Home   User Center   Featured   Recent   Search   Submission   Manuscripts  

Journal of Nature and Science (JNSCI), Vol.1, No.5, e101, 2015
Abstract  Full Text (PDF)  Cite this article

Immunology

 

A Subset Apart? Th9 cell regulation and current limitations to study

 

Zihan Zheng*, Jiwon Kang

 

Department of Microbiology & Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA


Th9 cells are a CD4+ T helper subset that has garnered considerable attention recently, due to their ability to produce large amounts of the cytokine IL-9. Many functional and regulatory roles associated with them are currently not fully understood. In this paper, we attempt to study several of the distinguishing features of Th9 cells to lead to a firmer understanding of the subset and its role in immunity. We consider and discuss some of the current limitations to studying Th9s, as well other Th9 properties of interest, on the levels of Th9 stimulation/differentiation, transcription factor interplay/ internal protein regulation, and cytokine secretion. In particular, we highlight the role of some potential co-stimulatory factors, as well as the utility of confirming a lineage-defining transcription factor. Journal of Nature and Science, 1(5):e101, 2015

 

Th9 | IL-9 | T helper differentiation | lineage-defining transcription factor

 

Introduction

T helper cells play a critical role in mammalian adaptive immunity, with the capability of shaping the immune response in many ways. Through their recognition of antigens caught and processed by antigen-presenting dendritic cells and macrophages (APCs), T helper cells can stimulate the activation of B cells and subsequent antibody production. T helper cells may also recruit many other immune cells to specific sites of inflammation through the release of potent chemoattractants, and also release cytokines to regulate immune cell function. CD4+ T helper cells undergo differentiation in response to environmental signals, with 6 subtypes (Th1, Th2, Th17, Th9, Treg, Tfh) being currently recognized commonly, and some (most notably Th22, but also Tfr) posited.[1] Each of these subsets has important, non-redundant functions in immune responses.[2] One of these subsets is T helper 9 (Th9), a phenotype which is deregulated in several inflammatory diseases, but which also plays an important role in the clearance of certain pathogens. In this review, we discuss in detail this phenotype’s differences from other T helper phenotypes on several different levels, as well as potential information that may permit us to more fully understand the phenotype under scrutiny.

 

Th9 Stimulation

One of the key aspects of study regarding T helper cells is the manner by which they are induced, and consequently the scenarios in which they may arise. Understanding the cytokines necessary for stimulating differentiation can also provide vital information for conducting useful in vitro experiments, and give hints as to the signal transduction networks that are critically involved in the cell type. Generally however, each cell type can be stimulated from a variety of different conditions, making such analysis difficult. For instance, Th17 cells are commonly induced by a combination of tumor growth factor-β (TGF-β) and interleukin-6 (IL-6).[3,4,5] Cells induced in such a manner are positive for the “lineage-defining” transcription factor retinoic acid-related orphan receptor gamma t (RORγt), and secrete substantial amounts of IL-17.[6,7,8] However, RORγt+  IL-17 producing  cells may also be induced without TGF-β, in the presence of  IL-6, IL-23, and IL-1b.[9,10,11] Interestingly, cells induced in the latter fashion may also be more pathogenic than those stimulated in the classical manner.[12] Similarly, Th9 cells are also subject to distinct differentiation conditions that may have important ramifications for their effects. Typically differentiated using a combination of IL-4 and TGF-β, Th9 cells have also been shown to favorably arise in the presence of IL-21, IL-6, and IL-21.[13,14,15,16] No clear consensus differences have currently been found recognized between these alternate polarizations. As such, it seems clear that a confluence of different factors may drive differentiation, and that the differentiation can follow from the effect of distinct pathways. The involvement of TGF- β is also additionally interesting due to the known role of Th9 cells in combatting helminthes. One paper has suggested that helminthes may secrete a compound that can mimic the effect of TGF- β to suppress T effector function—the possibility that Th9 cells may also be able to use the signal as an evolutionary defense against helminthes is quite fascinating.[17]

The classification of these pathways is also particularly intriguing in the context of understanding the effects the various T helper subtypes may have. Prior to the discovery of Th17 cells, T helpers were classified into a Th1/Th2 dichotomy, in which Th1 cells were to be mainly responsible for mediating inflammation, while Th2 cells were to protect against such inflammation.[18,19] While the discovery of additional subtypes has obviously complicated the picture, it is nonetheless still tempting to align the other subsets into that model. After all, some signaling pathways such as the IL-12 pathway that drives interferon gamma (IFNγ) production by Th1 cells seem to be clearly aligned to either be pro- or anti-inflammatory. [20,21] The standard pathway of Th9 cell differentiation challenges that clarity however, as both the IL-4 and TGF-β pathways are normally recognized as anti-inflammatory, yet drive the production of the pro-inflammatory IL-9. This result is particularly interesting given that IL-4 is a Th2 produced cytokine. [22,23] Further work may identify additional pathways that may lead to Th9 differentiation.

