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Several lines of evidence have documented the existence of a family of innate lymphoid cells (ILCs) that seem to be derived from a com-mon precursor cell dependent on the transcription inhibitor Id2. ILCs participate in diverse immunological processes, including host defense, the organogenesis of lymphoid tissues, and tissue remodeling and repair1. Those cells have been proposed to represent the innate counterparts of distinct types of CD4+ helper T lymphocytes on the basis of their production of the specific effector cytokines interferon-γ (IFN-γ), interleukin 5 (IL-5), IL-13, IL-17 or IL-22. Consistent with that proposal, the development and function of particular ILCs, des-ignated type 1, 2 or 3, appears to be dependent on key transcription factors that control the differentiation of the corresponding helper T cell subsets1. Given those findings, it is likely that conserved gene-regulatory networks are used to orchestrate functionally coherent patterns of expression of cytokine-encoding genes in cognate pairs of innate and adaptive lymphocytes.

The transcription factor Gfi1 is broadly expressed in the hematopoi-etic system and has critical roles in the self-renewal of hematopoietic stem cells2,3, neutrophil differentiation4–6 and B lymphopoiesis as well as T lymphopoiesis7,8. Moreover, Gfi1 functions in type 2 immune responses by controlling the IL-2-dependent population expansion of CD4+ type 2 helper T cells (TH2 cells) 9. Gfi1 also promotes TH2 polar-ization by antagonizing signaling by transforming growth factor-β (TGF-β) and therefore blocks the generation of the TH17 subset of helper T cells and regulatory T cells10. However, the corresponding

and potentially conserved functions of Gfi1 in the development and activation of innate lymphocytes that participate with TH2 cells in orchestrating type 2 immune responses remain to be explored.

Here we found that Gfi1 functioned in a cell-intrinsic manner to control the development, activation and proper specification of type 2 ILCs (ILC2 cells). Accordingly, inflammation elicited by IL-33, Nippostrongylus brasiliensis or protease allergen was severely impaired in the absence of Gfi1. Genetic and molecular analysis of ILC2 cells revealed a stringent requirement for Gfi1 in response to signaling by IL-33, but not to signaling by IL-25, as Gfi1 targeted and activated Il1rl1, the gene that encodes the ligand-binding subunit of the IL-33 receptor (ST2). Loss of Gfi1 in activated ILC2 cells resulted in a unique hybrid effector state characterized by derepression of the IL-17 inflammatory program, including the genes Rorc, Sox4, Il17a, Il17f, as well as those encoding the receptors IL-23R, TGF-βR3 and IL-1R1, which transduce signals known to promote the induction of TH17 cells. Notably, Gfi1-null ILC2 cells retained expression of many genes associated with type 2 inflammation, including IL-13. Given that Gfi1 is linked to reciprocal regulation of the fates of TH2 cells and TH17 cells, our results identify it as a conserved regulatory component that functions in cells of both the innate immune system and the adaptive immune system to sustain a type 2 cytokine response while repressing the IL-17 effector state. Our findings may have implications for the pathophysiology of severe asthma, which manifests deregulation of those two inflammatory states in a subset of patients.

1Department of Discovery Immunology, Genentech, South San Francisco, California, USA. 2Translational Immunology, Genentech, South San Francisco, California, USA. 3Institute for Systems and Genomics Biology and Department of Human Genetics, The University of Chicago, Chicago Illinois, USA. 4Department of Bioinformatics, Genentech, South San Francisco, California, USA. 5Department of Molecular Biology, Genentech, South San Francisco, California, USA. 6Center for Advanced Light Microscopy, Genentech, South San Francisco, California, USA. 7Department of Pathology, Genentech, South San Francisco, California, USA. 8Present address: Division of Immunobiology and the Center for Systems Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA. Correspondence should be addressed to C.J.S. (spooner.chauncey@gene.com) or H.S. (harinder.singh@cchmc.org).

Received 10 July; accepted 19 September; published online 20 October 2013; doi:10.1038/ni.2743

Specification of type 2 innate lymphocytes by the transcriptional determinant Gfi1Chauncey J Spooner1, Justin Lesch2, Donghong Yan2, Aly A Khan3, Alex Abbas4, Vladimir Ramirez-Carrozzi1, Meijuan Zhou2, Robert Soriano5, Jeffrey Eastham-Anderson6, Lauri Diehl7, Wyne P Lee2, Zora Modrusan5, Rajita Pappu1, Min Xu2, Jason DeVoss2 & Harinder Singh1,8

Type 2 innate lymphoid cells (ILC2 cells) participate in host defense against helminth parasites and in allergic inflammation. Given their functional relatedness to type 2 helper T cells (TH2 cells), we explored whether Gfi1 acts as a shared transcriptional determinant in ILC2 cells. Gfi1 promoted the development of ILC2 cells and controlled their responsiveness during infection with Nippostrongylus brasiliensis and protease allergen–induced lung inflammation. Gfi1 ‘preferentially’ regulated the responsiveness of ILC2 cells to interleukin 33 (IL-33) by directly activating Il1rl1, which encodes the IL-33 receptor (ST2). Loss of Gfi1 in activated ILC2 cells resulted in impaired expression of the transcription factor GATA-3 and a dysregulated genome-wide effector state characterized by coexpression of IL-13 and IL-17. Our findings establish Gfi1 as a shared determinant that reciprocally regulates the type 2 and IL-17 effector states in cells of the innate and adaptive immune systems.

