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Although the cytokine interleukin 9 (IL-9) was discovered decades ago, it remains one of the most enigmatic cytokines identified so far, in particular because its functional activities remain far from clear. Breakthroughs made through the use of IL-9 reporter mice have allowed the identification of cell types that produce IL-9 in vivo and, contrary to expectations based on previous results obtained in vitro, it is not T cells but instead a previously unknown type of innate lymphoid cell, called the ‘ILC2 cell’, that is the main cell type that expresses IL-9 in vivo. In this perspective, we put forward a hypothesis about the potential biological functions of IL-9 in the immune system and beyond.

Before the advent of modern molecular biology and genome sequenc-ing, biological sciences, including immunology, were based on a combination of fortuitous functional observations and sophisticated protein-purification technology. This was true for the discovery of interferons, hematopoietic growth factors and the first interleukins identified. The discovery of interleukin 9 (IL-9) followed this track, as it was identified as a growth factor, initially called ‘p40’, for a T cell line established from lymph nodes of antigen-primed mice after repeated in vitro culture in the presence of antigen1.

Subsequently a molecule with a similar activity, called TCGFIII (for ‘T cell growth factor III’)2, and a factor that stimulates the survival of mast cell lines, called MEA (for ‘mast cell-enhancing activity’)3, were found to be identical to p40. IL-9 thus started its ‘career’ as a growth factor for T cells and mast cells.

The cDNA that encodes mouse IL-9 was cloned from one of the helper T cell lines mentioned above and was one of the rare inter-leukins that was also completely sequenced at the protein level4. Contrary to mouse IL-9, human IL-9 was identified by cDNA-cloning procedures either by cross-hybridization with the mouse cDNA or by expression cloning of a factor with growth-stimulating activity for a megakaryoblastic leukemia cell line5. Both the human and mouse protein have been synthesized as 144–amino acid precursors with an 18–amino acid signal peptide. They are characterized by an abundance of cationic residues and four glycosylation sites; the latter explains the discrepancy between the size of the natural protein and

its predicted mass of 14,150 Daltons. Human IL-9 is encoded by a single-copy gene that is located in chromosome 5 in the 5q31-q35 region6 and is thus in the vicinity of the genes that encode IL-4, IL-13, IL-5, IL-3 and granulocyte-monocyte colony-stimulating factor. In the mouse, the gene encoding IL-9 is located on chromosome 13 (ref. 7) and thus is distant from the genes encoding the cytokines mentioned above, which are clustered on mouse chromosome 11. This may be an evolutionary ‘anecdote’, because so far differences in the expression of the genes encoding human and mouse IL-9 have not been reported.

The receptor for IL-9 (IL-9R) was identified through expression cloning and IL-9-binding procedures as a member of the superfamily of hematopoietin receptors with the canonical Trp-Ser-Glu-Trp-Ser motif in the extracellular domain that is shared by all members of this family8. The initial detection of IL-9R by the binding of radiolabeled IL-9 showed that IL-9R is expressed on mast cells, not on resting or freshly activated T cells, but on some long-term cultured T cell lines and on transformed T cells.

Immunological function of IL-9A wide spectrum of functions in both hematopoietic and nonhemato-poietic cells has been attributed to IL-9. In addition to its effects on the survival and proliferation of T cells and mast cells4,9, its postulated activities include the modulation of B cell responses10,11, as well as antiapoptotic effects on neurons12 and the induction of chemokines in epithelial and muscle cells13,14. IL-9 serves its diverse biologic func-tions via IL-9R, a cytokine receptor that consists of a ligand-specific α-subunit and a common γ-chain that is shared with the IL-2, IL-4, IL-7, IL-15 and IL-21 receptor complexes. The binding of IL-9 to IL-9R increases heterodimerization of the IL-9R α-subunit with the common γ-chain and induces activation of the kinases Jak1 and Jak3, which results in phosphorylation of IL-9R on a single tyrosine residue; that residue then acts as a docking site for transcription fac-tors STAT1, STAT3 and STAT5, which are then activated by the Jak kinases associated with the receptor. Studies of a set of mutations in the gene encoding IL-9R have elucidated the contributions of those STAT factors to various in vitro IL-9 effects, such as cell proliferation and prevention of apoptosis15.