In addition, it must be noted that many other environmental factors beyond cytokines may also play important roles in controlling Th9 differentiation. In particular, metabolism-related factors could be a special interest to T helper cell stimulation.[24] After all, factors as diverse amino acid starvation, succinate, and high concentration of sodium have been shown to potently affect Th17 cells in opposite ways and in a manner distinct from other T helper subtypes.[25,26,27,28,29] Some degree of hypoxia has also been shown to help drive Th17 differentiation over differentiation into other phenotypes.[30,31] Tregs have similarly been shown to react differently than other T helper subtypes in the face of non-cytokine stimuli.[32] In addition, the Treg phenotype has been shown to have a lower metabolic profile than T effector phenotypes, and would thus likely respond differently than the other phenotypes to antimetabolic stimuli.[33,34] It is thus quite likely that Th9 cells will also exhibit some distinct responses to certain non-specific environmental stimuli than other T helper phenotypes. In fact, one such differential response has already been reported; Th9 cells have been found to have increased expression in the presence of nitric oxide (NO), while Th17 cells were previously found to be significantly suppressed by NO.[35,36] Of additional interest is the observation by some groups that thymic stromal lymphopoietin (TSLP) may actually encourage Th9 differentiation, despite TSLP being generally regarded as a factor suppressing T effector differentiation.[37,38] There may also be an additional time-related factor to Th9 differentiation that is distinct from other T helper cells. After all, while T helper phenotypes are generally polarized in vitro for three days in the presence of the polarizing cytokines prior to experimentation, several papers dealing with Th9 cellshave used a double polarization process. During this process, Th9 cells are first cultured in the presence of IL-4 and TGF-β for three days, and then re-stimulated for an additional 2-3 days prior to experimentation.[35,39] It is not entirely clear why the double polarization process leads to clearer results, especially since other T helper subtypes generally begin to undergo apoptosis at that point due to a number of reasons.[40] It is possible that the additional polarization can add a step of selection. Further study may yield a more complete explanation.

 

Transcription Factor Interplay

After T helper cells are polarized, the next level at which they are typically studied is the protein level. The polarization process can be observe through investigation of the signal transduction networks inside the cell, as it undergoes chromatin remodeling and begins the production of its functional proteins. Transcription factors play a critical role in managing this process, as they regulate the expression of different proteins. As such, analysis of the transcription factors active in T helper cells has been an integral part of their study. Each T helper subtype is generally recognized to possess some transcription factor(s) that are unique to its lineage, as distinct from those shared among all subtypes. These transcription factors have been identified to be T-bet, GATA-3, RORγt, Foxp3, and Bcl6 for Th1, Th2, Th17, Treg, and Tfh, respectively.[41,42,43,44] In short, these transcription factors respond to the signaling from the stimulating cytokines to drive the cell to one fate, while suppressing the possibility of others. It should be noted, however, that such suppression is not by any means absolute.

The identification of these unique transcription factors is extremely useful to conducting subtype research, for they essentially serve as unique subtype markers. For instance, Th17 cells can be determined to exist through evidence on many different levels, including, but not limited to, qPCR for RORγt and IL-17 mRNA, flow cytometry using IL-17 and RORγt gates, ELISA for IL-17 protein, and western blotting for RORγt protein. Based on gene expression profile studies, Th9 cells are somewhat similar to Th2s and more distantly related to Treg, but also display many novel elements.[39] Unfortunately, no clear lineage-defining transcription factor has been found for Th9 cells. As such, current options for studying Th9 cells are more limited, and largely rely on detecting IL-9. Although 2 proteins (interferon regulatory factor 4 (IRF4) and Pu.1) have been posited as potential candidates, neither appears to be fully consistent with characteristics of the other transcription factors.[15,45] After all, IRF4 is broadly expressed by all T helper subtypes through the course of differentiation, and IRF4-/- mice are generally immunodeficient.[46,47,48] Pu.1 is more promising, as it is not so highly expressed in other T helper phenotypes, but is still highly expressed by myeloid and B cells during the course of their development.[49,50,51] As such it seems that IRF4 and Pu.1 are less specific, but still vital proteins for Th9 cells, leaving open the possibility of the existence of other transcription factors that are lineage defining. This analysis holds when the other common characteristic shared by lineage-defining transcription factors is considered, namely that they can bind to the promoter regions of the signature cytokines that the cells secrete.[52,53,54,55] Both IRF4 and Pu.1 fulfill that characteristic, being capable of binding to the IL-9 promoter.[16,56] However, eukaryotic promoter regions are long regions that can attract the binding of many different proteins. As such, there may well exist some other protein that binds to the IL-9 promoter and is also specific to the Th9 lineage. However, given that the current understandings have shifted from declaring proteins “master regulators” to merely identifying them as “lineage-defining”, it may be only necessary that a protein be a marker of some sort.

Beyond the search for a lineage-defining transcription factor, the interplay of various transcription factors in Th9 cells may yield interesting mechanistic insights. For instance, the potent suppressing protein Bcl6 has been shown to transiently downregulate during the process of Th9 differentiation, raising questions as to the means by which that downregualtion is induced.[57] The curious expression pattern of Bcl6 also raises the possibility that its re-emergence after several days is in fact marking the onset of apoptosis/exhaustion for the subtype. In addition, similar to how other STAT family members have been shown to aid in T helper activity, STAT6 has been demonstrated to enhance IL-9 transcription and influence Th9 activity in several ways.[58,59,60,61] The Notch and Smad pathways have also been identified to have a role in Th9 differentiation.[62,63] IRF1 has also been shown to play an important role in Th9, especially in causing it to also produce IL-21.[64] The effects of these proteins and others on Th9 cells have been mostly reviewed in-depth elsewhere.[65,66,67,68,69] Post-translational modifications (PTMs) on these proteins, and their subsequent impact on interactions between other proteins that strongly interact with modified residues may also yield curious results. After all, it is currently understood that IRF4 and Pu.1 binding along the IL-9 promoter induce changes in chromatin modeling, via proteins such as the histone acetyltransferase Gcn5.[70,71] As such, there may be some PTMs (and the corresponding PTM-inducing proteins) that are unique to Th9 cells and which are useful for their study.