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RESULTSGfi1 regulates ILC2 cell developmentEvidence suggests that common lymphoid progenitors (CLPs) func-tion as developmental intermediates of ILC2 cells11,12. Given that Gfi1 functions in early lymphopoiesis2,5,7 and is expressed in CLPs2, we investigated whether Gfi1 is important for the development of ILC2 cells in the bone marrow. To examine the expression and function of Gfi1 in the development and activation of ILC2 cells, we used mice with sequence encoding green fluorescent protein (GFP) knocked in to the Gfi1 coding region, which served as a reporter and as a loss-of-function mutation (Gfi1GFP)13. Gfi1 was expressed in precursors of ILC2 cells (‘ILC2 precursors’ (ILC2Ps)) in the bone marrow of Gfi1+/GFP mice (Fig. 1a). Moreover, Gfi1 expression was higher in ILC2Ps than in CLPs (Fig. 1b). The transition from CLP to ILC2P is associated with induction of expression of ST2 (the IL-33 receptor) and GATA-3 (a transcription factor required for the development of ILC2 cells)11,14. Accordingly, the upregulation of Gfi1 in ILC2Ps was correlated with the induction of Gata3 (GATA-3) and Il1rl1 (ST2) transcripts (Fig. 1b). IL-33 has been shown to promote the generation

of ILC2 cells from CLPs in vitro11. In keeping with those findings, the administration of IL-33 promoted the development of ILC2 cells in the bone marrow with higher expression of ST2 and of KLRG1, a maturation marker of ILC2 cells14 (Fig. 1c,d). Notably, Gfi1 expres-sion occurred in a graded manner in a developmental continuum spanning CLPs, ILC2Ps and ILC2 cells and was positively correlated with ST2 expression (Fig. 1e). This suggested a functional relation-ship between Gfi1 and signaling by IL-33 in the development of ILC2 cells.

Given the dynamic expression of Gfi1 during the development of ILC2 cells, we analyzed the function of Gfi1 in the generation of ILC2 cells during steady-state hematopoiesis and in response to IL-33. The bone marrow of Gfi1GFP/GFP mice (called ‘Gfi1−/− mice’ here) had fewer ILC2Ps than did the bone marrow of Gfi1+/+ mice (Fig. 1f,g and Supplementary Fig. 1a). The administration of IL-33, which promoted the generation of ILC2 cells in Gfi1+/+ mice, had a negligible effect on the development of ILC2 cells in the absence of Gfi1 (Fig. 1f–h and Supplementary Fig. 1a). To determine if Gfi1 functioned in a cell-autonomous manner in regulating the development of ILC2

Figure 1 Gfi1 regulates the development of ILC2 cells. (a) GFP expression in ILC2Ps (Lin−IL-7rα+ST2+Sca-1+ICOS+) from the bone marrow of naive Gfi1+/+ or Gfi1+/GFP mice, assessed by flow cytometry. (b) Expression of Gfi1, Gata3 and Il1rl1 in CLPs (Lin−IL-7rα+ST2−ICOS−) and ILC2Ps of naive Gfi1+/+ mice (n = 5 (pooled) per sample). ND, not detectable. (c,d) Expression of ST2 (c) and KLRG1 (d) on Lin−IL-7rα+ bone marrow cells from Gfi1+/+ mice treated with PBS or IL-33. Isotype, isotype-matched control antibody. (e) GFP fluorescence in bone marrow GFP− ILC2Ps from Gfi1+/+ mice (Control) and bone marrow CLPs (Lin−IL-7rα+ST2−), ILC2Ps (Lin−IL-7rα+ST2+) and IL-33-elicited ILC2 cells (Lin−IL-7rα+ST2hi) from Gfi1+/GFP mice (n = 5 per group), presented in arbitrary units (AU) as median fluorescence intensity (MFI). (f) Expression of ICOS and Sca-1 on Lin−ST2+ bone marrow cells from Gfi1+/+ or Gfi1−/− mice treated with PBS or IL-33, assessed by flow cytometry. Numbers in top right corners indicate percent ICOS+Sca-1+ (ILC2P) cells. (g,h) Quantification of Lin−IL-7rα+ST2+Sca-1+ ICOS+ ILC2Ps (g) and Lin−IL-7rα+ST2hi Sca-1+ICOS+ ILC2 cells (h) in the bone marrow of Gfi1+/+ and Gfi1−/− mice treated with PBS (n = 6 mice per group) or IL-33 (n = 12 mice per group). (i) Flow cytometry of bone marrow cells (gated on Lin−IL-7rα+Sca-1+ICOS+ cells) from CD45.1+ congenic mice reconstituted with mixed bone marrow from wild-type (CD45.1+) mice and Gfi1+/− (left) or Gfi1fl/− (right) CD45.2+ mice expressing tamoxifen-inducible Cre (ERT2-Cre) (n = 10 mice per group) and left untreated (UT) or treated with tamoxifen alone (Tam) or tamoxifen and IL-33 (Tam + IL-33). Numbers in outlined areas indicate percent CD45.1+CD45.2− cells (top left) or CD45.1−CD45.2+ cells (bottom right). (j) Summary of the results in i. Each symbol (a,g,h,j) represents an individual mouse; small horizontal lines indicate the mean (± s.d.). NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (Student’s t-test). Data are representative of three independent experiments (a), one experiment with three samples (b), two independent experiments (c,d,f–j) or one experiment with five mice per genotype and condition (e; mean and s.d. in b,g,h,j).

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cells, we reconstituted lethally irradiated CD45.1+ congenic mice with mixed bone marrow from wild-type (CD45.1+) mice and CD45.2+ mice with expression of a tamoxifen-inducible Cre recombinase fusion protein and a Gfi1GFP allele plus either a wild-type Gfi1 allele (Gfi1+/GFP) or a loxP-flanked Gfi1 allele (Gfi1GFP/fl) (Supplementary Fig. 1b). This experimental design allowed us to avoid the hemato-poietic stem cell defect of Gfi1−/− mice, which are impaired in the ability to repopulate the hematopoietic system2,3. Consistent with the defect in the development of ILC2 cells observed in the bone marrow of Gfi1−/− mice, tamoxifen-inducible deletion of Gfi1 led to a significant reduction in the frequency of ILC2Ps (Fig. 1i,j). Furthermore, the defect in the development of ILC2 cells associ-ated with the loss of Gfi1 was exacerbated by the administration of IL-33 (Fig. 1i,j), consistent with a failure of preexisting Gfi1-deficient ILC2Ps to respond to IL-33. Together, these findings demonstrated a cell-intrinsic role for Gfi1 in regulating the development of ILC2 cells during steady-state hematopoiesis as well as in an IL-33-instigated stress response.