The functional importance of IL-9 in the pathogenesis of dis-eases believed to be driven by a ‘classical’ type 2 response was ini-tially demonstrated in mice with transgenic expression of IL-9. Mice with systemic overexpression of IL-9 develop intestinal mastocytosis and enhanced production of immunoglobulin E and are resistant to intestinal infection by helminths16,17. Lung-selective overexpression of IL-9 results in spontaneous airway inflammation characterized by eosinophilia, mast-cell infiltration, enhanced mucus production and airway hyper-reactivity, which resembles the classical features of

The many lives of IL-9: a question of survival?Christoph Wilhelm1,3, Jan-Eric Turner1, Jacques Van Snick2 & Brigitta Stockinger1

1Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, London, UK. 2Ludwig Institute for Cancer Research, Brussels Branch, and Cellular Genetics, Experimental Medicine Units, Christian de Duve Institute of Cellular Pathology, Université de Louvain, Brussels, Belgium. 3Present address: Mucosal Immunology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. Correspondence should be addressed to B.S. (bstocki@nimr.mrc.ac.uk).

Published online 19 June 2012; doi:10.1038/ni.2303

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human asthma18,19. In contrast to those results obtained with IL-9- overexpression models, genetic deficiency in IL-9 is not protective in models of allergen-induced airway inflammation20 and does not prevent expulsion of the intestinal nematode Nippostrongylus brasiliensis21. Administration of a neutralizing antibody to IL-9, how-ever, ameliorates ovalbumin-induced airway inflammation in mice22,23 and results in less muscle contractility and worm expulsion after infection with Trichuris muris24. Although they remain unresolved, these inconsistencies might be related to the genetic background of the mice used and the background-specific bias toward the develop-ment of type 2 responses.

Other discrepancies also remain unresolved. For example, deficiency in IL-9R is reported to either ameliorate or worsen experimental autoimmune encephalomyelitis via effects on regula-tory T cells (Treg cells) and the TH17 subset of helper T cells25,26. IL-9 expression is induced in T cells cultured under TH17 cell– inducing conditions. Conversely, analysis of T cells infiltrating the spinal cord and draining lymph nodes of IL-9-fate-reporter mice with experimental autoimmune encephalomyelitis has demonstrated the absence of any cells that express the gene encoding IL-9 in the acute or chronic stage of the disease (C.W., J.-E.T., J.V.S. and B.S., unpublished data).

Is IL-9 a T helper type 2 cytokine?The discovery of IL-9 as a T cell growth factor present in the superna-tants of stimulated T cell lines suggested that T cells might be major producers of this cytokine. Subsequently, IL-9 was attributed to T helper type 2 (TH2) cells on the basis of experimental findings that linked it to immune responses associated with TH2 cells, such as helminth infection and allergic reactions18,27. Also, the close chro-mosomal association of the gene encoding IL-9 with those encoding IL-4, IL-5, and IL-13 (albeit only in humans) suggested such a con-nection. On the basis of those data, an antibody to human IL-9 was developed and is now in clinical trials for the treatment of asthma, but its efficacy has not yet been established28. Nevertheless, the link between IL-9 and TH2 cells has never been formally proven and it is now known that IL-9 is not a prototypical TH2 cytokine. In fact, the complexities encountered in the course of identifying the types of cells that secrete IL-9 in vivo have provided a valuable lesson for cytokine research in the future.

Because of the lack of a commercially available antibody that would allow intracellular staining for IL-9, the cytokine has been detected either by analysis of mRNA in mixed-cell populations or in culture supernatants by enzyme-linked immunosorbent assay. For analysis of the functionality of IL-9, cell populations from IL-9-deficient mice have been used. Neither of those approaches would allow unequivocal attri-bution of cytokine production on the single-cell level and, as a conse-quence, IL-9 production has been associated with several T cell subsets in addition to TH2 cells, such as Treg cells29,30 and TH17 cells25.