 

Cytokine Secretion

From the complex interplay of the various transcription factors and other proteins in regulating Th9 cells arises the secretion of large amounts of interleukin 9 (IL-9), the signature of the Th9 phenotype. In fact, as another group has suggested, the existence of CD4+ IL-9+ is perhaps the most convincing evidence that the subtype does indeed exist.[58,72,73] After all, although Th2 and Th17 have some capability of secreting IL-9, they cannot produce it at such a high level as Th9.[74] Besides IL-9, Th9 cells have also been shown to be capable of producing some amounts of IL-21 and IL-10.[75] Study of the impact of these other cytokines in affecting Th9 cell function is more difficult, however, since Th9 cells have fewer known markers than other T helper subtypes. This leads to a complication in fully comprehending the Th9 phenotype because of its wide range and irregularity of function in comparison to other T helper subsets.  As such, most of the work done on the effects of Th9 cytokine secretion thus far has been focused on IL-9.

Signaling via the IL-9 receptor, IL-9 can transduce effects through a JAK/STAT pathway to alter the expression of many genes in a variety of immune-related cell types.[76,77] In particular, the role of IL-9 in recruiting mast cells and enhancing the resulting release of histamines has been noted.[78] IL-9 has been shown to play an important role in several diseases, acting as an antagonist in asthma/bronchial hyperresponsiveness, atopic dermatitis (AD), and ulcerative colitis (UC), among others.[79,80,81,82] Beyond the obvious link between AD and asthma that has been previously noted, it is quite interesting that each of these autoimmune disorders also share the similarity of being Th2-related. As such, the common models used for studying Th9 cells in vivo are often based on oxazolone or ovalbumin, instead of other models such as DSS that more likely lead to Th1/Th17 responses.[83,84,85,86] This result is not surprising, given that Th9 cells require the presence of large amounts of IL-4 to differentiate, and IL-4 is commonly produced by Th2 cells.[87] However, it does complicate the study of Th9 cells significantly, especially in the context of using compounds to suppress their function. As such, Th9 cells might be better understood as a subtype that may arise slightly later, after Th2 cells have already formed at the site. Time-lapse experiments may be able to verify the authenticity of this hypothesis.

Regardless of the precise time that Th9 cells arise, it is clearly non-trivial that beyond the obvious need to clarify that a compound’s effect in a certain animal model is suppressing IL-9 from being produced by Th9 cells instead of Th2 or Th17 cells. Th2 cell production of IL-4 would also have to be monitored in tandem. This additional factor also introduces the possibility of divergence between in vivo and in vitro experiments, as in vitro experiments would hold the level of IL-4 constant even if its secretion fluctuated in vivo. However, it is also possible that there may exist some sort of double positive population of Th2/Th9 cells, due to plasticity between different T helper subtypes—a recently discovered population of Th2/Th17 double positive cells has been shown to be important in some diseases.[88] Beyond that concern, it is also unclear exactly how long Th9 cells are retained in tissue in vivo, with a recent report suggesting that they may have shorter effects than other T helper subtypes.[89] Such an observation also conflicts slightly with aforementioned techniques in in vitro experiments that culture the cells for longer than usual.  Greater rigor is thus necessary in studying Th9 cells than there may appear at first analysis.   

In addition, it is important to also recognize that IL-9 is also beneficial in responding to certain pathogens when properly regulated. For instance, IL-9 has been shown to have a strong protective capability in countering parasites such as helminthes, and such infections are endemic in many areas of the world.[90,91,92,93] In addition, IL-9 is also capable of attacking tumors, with that function being first detected in a study of melanoma.[94,95,96] Upregulation of IL-9 and Th9 may thus be beneficial in certain other conditions as well.[97,98] Th9 cell production of IL-3 may also be an important beneficial effect, as IL-3 has been shown to prolong DC survival.[99] This latter result may also be a feedback mechanism that Th9 cells can potentially use to survive for longer periods of time in tissue. That persistence is likely a key contributing factor to asthma.

 

Conclusion

Th9 cells are an important T helper subset with unique characteristics that defy the standard classification scheme for T helpers. In this paper, we discuss several of those distinguishing features in regards to Th9 stimulation, transcription factors involved, and Th9 cytokine secretion. We note some of questions that have yet to be answered regarding Th9 stimulation, including in terms of non-cytokine factors such as time. We also analyze the issue of Th9 cells currently lacking an accepted lineage-defining transcription factor, and the consequent difficulties that result. Further studies on Th9s may lead to new information that will eliminate the current limitations, and yield valuable data on potential therapies involving Th9s.


 

 

 


[1] Geginat J, Paroni M, Maglie S, Alfen JS, Kastirr I, Gruarin P, De Simone M, Pagani M, Abrignani S. Plasticity of human CD4 T cell subsets. Front Immunol. 2014 Dec 16;5:630.

[2] Kaiko GE, Horvat JC, Beagley KW, Hansbro PM. Immunological decision-making: how does the immune system decide to mount a helper T-cell response? Immunology. 2008 Mar;123(3):326-38.

[3] Mangan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, Hatton RD, Wahl SM, Schoeb TR, Weaver CT. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006, 441(7090): 231-234.

[4] O'Garra A, Stockinger B, Veldhoen M. Differentiation of human T(H)-17 cells does require TGF-beta! Nat Immunol 2008, 9(6): 588-590.