Gfi1 controls protective and pathological ILC2 cell responsesDespite the developmental defect caused by loss of Gfi1, ILC2 cells were detectable in the lungs and mesenteric lymph nodes (MLNs) of Gfi1−/− mice, with normal numbers in the latter anatomical

location (Fig. 2). Through the use of the Gfi1GFP allele, we noted that lung and MLN ILC2 cells exhibited equivalent and high expression of GFP (Supplementary Fig. 2a,b). We next determined if Gfi1−/− ILC2 cells underwent activation in the context of infection with the parasitic intestinal nematode N. brasiliensis, which elicits a potent ILC2 cell response that is required for worm expulsion15,16. In the absence of Gfi1, ILC2 cells in the lungs failed to proliferate in response to infection (Fig. 2a and Supplementary Fig. 3a). Consistent with those findings, the concentrations of IL-5 and IL-13 were significantly lower in bronchoalveolar lavage (BAL) fluid from N. brasiliensis– infected Gfi1−/− mice than in that from their Gfi1+/+ counterparts (Fig. 2b,c). Accordingly, recruitment of eosinophils and goblet-cell hyperplasia, two prominent features of the activity of IL-5 and IL-13, respectively, were diminished in the lungs of N. brasiliensis–infected Gfi1−/− mice relative to that in their Gfi1+/+ counterparts (Fig. 2d,e and Supplementary Fig. 3b,d).

In contrast to their counterparts in the lungs, Gfi1−/− ILC2 cell populations in the MLNs expanded in response to infection with N. brasiliensis, albeit to a lesser extent than did their wild-type coun-terparts (Fig. 2f). The defects in the activation of ILC2 cells in Gfi1−/− mice were associated with a severe impairment in worm clearance (Fig. 2g). We noted that loss of Gfi1 also attenuated additional com-ponents of the type 2 inflammatory response elicited by N. brasiliensis,

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Figure 2 Gfi1 regulates the activation of ILC2 cells during infection with N. brasiliensis. (a) Quantification of ILC2 cells (Lin−IL-7rα+ Sca-1+ICOS+) in the lungs of uninfected (UI) and N. brasiliensis–infected (N. bras) Gfi1+/+ mice (n = 5 per group) and Gfi1−/− mice (n = 5 per group). (b,c) Concentration of IL-5 (b) and IL-13 (c) in BAL fluid from Gfi1+/+ and Gfi1−/− mice left uninfected (n = 5 mice per group) or infected with N. brasiliensis (n = 10 mice per group), assessed 7 d after infection. (d,e) Histological analysis of sections of lungs from uninfected or N. brasiliensis–infected Gfi1+/+ and Gfi1−/− mice (n = 5 per group), stained with hematoxylin and eosin or periodic acid–Schiff. Arrows (e) indicate mucin- positive cells. Original magnification, ×2.5 (d) or ×10 (e). (f) Quantification of ILC2 cells (Lin−IL-7rα+Sca-1+ICOS+) in the MLNs of uninfected and N. brasiliensis–infected Gfi1+/+ mice (n = 5 per group) and Gfi1−/− mice (n = 5 per group). (g,h) Worm burden in the small intestine of Gfi1+/+ and Gfi1−/− mice (g) and of Gfi1fl/fl mice and Gfi1+/fl or Gfi1fl/fl mice expressing Cre engineered for conditional inactivation of Gfi1 in the hematopoietic system (Vav-Cre) (h), assessed 10 d after infection with N. brasiliensis. (i) Live cells among Lin−IL-7rα+ Sca-1+ICOS+ cells isolated from the MLNs of N. brasiliensis–infected Gfi1+/+ mice (n = 4) or Gfi1−/− mice (n = 2) and cultured for 6 d in vitro. (j) ST2 expression on cells cultured as in i. (k) Intracellular IL-5 and IL-13 in ILC2 cells cultured as in i. Numbers in quadrants indicate percent IL-5+IL-13+ cells (top right) or IL-5− IL-13+ cells (bottom right). Each symbol (a–c,f–h) represents an individual mouse; small horizontal lines indicate the mean (± s.d.). NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (Student’s t-test). Data are from two independent experiments (a–c,i–k; mean and s.d. in i), one experiment (d–g) or two experiments (h).

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including reduced numbers of CD4+ T cells (Supplementary Fig. 3c,e,f). It has been established that both ILC2 cells and TH2 cells contribute to worm expulsion16–18. Thus, the failure of Gfi1−/− mice to clear the worm infection was probably a consequence of reduced numbers of activated ILC2 cells as well as TH2 cells. Given that Gfi1 has been shown to be required for the differentiation of intestinal epithelial cells19, it was possible that a defect in the epithelial com-partment also affected worm clearance. However, this was unlikely, as conditional inactivation of Gfi1 in the hematopoietic system still manifested a severe impairment in worm clearance (Fig. 2h). Notably, cultured Gfi1−/− ILC2 cells isolated from the MLNs of N. brasiliensis–infected mice exhibited lower expression of ST2, which correlated with a reduced proliferative capacity (Fig. 2i,j). Although Gfi1−/− ILC2 cells had IL-13 expression similar to that of Gfi1+/+ ILC2 cells, their IL-5 expression was lower than that of their Gfi1+/+ counterparts (Fig. 2k). Thus, Gfi1 regulated the population expansion of ILC2 cells and specified their cytokine output.

To analyze the role of Gfi1 in regulating ILC2 cell–mediated inflam-mation that is independent of an adaptive T cell response, we used a protease allergen–induced lung-inflammation model, which has been

shown to induce eosinophil infiltration and mucous hyperproduction in the lung20. Intranasal instillation of papain into Gfi1−/− mice failed to induce activation of ILC2 cells, eosinophilia or the production of IL-5 or IL-13 in the lungs (Fig. 3). This was consistent with the failure of lung ILC2 cells to undergo population expansion in N. brasiliensis– infected Gfi1−/− mice. Thus, both protective and pathologic ILC2 cell–mediated responses were attenuated in the absence of Gfi1.

