoic acid and TGFb prime CD11c C CD11b ¡ CD103 C DCs and macrophages to induce FoxP3 C T cells. On the other hand, murine lung and gut lamina propria CD11c C CD11b C CD103 ¡ interferon regulatory factor (IRF)-dependent DCs (equivalent to human IRF-dependent CD1c C DCs and inflammatory BDCA C CD16 ¡ DCs of cancer patients, and synovial fluid from rheumatoid arthritis patients) induce Th17 cells. MDSCs produce immunomodulatory factors that can induce both Th17 cells and T reg cells. This function depends on MDSC activation, and regulates the balance and plasticity of Th17-T reg cells. MDSC-primed na€ ıve CD4 C T cells can either differentiate into FoxP3 C cells or IL17-producing cells, and a fraction of the two can convert from one to the other through IL17 C FoxP3 C and/or double negative intermediates. COX2/PGE 2 feedback is central in controlling the biology of MDSCs and can lead to both Th17 or T reg cell dominance. While iNOS/NO induces human Th17 cell differentiation, IDO1 promotes T reg cell induction.
IL10, all of which are implicated in both the induction of Th17 and T reg cells. In addition, they produce IL23 and NO following CD40 stimulation. 3 The different activation status of MDSCs thereby explain the dual nature of MDSC-derived factors. IDO and iNOS both function as critical molecular "switches" regulating Th17-T reg balance. IDO maintains T reg cells in their normal, potently suppressive state, but when blocked, it allows for IL6mediated T reg cell conversion into a nonsuppressive, pro-inflammatory Th17 phenotype. 48 Contrarily, iNOS/NO from MDSCs induces Th17 responses 3 and its inhibition abrogates IL17 production and results in TGFb-mediated FoxP3 C T reg cell induction. 43 Clinical Picture of Th17-FoxP3 C T Cell Inter-conversion Cancer presents a state of chronic sterile inflammation, where the production of pro-inflammatory cytokines by FoxP3 C cells underlies a pathogenic phenotype of ex-FoxP3 C cells.
Despite a nominally immunosuppressive microenvironment, cancer presents a chronic inflammation infiltrated with high frequency of both Th17 and T reg cells. By virtue of their ability to control cancer-associated Th17 cell-mediated inflammation, FoxP3 ¡ and FoxP3 C T reg cells impede cancer progression in an IL10-dependent manner. 49 However, their ability to control inflammation is lost in the course of disease and T reg cells shift from a protective IL10-producing anti-inflammatory to a IL17-producing cancer-promoting pro-inflammatory phenotype, with preserved capacity to suppress protective antitumor immune responses. Blatner et al. found preferential expansion of FoxP3 C in human colon cancer that can suppress T cells but are not anti-inflammatory like classic T reg . These FoxP3 C RORgt C IL17-producing pathogenic T reg cells are directly associated with inflammation and influence disease outcome in colon cancer. 14 Ablating RORgt specifically in T reg stabilizes their anti-inflammatory properties and enhances polyp specific immunity. In analogy, Keerthivasan et al. showed that activation of Wnt/b-catenin signaling in both Th17 and T reg cells is associated with enhanced pro-inflammatory cytokines and correlates with the progression of colitis and colon cancer, which could be reversed in RORgt ¡/¡ mice. 50

Conclusions
Herein discussed data provide a rationale to target Th17-T reg commensalism (and not T reg cells alone) with novel interventional approaches (see Fig. 2). Combinatorial targeting of Th17-T reg interrelationship to promote IFNg-producing Th1/17 cells will allow for an enhanced antitumor immunity. 51 It will also be valuable to evaluate the Th17-T reg balance together with the plastic potential of Th17 and FoxP3 C cells as prognostic factors.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Figure 2. IL17 C FoxP3 C cells present a new therapeutic target in cancer. Simultaneous promotion of IL6 production with an IDO inhibitor and targeting of T reg cells with low-dose cyclophosphamide can increase intra-tumoral accumulation of CD8 C T cells and thereby enhance antitumor immunity. Another combinatorial Th17-T reg targeting approach of potential translational relevance may be RORgt inhibition with digoxin and enhancement of intra-tumoral IL12 concentrations to promote IFNg-producing Th1/17 cells. e1040217-4 Volume 5 Issue 1 OncoImmunology