[5] Kimura A, Naka T, Kishimoto T. IL-6-dependent and -independent pathways in the development of interleukin 17-producing T helper cells. Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):12099-104.

[6] Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, Cua DJ, Littman DR.    The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006 Sep 22;126(6):1121-33.

[7] Park TY, Park SD, Cho JY, Moon JS, Kim NY, Park K, Seong RH, Lee SW, Morio T, Bothwell AL, Lee SK. RORγt-specific transcriptional interactomic inhibition suppresses autoimmunity associated with TH17 cells. Proc Natl Acad Sci U S A. 2014 Dec 30;111(52):18673-8.

[8] Wilson NJ, Boniface K, Chan JR, McKenzie BS, Blumenschein WM, Mattson JD, Basham B, Smith K, Chen T, Morel F, Lecron JC, Kastelein RA, Cua DJ, McClanahan TK, Bowman EP, de Waal Malefyt R.. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol. 2007 Sep;8(9):950-7.

[9] Korn T, Bettelli E, Gao W, Awasthi A, Jäger A, Strom TB, Oukka M, Kuchroo VK. IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells. Nature 2007, 448(7152): 484-487.

[10] Brüstle A, Heink S, Huber M, Rosenplänter C, Stadelmann C, Yu P, Arpaia E, Mak TW, Kamradt T, Lohoff M.. The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4. Nat Immunol. 2007 Sep;8(9):958-66.

[11] Xu L, Kitani A, Fuss I, Strober W. Cutting edge: regulatory T cells induce CD4+CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-beta. J Immunol. 2007 Jun 1;178(11):6725-9.

[12] Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med. 2005, 201(2): 233-240.

[13] Liao W, Spolski R, Li P, Du N, West EE, Ren M, Mitra S, Leonard WJ. Opposing actions of IL-2 and IL-21 on Th9 differentiation correlate with their differential regulation of BCL6 expression. Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):3508-13.

[14] Beriou G, Bradshaw EM, Lozano E, Costantino CM, Hastings WD, Orban T, Elyaman W, Khoury SJ, Kuchroo VK, Baecher-Allan C, Hafler DA. TGF-beta induces IL-9 production from human Th17 cells. J Immunol. 2010 Jul 1;185(1):46-54.

[15] Wong MT, Ye JJ, Alonso MN, Landrigan A, Cheung RK, Engleman E, Utz PJ. Regulation of human Th9 differentiation by type I interferons and IL-21. Immunol Cell Biol. 2010 Aug;88(6):624-31.

[16] Staudt V, Bothur E, Klein M, Lingnau K, Reuter S, Grebe N, Gerlitzki B, Hoffmann M, Ulges A, Taube C, Dehzad N, Becker M, Stassen M, Steinborn A, Lohoff M, Schild H, Schmitt E, Bopp T. Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells. Immunity. 2010 Aug 27;33(2):192-202.

[17] Grainger JR, Smith KA, Hewitson JP, McSorley HJ, Harcus Y, Filbey KJ, Finney CA, Greenwood EJ, Knox DP, Wilson MS, Belkaid Y, Rudensky AY, Maizels RM. Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-β pathway. J Exp Med. 2010 Oct 25;207(11):2331-41.

[18]Mucida D, Cheroutre H. The many face-lifts of CD4 T helper cells. Adv Immunol. 2010;107:139-52.

[19] A. Berger. Th1 and Th2 responses: what are they? . BMJ, 2000;321:424

[20] Piccotti JR, Chan SY, Li K, Eichwald EJ, Bishop DK. Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-gamma-producing cells. J Immunol. 1997 Jan 15;158(2):643-8.

[21] Yang DD, Conze D, Whitmarsh AJ, Barrett T, Davis RJ, Rincón M, Flavell RA. Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2. Immunity. 1998 Oct;9(4):575-85.

[22] Swain SL, McKenzie DT, Dutton RW, Tonkonogy SL, English M. The role of IL4 and IL5: characterization of a distinct helper T cell subset that makes IL4 and IL5 (Th2) and requires priming before induction of lymphokine secretion. Immunol Rev. 1988 Feb;102:77-105.

[23] Torbett BE, Laxer JA, Glasebrook AL. Frequencies of T cells secreting IL-2 and/or IL-4 among unprimed CD4+ populations. Evidence that clones secreting IL-2 and IL-4 give rise to clones which secrete only IL-4. Immunol Lett. 1990 Jan;23(3):227-33.

[24] Ghesquière B, Wong BW, Kuchnio A, Carmeliet P. Metabolism of stromal and immune cells in health and disease. Nature. 2014 Jul 10;511(7508):167-76.

[25] Carlson TJ, Pellerin A, Djuretic IM, Trivigno C, Koralov SB, Rao A, Sundrud MS. Halofuginone-induced amino acid starvation regulates Stat3-dependent Th17 effector function and reduces established autoimmune inflammation. J Immunol. 2014 Mar 1;192(5):2167-76.

[26] Tannahill GM, Curtis AM, Adamik J, Palsson-McDermott EM, McGettrick AF, Goel G, Frezza C, Bernard NJ, Kelly B, Foley NH, Zheng L, Gardet A, Tong Z, Jany SS, Corr SC, Haneklaus M, Caffrey BE, Pierce K, Walmsley S, Beasley FC, Cummins E, Nizet V, Whyte M, Taylor CT, Lin H, Masters SL, Gottlieb E, Kelly VP, Clish C, Auron PE, Xavier RJ, O'Neill LA. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature. 2013 Apr 11;496(7444):238-42.