Gfi1 regulates the responsiveness of ILC2 cells to IL-33IL-33 signaling has been shown to serve an important role in the acti-vation of ILC2 cells in the lungs of helminth-infected mice21. In con-trast, both IL-33 and IL-25 are known to contribute to the activation of ILC2 cells in the gut16. Thus, we investigated whether Gfi1 controls the responsiveness of ILC2 cells to IL-33 and IL-25 differently, given the distinct responses of ILC2 cells in the lungs and the MLNs of N. brasiliensis–infected Gfi1−/− mice. To test our proposition, we ini-tially used the Gfi1GFP reporter mice described above to correlate Gfi1 expression with the responsiveness of ILC2 cells to IL-33 or IL-25. Administration of each cytokine induced a significant increase (20- to 80-fold) in ILC2 cells that expressed Gfi1 (Fig. 4a,b). Notably,

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Figure 3 Elicitation of ILC2 cells by papain challenge is impaired in the lungs of Gfi1−/− mice. (a) Flow cytometry of ILC2 cells (gated on Lin−ST2+Sca-1+ cells) in the lungs of papain-treated Gfi1+/+ and Gfi1−/− mice (n = 5 per group). Numbers in quadrants indicate percent cells in each. (b,c) Quantification of lung ILC2 cells (Lin−IL-7rα+ST2+Sca-1+ICOS+) (b) or eosinohils (CD45+Siglec-F+F4/80int) (c) in papain-treated Gfi1+/+ and Gfi1−/− mice. (d,e) Concentration of IL-5 (d) and IL-13 (e) in BAL fluid from papain-treated Gfi1+/+ and Gfi1−/− mice. Each symbol (b–e) represents an individual mouse; small horizontal lines indicate the mean (± s.d.). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (Student’s t-test). Data are from one experiment (a,d,e) or two experiment (b,c).

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Figure 4 Gfi1 expression is correlated with the responsiveness of ILC2 cells to IL-33. (a) Expression of ICOS and IL-7rα in cells in MLNs from Gfi1+/+ and Gfi1+/GFP mice treated with PBS, IL-33 or IL-25 (left margin), assessed by flow cytometry (gated on Lin− cells). (b) Quantification of IL-7rα+Sca-1+ICOS+ cells in MLNs from Gfi1+/+ mice (Lin−), Gfi1+/GFP mice (Lin−GFP+ gate) or Gfi1+/GFP mice (Lin−GFP− gate) treated with PBS, IL-33 or IL-25. Each symbol represents an individual mouse (n = 5–7 per group); small horizontal lines indicate the mean (± s.d.). (c) Quantification of live cells among Lin−GFP− or Lin−GFP+ cells sorted from the MLNs of IL-33-treated Gfi1+/GFP mice and cultured in vitro with IL-2, IL-7 and IL-33, assessed on days 2, 4 and 6. (d,e) Expression of c-Kit and Sca-1 (d) or intracellular IL-5 and IL-13 (e) in Lin−GFP+ cells cultured for 6 d in vitro with IL-2, IL-7 and IL-33, assessed by flow cytometry. Numbers in quadrants (a,d,e) indicate percent cells in each. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (Student’s t-test). Data are representative of three experiments (a,b) or two experiments (c,d,e; mean ± s.d. in c; n = 4 samples in d,e).

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results demonstrated a selective and critical role for Gfi1 in eliciting ILC2 cells in response to IL-33 signaling.

Gfi1 specifies and maintains the ILC2 cell effector stateTo gain molecular insight into the function of Gfi1 in ILC2 cells, we performed genome-wide expression analysis of cultured cells elicited from Gfi1+/+ and Gfi1−/− mice with IL-25 (Fig. 6a). Notably, Gfi1−/− ILC2 cells had a lower abundance of Il1rl1 transcripts than did Gfi1+/+ ILC2 cells (Fig. 6a and Supplementary Fig. 4a). As a consequence, fewer Gfi1−/− ILC2 cells than Gfi1+/+ ILC2 cells expressed ST2 on the cell surface (Supplementary Fig. 4b). Accordingly, phosphor-ylation of the p65 subunit of the transcription factor NF-κB, which is induced in response to IL-33, was impaired in Gfi1−/− ILC2 cells (Supplementary Fig. 4c). Thus, the diminished proliferative capac-ity of Gfi1−/− ILC2 cells in response to IL-33 in vivo (Fig. 5a–c) and in vitro (Fig. 5d and Supplementary Fig. 4d) was most probably attributable to diminished expression of and signaling via ST2. Gfi1 also positively regulated Crlf2 (Fig. 6a), which encodes the receptor for thymic stromal lymphopoietin (TSLP), shown to elicit the activa-tion of ILC2 cells22. Notably, Gfi1−/− ILC2 cells had higher expression of Il17rb, which encodes a subunit of the IL-25 receptor, than