Naive CD4+ T cells have been shown to differentiate into IL-9-secreting cells in vitro with the combination of IL-4 and transform-ing growth factor-β (TGF-β)31. The use of a polyclonal antibody that works for intracellular staining has allowed the identification of which CD4+ T cell subset produces IL-9. In agreement with another publication reporting that the same cytokine combination is active in supporting the generation of cells of the ‘TH9’ subset of helper T cells32, it was clear that those IL-9-secreting cells were distinct from any other CD4+ T cell subset, including TH2, TH17 or Treg cells. Initially, IL-9 was identified as a candidate cytokine produced by TH17 cells in a microarray study. However, careful cell purification, as well as the advantage of intracellular staining, has identified cells

that produce IL-9 as being distinct from TH17 cells. Interestingly, polarized TH2 cells can be deviated into TH9 cells by exposure to TGF-β, which results in downregulation of the transcription factor GATA-3 and loss of IL-4 and IL-5 production; this suggests that TH9 cells might have a developmental connection to TH2 cells. Although the transcription factors PU.1 and IRF4 have been suggested to direct TH9 development, it is unlikely that these can be considered lineage- specifying factors, as they have wide expression in many T cell sub-sets33,34. Furthermore, these data were all obtained in vitro, and it has proven difficult so far to obtain reliable intracellular staining of IL-9 in T cells isolated ex vivo. Thus, direct evidence for the existence of a TH9 T cell subset in vivo is still missing. In addition, it has been observed that even in vitro, IL-9 production seems transient, with maximal detection in the first 48 h after initiation of culture and a gradual decrease over time35,36.

Because of the potentially short-lived production of this cytokine, a ‘fate reporter’ for IL-9 has been generated that allows the detection of cells that had initiated transcription of the gene encoding IL-9 regardless of whether they were still producing the cytokine at the time of analysis. These mice with transgenic expression of a bacterial artificial chromosome encoding an IL-9 fate reporter express Cre recombinase under the control of the endogenous IL-9 locus (Il9Cre mice). For visualization of Cre activity, Il9Cre mice were bred with reporter mice expressing enhanced yellow fluorescent protein (eYFP) under the control of the endogenous Rosa 26 promoter (R26ReYFP mice). In the resultant Il9CreR26ReYFP progeny, the fluorescent reporter permanently labels cells that had expressed the gene encod-ing IL-9 regardless of the present production status of this cytokine. Analysis of these mice has provided further surprises.

Innate lymphoid cells produce IL-9 in vivoNaive IL9CreR26ReYFP mice do not express any eYFP in lymphoid organs or peripheral tissues, but after exposure to papain (which induces airway inflammation), IL-9 protein is readily detected in bronchoalveolar lavage fluid and eYFP+ cells can be isolated from lung tissue36. Surprisingly, however, most eYPF+ cells are present in a population of innate lymphoid cells (ILCs) that lack lineage mark-ers for other subsets of hematopoietic cells of the immune system. The number of IL-9-producing ILCs dwarfs that of IL-9-producing T cells, and most IL-9 protein also stems from ILCs and not T cells. Nevertheless, eYFP reporter activity is readily induced in T cells in vitro and is also detectable (albeit in very small amounts) in T cells from the lung tissue of mice repeatedly exposed to ovalbu-min by intranasal challenge, which indicates that there is no intrinsic problem with the reporting of IL-9 expression in T cells. In several other experimental models, including infection with Trichuris muris, Heligmosomoides polygyrus or Nippostrongylus brasiliensis, little if any eYFP expression has been observed in T cells (C.W., J.-E.T., J.V.S. and B.S., unpublished data). In contrast, ILCs expressing eYFP are readily detectable. These ILCs are similar to cells described as ‘natural helper cells’ or ‘nuocytes’, and according to present thinking, we call these ‘ILC2 cells’37.

Regulation of IL-9 productionElucidating the sequence of events that lead to the induction of IL-9 in ILC2 cells, as well as the downstream consequences of IL-9 produc-tion, has confirmed earlier findings about the inflammatory mediators connected with IL-9 but has also linked these events into a distinctly different molecular sequence.