[27] Sinclair LV, Rolf J, Emslie E, Shi YB, Taylor PM, Cantrell DA. Control of amino-acid transport by antigen receptors coordinates the metabolic reprogramming essential for T cell differentiation. Nat Immunol. 2013 May;14(5):500-8.

[28] Sundrud MS, Koralov SB, Feuerer M, Calado DP, Kozhaya AE, Rhule-Smith A, Lefebvre RE, Unutmaz D, Mazitschek R, Waldner H, Whitman M, Keller T, Rao A. Halofuginone inhibits TH17 cell differentiation by activating the amino acid starvation response. Science. 2009 Jun 5;324(5932):1334-8.

[29] Kleinewietfeld M, Manzel A, Titze J, Kvakan H, Yosef N, Linker RA, Muller DN, Hafler DA. Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells. Nature. 2013 Apr 25;496(7446):518-22

[30] Wang H, Flach H, Onizawa M, Wei L, McManus MT, Weiss A. Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210. Nat Immunol. 2014 Apr;15(4):393-401.

[31] Shi LZ, Wang R, Huang G, Vogel P, Neale G, Green DR, Chi H. HIF1alpha-dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells. J Exp Med. 2011 Jul 4;208(7):1367-76.

[32] Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M, Glickman JN, Garrett WS. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science. 2013 Aug 2;341(6145):569-73.

[33] Byersdorfer CA, Tkachev V, Opipari AW, Goodell S, Swanson J, Sandquist S, Glick GD, Ferrara JL. Effector T cells require fatty acid metabolism during murine graft-versus-host disease. Blood. 2013 Oct 31;122(18):3230-7.

[34] Michalek RD1, Gerriets VA, Jacobs SR, Macintyre AN, MacIver NJ, Mason EF, Sullivan SA, Nichols AG, Rathmell JC. Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets. J Immunol. 2011 Mar 15;186(6):3299-303.

[35] Niedbala W, Besnard AG, Nascimento DC, Donate PB, Sonego F, Yip E, Guabiraba R, Chang HD, Fukada SY, Salmond RJ, Schmitt E, Bopp T, Ryffel B, Liew FY. Nitric oxide enhances Th9 cell differentiation and airway inflammation. Nat Commun. 2014 Aug 7;5:4575.

[36] Jianjun Yang, Zhang R, Lu G, Shen Y, Peng L, Zhu C, Cui M, Wang W, Arnaboldi P, Tang M, Gupta M, Qi CF, Jayaraman P, Zhu H, Jiang B, Chen SH, He JC, Ting AT, Zhou MM, Kuchroo VK, Morse HC 3rd, Ozato K, Sikora AG, Xiong H. T cell–derived inducible nitric oxide synthase switches off Th17 cell differentiation. J Exp Med. 2013 Jul 1;210(7):1447-62.

[37] Froidure A, Shen C, Gras D, Van Snick J, Chanez P, Pilette C. Myeloid dendritic cells are primed in allergic asthma for thymic stromal lymphopoietin-mediated induction of Th2 and Th9 responses. Allergy. 2014 Aug;69(8):1068-76.

[38] Yao W, Zhang Y, Jabeen R, Nguyen ET, Wilkes DS, Tepper RS, Kaplan MH, Zhou B. Interleukin-9 is required for allergic airway inflammation mediated by the cytokine TSLP. Immunity. 2013 Feb 21;38(2):360-72.

[39] Jabeen R, Goswami R, Awe O, Kulkarni A, Nguyen ET, Attenasio A, Walsh D, Olson MR, Kim MH, Tepper RS, Sun J, Kim CH, Taparowsky EJ, Zhou B, Kaplan MH. Th9 cell development requires a BATF-regulated transcriptional network. J Clin Invest. 2013 Nov 1;123(11):4641-53.

[40] Huang YH, Zhu C, Kondo Y, Anderson AC, Gandhi A, Russell A, Dougan SK, Petersen BS, Melum E, Pertel T, Clayton KL, Raab M, Chen Q, Beauchemin N, Yazaki PJ, Pyzik M, Ostrowski MA, Glickman JN, Rudd CE, Ploegh HL, Franke A, Petsko GA, Kuchroo VK, Blumberg RS. CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature. 2015 Jan 15;517(7534):386-90.

[41] Mullen AC, High FA, Hutchins AS, Lee HW, Villarino AV, Livingston DM, Kung AL, Cereb N, Yao TP, Yang SY, Reiner SL. Role of T-bet in commitment of TH1 cells before IL-12-dependent selection. Science. 2001 Jun 8;292(5523):1907-10.

[42] Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell. 1997 May 16;89(4):587-96.

[43] Yu D, Rao S, Tsai LM, Lee SK, He Y, Sutcliffe EL, Srivastava M, Linterman M, Zheng L, Simpson N, Ellyard JI, Parish IA, Ma CS, Li QJ, Parish CR, Mackay CR, Vinuesa CG. The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment. Immunity. 2009 Sep 18;31(3):457-68.

[44] Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003 Dec 15;198(12):1875-86.

[45] Huber M, Lohoff M. IRF4 at the crossroads of effector T-cell fate decision. Eur J Immunol. 2014 Jul;44(7):1886-95.

[46] Gökmen MR, Dong R, Kanhere A, Powell N, Perucha E, Jackson I, Howard JK, Hernandez-Fuentes M, Jenner RG, Lord GM. Genome-wide regulatory analysis reveals that T-bet controls Th17 lineage differentiation through direct suppression of IRF4. J Immunol. 2013 Dec 15;191(12):5925-32.