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Figure 5 Gfi1 is required for the responsiveness of ILC2 cells to IL-33. (a) Flow cytometry of ILC2 cells (Lin−Sca-1+) in MLNs of Gfi1+/+ or Gfi1−/− mice treated with PBS, IL-33 or IL-25 (left margin). Numbers in quadrants indicate percent cells in each. (b) Incorporation of BrdU by ILC2 cells (Lin−IL-7rα+Sca-1+ICOS+) in MLNs of Gfi1+/+ or Gfi1−/− mice (n = 5 per group) treated PBS, IL-33 or IL-25. (c) Quantification of ILC2 cells from MLNs of Gfi1+/+ or Gfi1−/− mice (n = 5–8 per group). (d) Quantification of Lin− cells sorted from the MLNs of IL33-treated Gfi1+/+ mice (n = 5) or Gfi1−/− mice (n = 3) and cultured in vitro in IL-2, IL-7 and IL-33. (e) Concentration of IL-5 in the peritoneal cavity of Gfi1+/+ or Gfi1−/− mice (n = 12 per group) treated with PBS, IL-33 or IL-25. (f) Quantification of eosinophils (SSChiSiglec-F+F4/80int) in the peritoneum of mice treated with PBS (n = 11–12 mice per group), IL-33 (n = 6 mice per group) or IL-25 (n = 6 mice per group). (g) Periodic acid–Schiff staining of goblet cells in small intestine of Gfi1+/+ or Gfi1−/− mice treated PBS, IL-33 or IL-25. Scale bars, 100 µm. (h) Quantification of Periodic acid–Schiff–positive (PAS+) cells in g. Each symbol (c,e,f,h) represents an individual mouse; small horizontal lines indicate the mean (± s.d.). NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (Student’s t-test). Data are representative of three independent experiments (a,c), one experiment (b), two independent experiments (d; mean ± s.d.), four independent experiments (e,f) or two independent experiments with six mice per group (h).

the responsiveness of ILC2 cells to IL-33 in vivo was restricted to cells that expressed Gfi1 (Fig. 4a,b). Moreover, sorted GFP+ cells nega-tive for lineage markers (Lin−GFP+ cells), but not their Lin−GFP− counterparts, rapidly proliferated in vitro (Fig. 4c) and gave rise to ILC2 cells that expressed IL-5 and IL-13 (Fig. 4d,e). However, the administration of IL-25 elicited both GFP+ and GFP− ILC2 cells in the MLNs (Fig. 4a,b), consistent with an ability to elicit ILC2 cells in the gut in a Gfi1-independent manner. These findings suggested that Gfi1 activity may be more stringently needed to mediate signaling by IL-33 rather than signaling by IL-25.

To genetically assess whether Gfi1 regulated ILC2 cell activation differently in response to IL-33 and IL-25, we administered those cytokines to Gfi1+/+ and Gfi1−/− mice. As Gfi1−/− mice had normal numbers of ILC2 cells in the MLNs (Fig. 2f), this allowed strict com-parison of their activation properties relative to those of their wild-type counterparts. Notably, the activation of Gfi1−/− ILC2 cells was considerably impaired in response to IL-33, as shown by the lack of any appreciable population expansion of ILC2 cells and diminished incorporation of the thymidine analog BrdU in vivo (Fig. 5a–c). In contrast, administration of IL-25 induced the population expan-sion of Gfi1−/− ILC2 cells, albeit to a lesser extent than that of Gfi1+/+ ILC2 cells (Fig. 5a–c). Consistent with the in vivo findings, Lin− cells isolated from the MLNs of IL-33-treated Gfi1−/− mice were unable to proliferate in vitro (Fig. 5d), unlike their counterparts exposed to IL-25 (discussed below). Induction of IL-5, eosinophilia and goblet-cell hyperplasia were attenuated in Gfi1−/− mice relative to that in Gfi1+/+ mice after the administration of either IL-33 or

IL-25 (Fig. 5e–h). That was most probably a consequence of the reduced number of activated ILC2 cells in Gfi1−/− mice (Fig. 5a–c) that had diminished expression of IL-5 (Fig. 2k). Together, these

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compared the genome-wide expression pattern of Gfi1−/− ILC2 cells with that of primary GATA-3+ ILC2 cells and RORγt+NKp46− ILCs14. Analysis with IPA software (Ingenuity pathway analysis) revealed a set of genes ‘preferentially’ expressed by GATA-3+ ILC2 cells that encode molecules that function in eosinophil inflammation that were downregulated in Gfi1−/− ILC2 cells (Supplementary Fig. 5c and Supplementary Table 1). Conversely, Gfi1−/− cells manifested derepression of an equivalent number of genes encoding molecules associated with neutrophil inflammation that were shared with RORγt+NKp46− ILCs (Supplementary Fig. 5c and Supplementary Table 2). Thus, Gfi1−/− ILC2 cells manifested an unusual hybrid cellular state characterized by the attenuation of some, but not all, components of the type 2 effector program and concomitant activa-tion of the IL-17 effector state.

To specifically assess the function of Gfi1 in activated ILC2 cells, we conditionally deleted Gfi1 in cultured cells through the use of a Gfi1fl allele and expression of tamoxifen-inducible Cre. That reca-pitulated the phenotypes that resulted from a germline mutation of Gfi1, including reduced cellular proliferation, diminished expression of ST2 and IL-5 and derepression of IL-17A expression (Fig. 6g–j). We obtained similar results with MLN ILC2 cells with a Gfi1fl allele that were elicited by either IL-33 or IL-25 (C.S. and H.S., data not shown). The decrease in ST2 expression after conditional dele-tion of Gfi1 was less pronounced than that in Gfi1−/− ILC2 cells (Supplementary Fig. 4b), which may have been due to the stability of residual Gfi1 and ST2. Nevertheless, these results demonstrated that Gfi1 was continuously required for maintenance of the type 2 effector state of ILC2 cells and for prevention of inappropriate derepression of the Il17 locus. In keeping with that conclusion, the restoration of

did Gfi1+/+ ILC2 cells (Fig. 6a and Supplementary Fig. 4a). This may have been due to compensation for the impaired signaling by IL-33 that was enabled by selective population expansion of Gfi1−/− cells with higher expression of the IL-25 receptor. Alternatively, Gfi1 might target Il17rb and negatively regulate its expression (discussed below). In addition to genes encoding cytokine receptors of ILC2 cells, Gfi1 also regulated the expression of genes encoding the effec-tor cytokines IL-5 and IL-6 (refs. 16,23) (Fig. 6a and Supplementary Fig. 4a,e). In contrast, expression of IL-13 was not dependent on Gfi1 (Supplementary Fig. 4e). The impairment in the gene-expression program of Gfi1−/− ILC2 cells was accompanied by reduced expression of the ILC2 cell transcriptional determinant GATA-3 (Fig. 6a and Supplementary Fig. 4a,f).