The induction of IL-9 in ILC2 cells is dependent on IL-2 provided by the adaptive T cell response, a phenomenon demonstrated before

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in vitro in human T cell lines38–40 and mouse primary T cell cul-tures41,42, although the possibility of ‘contamination’ of the latter cul-tures with ILC2 cells cannot be ruled out. Furthermore, IL-2-driven production of IL-9 requires prior activation, as ILC2 cells isolated from naive mice do not produce IL-9 after stimulation with IL-2. Studies suggest that IL-2 functions only in the presence of IL-33, as the ILC2 population induced by intrapulmonary IL-33 challenge is able to produce IL-9 after challenge with IL-2 (C.W., J.-E.T., J.V.S. and B.S., unpublished data). In line with that, culture of ILC2 cells isolated from naive mice with a combination of IL-2 and IL-33 induces IL-9 expres-sion (C.W., J.-E.T., J.V.S. and B.S., unpublished data). Interestingly, it has been reported that IL-33, as well as other cytokines that belong to the IL-1 family, can enhance IL-9 production by CD4+ T cells cul-tured with TGF-β, even in the absence of IL-4 (ref. 41). Whether TGF-β (and/or IL-4) also has an effect on the IL-9 production of ILC2 cells has not been studied so far. Together these data suggest certain similarities between T cells and ILC2 cells in the regulation of IL-9 production, which await further investigation (Fig. 1).

As mentioned above, another unique feature of IL-9 expression is its transient nature, which makes it exceptionally difficult to track IL-9-producing cells in vivo. As IL-9 reporter activity is induced in the context of papain-induced lung inflammation, a detailed time course analysis of IL-9 protein expression was done that showed it was detectable only shortly after an allergen challenge36. In addition, IL-9-producing cells (detected by intracellular cytokine staining) appeared as early as 6 h after the allergen rechallenge. However, by 24 h after the challenge, no IL-9+ cells were detected. This phenomenon explains the lack of a report showing substantial intracellular cytokine stain-ing of cells isolated ex vivo, even though a monoclonal antibody for intracellular staining is now available. Although fewer in number, IL-9-producing T cells can be detected during papain-induced lung inflammation, and IL-9 expression in T cells shows kinetics similar to those of its expression in ILC2 cells (C.W., J.-E.T., J.V.S. and B.S., unpublished data), which additionally explains the lack of robust data for ex vivo intracellular staining of IL-9 in T cells. The transient expression pattern of IL-9 indicates its critical function in the immune system that seems to require tight regulation, possibly because of the dangers inherent in its prolonged expression.

IL-9 regulates ILC functionThe function of IL-9 seems to be more complex in vivo. Studies of IL-9-fate-reporter mice have established that naive mice have almost no IL-9 expression, which suggests a limited function for IL-9 in the steady state. After lung inflammation is induced with papain, IL-9-producing ILC2 cells rapidly lose expression of IL-9 protein but continue to express IL-5 and IL-13. Furthermore, culture of ILC2 cells in the presence of IL-9 enhances the expression of IL-5 and IL-13, and depletion of IL-9 in vivo in the model of papain-induced lung inflam-mation results in lower expression of IL-5 and IL-13 in the lungs.

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Enhancing

IL-1α, IL-1βIL-18IL-33IL-25IL-9

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IL-9IL-10

IL-9IL-5

IL-13IL-6

Amphiregulin

TGF-βIL-4

IL-2

IL-33IL-25 (?)IL-7 (?)

Figure 1 Regulation of IL-9 production in T cells and ILCs. Although in vitro stimulation of T cells with TGF-β and IL-4 results in TH9 differentiation, the main IL-9-producing cell type detectable in vivo is the ILC2 cell. These cells are activated in vitro and in vivo by the epithelial cell–derived cytokine IL-33 and produce IL-9 after exposure to IL-2, a cytokine that also positively affects IL-9 production in T cells. A variety of other cytokines have been described as enhancing IL-9 production in T cells in vitro, including members of the IL-1 family and IL-25, and some also have a stimulatory effect on IL-9 production in ILC2 cells. TH9 cells differentiated in vitro produce IL-9 and IL-10 but not IL-4, IL-5 or IL-13, whereas IL-9-producing ILC2 cells secrete large amounts of IL-5 and IL-13. The transcription factors STAT6, IRF4 and PU.1 contribute to TH9 differentiation, whereas the transcription factors GATA-3 and RORα seem to be involved in ILC2 development.