[47] Bollig N, Brüstle A, Kellner K, Ackermann W, Abass E, Raifer H, Camara B, Brendel C, Giel G, Bothur E, Huber M, Paul C, Elli A, Kroczek RA, Nurieva R, Dong C, Jacob R, Mak TW, Lohoff M. Transcription factor IRF4 determines germinal center formation through follicular T-helper cell differentiation. Proc Natl Acad Sci U S A. 2012 May 29;109(22):8664-9.

[48] Biswas PS, Gupta S, Stirzaker RA, Kumar V, Jessberger R, Lu TT, Bhagat G, Pernis AB.  Dual regulation of IRF4 function in T and B cells is required for the coordination of T-B cell interactions and the prevention of autoimmunity. J Exp Med. 2012 Mar 12;209(3):581-96.

[49] Christie DA, Xu LS, Turkistany SA, Solomon LA, Li SK, Yim E, Welch I, Bell GI, Hess DA, DeKoter RP. PU.1 Opposes IL-7-Dependent Proliferation of Developing B Cells with Involvement of the Direct Target Gene Bruton Tyrosine Kinase. J Immunol. 2015 Jan 15;194(2):595-605.

[50] Carotta S, Willis SN, Hasbold J, Inouye M, Pang SH, Emslie D, Light A, Chopin M, Shi W, Wang H, Morse HC 3rd, Tarlinton DM, Corcoran LM, Hodgkin PD, Nutt SL. The transcription factors IRF8 and PU.1 negatively regulate plasma cell differentiation. J Exp Med. 2014 Oct 20;211(11):2169-81.

[51] Kang JW, Park YS, Kim MS, Lee DH, Bak Y, Ham SY, Song YS, Hong JT, Yoon DY. IL-32α down-regulates β2 integrin (CD18) expression by suppressing PU.1 expression in myeloid cells. Cell Signal. 2014 Jul;26(7):1514-22

[52] Kojima H, Takeda Y, Muromoto R, Takahashi M, Hirao T, Takeuchi S, Jetten AM, Matsuda T. Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ. Toxicology. 2015 Jan 9. pii: S0300-483X(15)00015-3

[53] Lovett-Racke AE, Rocchini AE, Choy J, Northrop SC, Hussain RZ, Ratts RB, Sikder D, Racke MK. Silencing T-bet defines a critical role in the differentiation of autoreactive T lymphocytes. Immunity. 2004 Nov;21(5):719-31.

[54] Miller MM, Akaronu N, Thompson EM, Hood SF, Fogle JE. Modulating DNA Methylation in Activated CD8+ T Cells Inhibits Regulatory T Cell-Induced Binding of Foxp3 to the CD8+ T Cell IL-2 Promoter. J Immunol. 2015 Feb 1;194(3):990-8. doi: 10.4049/jimmunol.1401762. Epub 2014 Dec 29.

[55] Li Z, Lin F, Zhuo C, Deng G, Chen Z, Yin S, Gao Z, Piccioni M, Tsun A, Cai S, Zheng SG, Zhang Y, Li B. PIM1 kinase phosphorylates the human transcription factor FOXP3 at serine 422 to negatively regulate its activity under inflammation. J Biol Chem. 2014 Sep 26;289(39):26872-81.

[56] Ramming A, Druzd D, Leipe J, Schulze-Koops H, Skapenko A. Maturation-related histone modifications in the PU.1 promoter regulate Th9-cell development. Blood. 2012 May 17;119(20):4665-74.

[57] Bassil R, Orent W, Olah M, Kurdi AT, Frangieh M, Buttrick T, Khoury SJ, Elyaman W. BCL6 controls Th9 cell development by repressing Il9 transcription. J Immunol. 2014 Jul 1;193(1):198-207.

[58] Goswami R, Jabeen R, Yagi R, Pham D, Zhu J, Goenka S, Kaplan MH. STAT6-dependent regulation of Th9 development. J Immunol. 2012 Feb 1;188(3):968-75.

[59] Mathur AN, Chang HC, Zisoulis DG, Stritesky GL, Yu Q, O'Malley JT, Kapur R, Levy DE, Kansas GS, Kaplan MH. Stat3 and Stat4 direct development of IL-17-secreting Th cells. J Immunol. 2007 Apr 15;178(8):4901-7.

[60] Zheng Y, Wang Z, Deng L, Zhang G, Yuan X, Huang L, Xu W, Shen L. Modulation of STAT3 and STAT5 activity rectifies the imbalance of Th17 and Treg cells in patients with acute coronary syndrome. Clin Immunol. 2015 Jan 5. pii: S1521-6616(14)00290-3.

[61] Backert I, Koralov SB, Wirtz S, Kitowski V, Billmeier U, Martini E, Hofmann K, Hildner K, Wittkopf N, Brecht K, Waldner M, Rajewsky K, Neurath MF, Becker C, Neufert C. STAT3 activation in Th17 and Th22 cells controls IL-22-mediated epithelial host defense during infectious colitis. J Immunol. 2014 Oct 1;193(7):3779-91.

[62] Elyaman W, Bassil R, Bradshaw EM, Orent W, Lahoud Y, Zhu B, Radtke F, Yagita H, Khoury SJ. Notch receptors and Smad3 signaling cooperate in the induction of interleukin-9-producing T cells. Immunity. 2012 Apr 20;36(4):623-34.