Various genes associated with IL-17-mediated inflammation, including Il1r1, Tgfbr3, Il23r, Sox4, Rorc, Il17a and Il17f, were dere-pressed in Gfi1−/− ILC2 cells (Fig. 6a and Supplementary Fig. 5a). In fact, Il17a and Il17f were two of the genes most derepressed in Gfi1−/− ILC2 cells (Fig. 6a). Notably, those genes were also dere-pressed in primary ILC2 cells isolated from the MLNs of IL-25-treated Gfi1−/− mice (Fig. 6b,c). Consistent with those findings, a large frac-tion of Gfi1−/− ILC2 cells that were either cultured in vitro or analyzed immediately after isolation (without culture) expressed IL-17A and IL-17F (Fig. 6d,e and Supplementary Fig. 5b). However, we did not observe substantial expression of IFN-γ in either of those groups of Gfi1−/− ILC2 cells (Fig. 6f and Supplementary Fig. 5b). That result was consistent with the lack of induction of Tbx21 (which encodes the transcription factor T-bet) in the absence of Gfi1 (Fig. 6a). Given that a large fraction of Gfi1−/− ILC2 cells coexpressed IL-13 along with IL-17A and IL-17F (Fig. 6d,e and Supplementary Fig. 5b), we

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*Figure 6 Loss of Gfi1 in ILC2 cells results in a hybrid effector state. (a) Genome-wide microarray analysis of ILC2 cells (Lin−IL-7rα+Sca-1+ICOS+c-Kit+GFP+) sorted from MLNs of IL-25-treated Gfi1+/+ and Gfi1−/− mice and cultured for 6 d in vitro, presented as a ‘volcano’ plot; colors indicate genes downregulated (pink) or upregulated (blue) in Gfi1−/− cells relative to their expression in Gfi1+/+ cells. (b,c) Quantitative PCR analysis of Il17a transcripts (b) and Il17f transcripts (c) in freshly isolated Lin−IL-7rα+Sca-1+ ICOS+c-Kit+ cells sorted from MLNs of IL-25-treated Gfi1+/+ or Gfi1−/− mice. (d) Intracellular expression of IL-17A and IL-13 in ILC2 cells cultured for 6 d in vitro. (e,f) Intracellular expression of IL-17A and IL-13 (e) or IFN-γ and IL-13 (f) in ILC2 cells sorted from MLNs of IL-25-treated Gfi1+/+ mice (n = 4) or Gfi1−/− mice (n = 4) and stimulated for 4 h with the phorbol ester PMA and ionomycin. (g) Live cells among ILC2 cells (Lin−IL-7rα+Sca-1+ICOS+c-Kit+GFP+) sorted from MLNs of IL-33-treated Gfi1fl/− mice (n = 4) and Gfi1fl/− mice that express tamoxifen-inducible Cre (ERT2-Cre) (n = 4), then cultured for 6 d in IL-2, IL-7, IL-25, IL-33 and tamoxifen (100 nM). (h) ST2 expression in cells from mice as in g. (i,j) Intracellular expression of IL-5 and IL-13 (i) or IL-17A and IL-13 (j) in cells treated as in g, then stimulated for 4 h with PMA and ionomycin in the presence of monensin (j only) before IL-17A detection. Numbers in quadrants (d–f,i,j) indicate percent cells in each. *P < 0.01 and **P < 0.001 (Student’s t-test). Data are representative of two experiments (a–d) or two independent experiments (e–j; mean and s.d. in b,c,g).

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Gfi1 expression in Gfi1−/− ILC2 cells resulted in repression of IL-17A expression (Supplementary Fig. 5d).

To determine which genes associated with type 2 and IL-17-dependent inflammation were directly activated or repressed, respectively, by Gfi1 in ILC2 cells, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). The Gfi1-binding-site motif was the most highly represented in sequence elements targeted by Gfi1 (Fig. 7a). Interestingly, Gfi1 targeted the promoter and an upstream element in the Il1rl1 locus (Fig. 7b), which demonstrated a direct role for Gfi1 in the control of IL-33 signaling in ILC2 cells. Thus, Gfi1 regulated the proliferation of ILC2 and TH2 cells by controlling pivotal cytokine signaling pathways, such as IL-33 and IL-2 (ref. 9), respectively. Notably, Gfi1 also targeted genes that encode key ele-ments of the type 2 inflammatory program (for example, Il5, Gata3, Crlf2 and Il17rb) and IL-17 inflammatory program (for example, Sox4, Il17a, Il17f, Il1r1, Tgfbr3 and Itgae) (Fig. 7b,c and Supplementary Fig. 6), which suggested that it functioned both as an activator and as a repressor, albeit in a context-dependent manner. These findings identified Gfi1 as a crucial transcriptional determinant that func-tioned at the nexus of type 2 and IL-17-dependent inflammatory pro-grams in lymphocytes of the innate and adaptive immune systems.

DISCUSSIONIn this study we identified a cell-intrinsic role for Gfi1 in regulating the development of ILC2 cells, including the transition from CLP to ILC2P and as ILC2Ps underwent further maturation. Gfi1 also regulated the differentiated state of ILC2 cells. Gfi1−/− ILC2 cells were impaired in their expression of IL-5. The defects in the development and dif-ferentiation of Gfi1−/− ILC2 cells were probably due in part to their impaired expression of GATA-3. Notably, Gfi1 also regulated the expres-sion of Il1rl1, which encodes the ST2 subunit of the IL-33 receptor. We observed that the transitions in the development and maturation

of ILC2 cells were accompanied by increased expression of the IL-33 receptor. Although a role for IL-33 signaling in the development of ILC2 cells in the bone marrow remains to be elucidated, it is possible that Gfi1-deficient mice have a defect in the generation of ILC2Ps partly as a consequence of an inability to properly induce expression of Il1rl1.