Epithelial cell damage

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IL-9R

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Figure 2 Hypothetical model for the function of IL-9 in the regulation of ILC2 cells. IL-9 production by ILC2 cells is induced by IL-33 from damaged epithelial cells. IL-9 enhances the survival and/or proliferation of ILC2 cells, as well as the production of IL-5 and IL-13 by ILC2 cells, in an autocrine manner. In the adaptive phase of the immune response, activated T cells provide IL-2, which further enhances the production of IL-9 by ILC2 cells. IL-5 and IL-13 promote classical features of the type 2 immune response, such as eosinophil recruitment, mucus production and mast-cell accumulation.

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It is not clear if such effects are mediated by promotion of the survival and/or proliferation of ILC2 cells or by an active influence on their cytokine expression. However, we must point out that in addition to serving as the main source of IL-9, ILC2 cells also seem to be an important target of IL-9, which suggests a previously unrecognized route of (possibly autocrine) regulation and fine tuning of the expres-sion of IL-13 and IL-5 by ILC2 cells (Fig. 2).

Support for that hypothesis has been provided by publications showing that both transgenic overexpression IL-9 and injection of recombinant IL-9 lead to the upregulation of IL-5 and IL-13 in the lungs19 and mesenteric lymph nodes43. Moreover, all the features of spontaneous airway inflammation noted in mice with transgenic expression of IL-9 are abrogated in the progeny of those mice crossed with IL-13-deficient mice44,45, as are features of IL-9-mediated upreg-ulation of genes in the intestinal mucosa46. This indicates that the function of IL-9 might be indirect (via IL-13 and/or IL-5), which supports the proposal that it has a regulatory role in the cytokine expression and survival and/or proliferation of cells that are able to produce type 2 cytokines. Furthermore, spontaneous airway inflam-mation is still evident in mice with transgenic expression of IL-9 on a background deficient in the RAG recombinase44, which emphasizes the idea that IL-9 may ‘preferentially’ target cells of the innate immune system rather than those of the adaptive immune system; for example, ILC2 cells in this inflammatory scenario.

Concluding remarks: IL-9 as a survival factor?Despite 20 years of active research, the function of IL-9 is not fully understood, and the literature frequently provides conflicting results of efforts to identify additional functions for IL-9. A recurrent feature and therefore perhaps a common denominator of the function of IL-9 seems to be its ability to promote the survival of an astonishingly large number of different cell types, including T cells4, mast cells3 and eosinophils47 (although here secondary effects via IL-5 might be important), as well as neurons12, tumors15 and epithelial cells48. If indeed one of the main functions of IL-9 is to mediate antiapoptotic effects in target cells49, it seems that many of the diverse effects of IL-9 might be a consequence of enhanced cell survival after exposure to IL-9. For example, a variety of helper T cells, including TH2, TH17 and Treg cells, respond to IL-9. Exposure of such effector cells to IL-9 either in vivo as a consequence of IL-2-mediated induction of IL-9 in ILC2 cells or during in vitro culture might result in improved survival. IL-9-induced survival could facilitate enhanced functional responses of all T cells that express IL-9R and thus mediate a diversity of differ-ent functions. Indeed, the reported enhanced suppressive activity of Treg cells cultured in the presence of IL-9 correlates with the improved survival of Treg cells26. If applied to other IL-9-responsive cell types, improved survival under the influence of IL-9 might help to explain diverse IL-9-mediated phenotypes such as enhanced antibody pro-duction by B cells and chemokine expression by epithelial cells, as well as enhanced protease expression by mast cells9. Such a ‘quantitative’ effect of IL-9 on immune processes would furthermore explain the puzzling lack of symmetry in the effects of IL-9 overexpression and IL-9 deficiency. In conclusion, we hypothesize that the main function of IL-9 may be to promote ‘signature functions’ of hematopoietic as well as nonhematopoietic types of target cells.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

published online at http://www.nature.com/doifinder/10.1038/ni.2303. reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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