[63] Tamiya T, Ichiyama K, Kotani H, Fukaya T, Sekiya T, Shichita T, Honma K, Yui K, Matsuyama T, Nakao T, Fukuyama S, Inoue H, Nomura M, Yoshimura A. Smad2/3 and IRF4 play a cooperative role in IL-9-producing T cell induction. J Immunol. 2013 Sep 1;191(5):2360-71.

[64] Végran F, Berger H, Boidot R, Mignot G, Bruchard M, Dosset M, Chalmin F, Rébé C, Dérangère V, Ryffel B, Kato M, Prévost-Blondel A, Ghiringhelli F, Apetoh L. The transcription factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells. Nat Immunol. 2014 Aug;15(8):758-66.

[65] Li P, Spolski R, Liao W, Leonard WJ. Complex interactions of transcription factors in mediating cytokine biology in T cells. Immunol Rev. 2014 Sep;261(1):141-56.

[66] Oh H, Ghosh S. NF-κB: roles and regulation in different CD4(+) T-cell subsets. Immunol Rev. 2013 Mar;252(1):41-51.

[67] Jabeen R, Kaplan MH. The symphony of the ninth: the development and function of Th9 cells. Curr Opin Immunol. 2012 Jun;24(3):303-7.

[68] Kaplan MH. Th9 cells: differentiation and disease. Immunol Rev. 2013 Mar;252(1):104-15.

[69] Zhao P, Xiao X, Ghobrial RM, Li XC. IL-9 and Th9 cells: progress and challenges. Int Immunol. 2013 Oct;25(10):547-51.

[70] Goswami R, Kaplan MH. Gcn5 is required for PU.1-dependent IL-9 induction in Th9 cells. J Immunol. 2012 Sep 15;189(6):3026-33.

[71] Jones CP, Gregory LG, Causton B, Campbell GA, Lloyd CM. Activin A and TGF-beta promote T(H)9 cell-mediated pulmonary allergic pathology. J Allergy Clin Immunol. 2012;129:1000–1010. e1003.

[72] Schmitt E, Klein M, Bopp T. Th9 cells, new players in adaptive immunity. Trends Immunol. 2014 Feb;35(2):61-8.

[73] Cortelazzi C, Campanini N, Ricci R, De Panfilis G. Inflammed skin harbours Th9 cells. Acta Derm Venereol. 2012 doi: 10.2340/00015555-1408.

[74] Nowak EC, Weaver CT, Turner H, Begum-Haque S, Becher B, Schreiner B, Coyle AJ, Kasper LH, Noelle RJ. IL-9 as a mediator of Th17-driven inflammatory disease. J Exp Med. 2009 Aug 3;206(8):1653-60.

[75]Araujo-Pires AC, Francisconi CF, Biguetti CC, Cavalla F, Aranha AM, Letra A, Trombone AP, Faveri M, Silva RM, Garlet GP. Simultaneous analysis of T helper subsets (Th1, Th2, Th9, Th17, Th22, Tfh, Tr1 and Tregs) markers expression in periapical lesions reveals multiple cytokine clusters accountable for lesions activity and inactivity status. J Appl Oral Sci. 2014 Jul-Aug;22(4):336-46.

[76] Osterfeld H, Ahrens R, Strait R, Finkelman FD, Renauld JC, Hogan SP. Differential roles for the IL-9/IL-9 receptor alpha-chain pathway in systemic and oral antigen-induced anaphylaxis. J Allergy Clin Immunol. 2010 Feb;125(2):469-476.e2.

[77] Hornakova T, Staerk J, Royer Y, Flex E, Tartaglia M, Constantinescu SN, Knoops L, Renauld JC. Acute lymphoblastic leukemia-associated JAK1 mutants activate the Janus kinase/STAT pathway via interleukin-9 receptor alpha homodimers. J Biol Chem. 2009 Mar 13;284(11):6773-81.

[78] Blankenhaus B, Reitz M, Brenz Y, Eschbach ML, Hartmann W, Haben I, Sparwasser T, Huehn J, Kühl A, Feyerabend TB, Rodewald HR, Breloer M. Foxp3 regulatory T cells delay expulsion of intestinal nematodes by suppression of IL-9-driven mast cell activation in BALB/c but not in C57BL/6 mice. PLoS Pathog. 2014 Feb 6;10(2):e1003913.

[79] Übel C, Graser A, Koch S, Rieker RJ, Lehr HA, Müller M, Finotto S. Role of Tyk-2 in Th9 and Th17 cells in allergic asthma. Sci Rep. 2014 Aug 11;4:5865.

[80] Ma L, Xue HB, Guan XH, Shu CM, Zhang JH, Yu J. Possible pathogenic role of T helper type 9 cells and interleukin (IL)-9 in atopic dermatitis. Clin Exp Immunol. 2014 Jan;175(1):25-31.

[81] Nalleweg N, Chiriac MT, Podstawa E, Lehmann C, Rau TT, Atreya R, Krauss E, Hundorfean G, Fichtner-Feigl S, Hartmann A, Becker C, Mudter J IL-9 and its receptor are predominantly involved in the pathogenesis of UC. Gut. 2014 Jun 23. pii: gutjnl-2013-305947.

[82] Yao X, Kong Q, Xie X, Wang J, Li N, Liu Y, Sun B, Li Y, Wang G, Li W, Qu S, Zhao H, Wang D, Liu X, Zhang Y, Mu L, Li H. Neutralization of interleukin-9 ameliorates symptoms of experimental autoimmune myasthenia gravis in rats by decreasing effector T cells and altering humoral responses. Immunology. 2014 Nov;143(3):396-405.