We demonstrated that Gfi1 was critical for the responsiveness of ILC2 cells to IL-33 signaling in peripheral tissues. The reduced expression of ST2 in Gfi1−/− ILC2 cells probably accounted for the profound defect in their population expansion in lungs and MLNs in response to IL-33. As stimulation with IL-33 in vivo led to increased expression of Gfi1 in ILC2 cells in the bone marrow, we propose that Gfi1 and ST2 function as components of an IL-33-dependent positive feedback loop to stimulate the activation of ILC2 cells. Notably, Gfi1 has been shown to function as a negative regulator of its own expres-sion13. That finding was supported by our observation of enhanced expression of GFP in Gfi1fl/GFP ILC2 cells after tamoxifen-induced conditional inactivation of Gfi1 in the bone marrow of chimeric mice (data not shown). On the basis of those results, we propose a Gfi1-dependent regulatory network that underlies IL-33-mediated ILC2 activation. The overall network architecture comprises a two-node feedback loop that juxtaposes a positive feedback loop between Gfi1 and the IL-33–ST2 signaling axis with autoinhibition by Gfi1. That architecture seems to be suitably configured to enable the rapid and robust proliferation of ILC2 cells in response to IL-33. Together our findings establish a new link between Gfi1 and a receptor system with a pivotal role in initiating type 2 immune responses. Moreover, Gfi1 probably has a recurrent role in regulating IL-33 responses, given that the IL-33 receptor is used by multiple cell types, such as mast cells and TH2 cells, that function in type 2 immunity24.

Our results have demonstrated that Gfi1 was needed to properly specify the type 2 effector program, as the expression of IL-5 and GATA-3 was impaired in the absence of Gfi1. We note that the reduc-tion in expression of both IL-5 and GATA-3 in Gfi1−/− ILC2 cells was greater than that of their transcripts, which suggested both tran-scriptional and post-transcriptional modes of Gfi1-mediated control. In keeping with those findings, Gfi1 has been linked to the stabiliza-tion of GATA-3 protein in TH2 cells by inhibiting its ubiquitin- and proteasome-dependent degradation25. Thus, Gfi1 regulates ILC2 cells by inducing the expression of type 2 cytokine receptors and effec-tor cytokines, as well as a cognate transcriptional determinant. Given that GATA-3 may positively regulate Gfi1 expression26, such a self- reinforcing feed-forward loop may be critical for the generation, popu-lation expansion and stabilization of a type 2 effector state in both innate and adaptive lymphocytes.

Notably, Gfi1 functioned in ILC2 cells to actively repress the IL-17 effector program. Those results paralleled the regulatory functions of Gfi1 in neutrophils, in which it targets promoters of lineage-specific genes with high expression, as well as those of macrophage genes that are repressed27. Genetic evidence has demonstrated a critical role for the transcription factor Sox4 in the activation of Rorc (which encodes the transcription factor RORγt) and the generation of IL-17-produc-ing γδ T cells28. Given that Gfi1 did not seem to target Rorc in ILC2 cells (data not shown), derepression of Sox4 may account, in part, for the greater abundance of Rorc transcripts and induction of the IL-17 inflammatory program in Gfi1−/− ILC2 cells. It is likely that the transcription-repressive activity of Gfi1 in ILC2 cells is mediated by the histone methyltransferase G9a, which interacts with Gfi1 (ref. 29) and serves a similar function in TH2 cells30. Notably, the TH1 effector state seems to be actively repressed in TH2 cells by the histone methyltrans-ferase SUV39H1 (ref. 31), but probably in a Gfi1-independent manner. We therefore hypothesize that Gfi1-dependent and Gfi1-independent

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Figure 7 Gfi1 targets and regulates genes encoding components of the type 2 and IL-17 expression programs. (a) Most highly represented motif in the Gfi1 cistrome in ILC2 cells, as assessed by the MEME (‘Multiple Em for Motif Elicitation’) suite of motif-based sequence-analysis tools and the TOMTOM motif-comparison tool; letter size indicates nucleotide frequency (presented as ‘bits’) at each position (horizontal axis). (b) Occupancy by Gfi1 at the Il1rl1, Il5, Sox4 and Il17a loci in ILC2 cells. (c) Regulatory network of genes targeted and regulated by Gfi1 in ILC2 cells, generated with the BioTapestry tool for analysis of genetic regulatory networks. Magenta lines indicate regulatory connections revealed by integration of gene expression and ChIP-seq analysis of Gfi1; solid and dashed black lines indicate previously reported connections in TH2 or γδ T cells. It remains to be determined if GATA-3 directly activates Gfi1 (dashed line).

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regulatory circuits mediate repression of the TH17 effector program and the TH1 effector program, respectively, in ILC2 cells and TH2 cells.

The dual role of Gfi1 in promoting the expression of TH2 cytokines while actively repressing the IL-17 program has implications for the patho-physiology of asthma, as a subset of patients with asthma display type 2 and IL-17-mediated inflammation in the lungs32. T cells that coexpress type 2 cytokines and IL-17 have been identified in experimental models of allergic inflammation33 and in asthmatic patients34. We propose that pathophysiological signals that antagonize the activity or expression of Gfi1 could enable an ongoing type 2 response initiated by ILC2 cells with IL-5-stimulated eosinophilic inflammation to evolve into a mixed inflam-matory phase that includes the IL-17-mediated recruitment of neutrophils. In that pathophysiological scenario, ILC2 cells could switch from express-ing IL-5 to expressing IL-17 while retaining expression of IL-13. Thus, Gfi1 would have a pivotal role in preventing ILC2 cells and TH2 cells from adopting the effector state characterized by IL-17-driven inflammation.