[83] Gerlach K, Hwang Y, Nikolaev A, Atreya R, Dornhoff H, Steiner S, Lehr HA, Wirtz S, Vieth M, Waisman A, Rosenbauer F, McKenzie AN, Weigmann B, Neurath MF. TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells. Nat Immunol. 2014 Jul;15(7):676-86

[84] Li H, Edin ML, Bradbury JA, Graves JP, DeGraff LM, Gruzdev A, Cheng J, Dackor RT, Wang PM, Bortner CD, Garantziotis S, Jetten AM, Zeldin DC. Cyclooxygenase-2 inhibits T helper cell type 9 differentiation during allergic lung inflammation via down-regulation of IL-17RB. Am J Respir Crit Care Med. 2013 Apr 15;187(8):812-22

[85] Lin JY, Chen JS, Hsu CJ, Miaw SC, Liu CY, Lee SJ, Chen PC, Wang LF. Epicutaneous sensitization with protein antigen induces Th9 cells. J Invest Dermatol. 2012 Mar;132(3 Pt 1):739-41.

[86] Okada Y, Tsuzuki Y, Sato H, Narimatsu K, Hokari R, Kurihara C, Watanabe C, Tomita K, Komoto S, Kawaguchi A, Nagao S, Miura S. Trans fatty acids exacerbate dextran sodium sulphate-induced colitis by promoting the up-regulation of macrophage-derived proinflammatory cytokines involved in T helper 17 cell polarization. Clin Exp Immunol. 2013 Dec;174(3):459-71.

[87] Kim HS, Chung DH. IL-9-producing invariant NKT cells protect against DSS-induced colitis in an IL-4-dependent manner. Mucosal Immunol. 2013 Mar;6(2):347-57.

[88] Wang YH, Voo KS, Liu B, Chen CY, Uygungil B, Spoede W, Bernstein JA, Huston DP, Liu YJ. A novel subset of CD4(+) T(H)2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma. J Exp Med. 2010 Oct 25;207(11):2479-91.

[89] Tan C, Aziz MK, Lovaas JD, Vistica BP, Shi G, Wawrousek EF, Gery I. Antigen-specific Th9 cells exhibit uniqueness in their kinetics of cytokine production and short retention at the inflammatory site. J Immunol. 2010 Dec 1;185(11):6795-801

[90] Licona-Limón P, Henao-Mejia J, Temann AU, Gagliani N, Licona-Limón I, Ishigame H, Hao L, Herbert DR, Flavell RA. Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection. Immunity. 2013 Oct 17;39(4):744-57.

[91] Anuradha R, George PJ, Hanna LE, Chandrasekaran V, Kumaran P, Nutman TB, Babu S. IL-4-, TGF-β-, and IL-1-dependent expansion of parasite antigen-specific Th9 cells is associated with clinical pathology in human lymphatic filariasis. J Immunol. 2013 Sep 1;191(5):2466-73.

[92] Angkasekwinai P, Srimanote P, Wang YH, Pootong A, Sakolvaree Y, Pattanapanyasat K, Chaicumpa W, Chaiyaroj S, Dong C. Interleukin-25 (IL-25) promotes efficient protective immunity against Trichinella spiralis infection by enhancing the antigen-specific IL-9 response. Infect Immun. 2013 Oct;81(10):3731-41.

[93] Turner JE, Morrison PJ, Wilhelm C, Wilson M, Ahlfors H, Renauld JC, Panzer U, Helmby H, Stockinger B. IL-9-mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation. J Exp Med. 2013 Dec 16;210(13):2951-65.

[94] Bu XN, Zhou Q, Zhang JC, Ye ZJ, Tong ZH, Shi HZ. Recruitment and phenotypic characteristics of interleukin 9-producing CD4+ T cells in malignant pleural effusion. Lung. 2013 Aug;191(4):385-9.

[95] Lu Y, Hong S, Li H, Park J, Hong B, Wang L, Zheng Y, Liu Z, Xu J, He J, Yang J, Qian J, Yi Q. Th9 cells promote antitumor immune responses in vivo. J Clin Invest. 2012 Nov 1;122(11):4160-71.

[96] Purwar R, Schlapbach C, Xiao S, Kang HS, Elyaman W, Jiang X, Jetten AM, Khoury SJ, Fuhlbrigge RC, Kuchroo VK, Clark RA, Kupper TS. Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells. Nat Med. 2012 Aug;18(8):1248-53.

[97] Vasanthakumar R, Mohan V, Anand G, Deepa M, Babu S, Aravindhan V. Serum IL-9, IL-17, and TGF-β levels in subjects with diabetic kidney disease (CURES-134). Cytokine. 2014 Dec 23.

[98] Mangus CW, Massey PR, Fowler DH, Amarnath S. Rapamycin resistant murine th9 cells have a stable in vivo phenotype and inhibit graft-versus-host reactivity. PLoS One. 2013 Aug 21;8(8):e72305

[99] Park J, Li H, Zhang M, Lu Y, Hong B, Zheng Y, He J, Yang J, Qian J, Yi Q. Murine Th9 cells promote the survival of myeloid dendritic cells in cancer immunotherapy. Cancer Immunol Immunother. 2014 Aug;63(8):835-45.


____________

Conflict of interest: No conflicts declared.

*Corresponding Author: Zihan Zheng. 7001 Juniper Valley Rd, Middle Village, NY 11379, USA. Phone: (646)-724-7378.

Email: zihanz@live.unc.edu

© 2015 by the Journal of Nature and Science (JNSCI).