Our findings also have evolutionary implications about the emer-gence of cells of the adaptive immune system, such as T cells, from their more primitive innate counterparts. Helper T cells seem to share many attributes with ILCs, including survival signals (for example, signaling by IL-2 and IL-7), transcriptional regulators that control distinct cell fates (RORγt, Ahr, GATA-3 and Gfi1) and cytokine production (IL-4, IL-5, IL-9, IL-13, IL-17 and IL-22)35. Thus, it is likely that preexisting regula-tory circuits operative in cells of the innate immune system were coopted by their counterparts in the adaptive immune system. Those adaptive cells could utilize the same cytokine-dependent effector mechanisms, thereby amplifying specific immune responses. Notably, the adaptive cells could respond to a more diverse set of pathogen components by exploiting a large combinatorial set of antigen receptors generated by somatic DNA rearrangement and retain memory of such encounters.

METHODSMethods and any associated references are available in the online version of the paper.

Accession codes. GEO: microarray data, GSE45621; ChIP-seq data, GSE50806.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACKnoWLEDgMEntSWe thank members of the Singh laboratory, as well as H. Xi, M. van Lookeren Campagne, D. Holmes, S. Lutz and W. Ouyang, for discussions; and the Mouse Genetics Department, FACS, microarray, DNA sequencing, microscopy, and oligonucleotide synthesis facilities at Genentech for assistance and reagents.

AUtHoR ContRIBUtIonSC.J.S. and H.S. designed and interpreted the experiments and wrote the manuscript; C.J.S., D.Y. and M.X. used the N. brasiliensis infection model; C.J.S., J.L., and J.D. did the experiments with administration of IL-25 and IL-33; C.J.S, M.Z. and W.P.L. used the papain lung-inflammation model; J.E.-A. and L.D. assisted with the animal pathology; V.R.-C. and R.P. assisted with the in vitro ILC2 cell cultures; R.S. and Z.M. did the microarray analysis; and A.A. and A.A.K. did the computational and statistical analysis of the microarray and ChIP-seq data, respectively.

CoMPEtIng FInAnCIAL IntEREStSThe authors declare competing financial interests: details are available in the online version of the paper.

reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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18. Voehringer, D., Reese, T.A., Huang, X., Shinkai, K. & Locksley, R.M. Type 2 immunity is controlled by IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate immune system. J. Exp. Med. 203, 1435–1446 (2006).

19. Shroyer, N.F., Wallis, D., Venken, K.J., Bellen, H.J. & Zoghbi, H.Y. Gfi1 functions downstream of Math1 to control intestinal secretory cell subtype allocation and differentiation. Genes Dev. 19, 2412–2417 (2005).

20. Halim, T.Y., Krauss, R.H., Sun, A.C. & Takei, F. Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation. Immunity 36, 451–463 (2012).

21. Yasuda, K. et al. Contribution of IL-33-activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice. Proc. Natl. Acad. Sci. USA 109, 3451–3456 (2012).

22. Kim, B.S. et al. TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation. Sci. Transl. Med. 5, 170ra116 (2013).

23. Moro, K. et al. Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. Nature 463, 540–544 (2010).

24. Smith, D.E. The biological paths of IL-1 family members IL-18 and IL-33. J. Leukoc. Biol. 89, 383–392 (2011).

25. Shinnakasu, R. et al. Gfi1-mediated stabilization of GATA3 protein is required for Th2 cell differentiation. J. Biol. Chem. 283, 28216–28225 (2008).

26. Zhu, J. et al. Growth factor independent-1 induced by IL-4 regulates Th2 cell proliferation. Immunity 16, 733–744 (2002).

27. Duan, Z. & Horwitz, M. Targets of the transcriptional repressor oncoprotein Gfi-1. Proc. Natl. Acad. Sci. USA 100, 5932–5937 (2003).

28. Malhotra, N. et al. A network of high-mobility group box transcription factors programs innate interleukin-17 production. Immunity 38, 681–693 (2013).

29. Duan, Z., Zarebski, A., Montoya-Durango, D., Grimes, H.L. & Horwitz, M. Gfi1 coordinates epigenetic repression of p21Cip/WAF1 by recruitment of histone lysine methyltransferase G9a and histone deacetylase 1. Mol. Cell Biol. 25, 10338–10351 (2005).

30. Lehnertz, B. et al. Activating and inhibitory functions for the histone lysine methyltransferase G9a in T helper cell differentiation and function. J. Exp. Med. 207, 915–922 (2010).

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ONLINE METHODSMice. Gfi1+/GFP and Gfi1+/fl mice, which have been described9,13, were from the University of Duisburg-Essen and the US National Institutes of Health, respectively. Mice were maintained in pathogen-free conditions in accordance with guidelines approved by the Institutional Animal Care and Use Committee at Genentech.

Cytokine administration. Gfi1+/+, Gfi1+/GFP (Gfi1+/−) or Gfi1GFP/GFP (Gfi1−/−) mice were given intraperitoneal injection of 500 ng of IL-25 or IL-33 (in-house) on days 0, 1, 2 and 3 as described15,16. MLNs, bone marrow, peri-toneal lavage fluid (peritoneal lavage fluid), serum and small intestines were harvested and analyzed on day 4. Gfi1+/+ or Gfi1−/− mice were given 500 ng of IL-25 or IL-33 on days 0, 1 and 3 intranasally as described36. BAL fluid and lungs were collected and analyzed on day 4. Labeling with BrdU in vivo was achieved by intraperitoneal injection of 1 mg BrdU (5-bromodeoxyuridine; BD Biosciences) on days 2 and 3 after elicitation of ILC2 cells. ILC2 cells were analyzed 24 h after the second BrdU administration.

Bone marrow chimeras. B6.SJL-PtprcaPepcb/BoyJ recipient mice (002014; Jackson Laboratories) were lethally irradiated with 1,050 rads with a 137Cs source (two doses of 525 rads ~4–5 h apart) and were reconstituted by intravenous injection of bone marrow from B6.SJL-PtprcaPepcb/BoyJ mice (2.5 × 106 cells) and Gfi1+/−Rosa26ERT2-Cre mice (2.5 × 106 cells), or B6.SJL-PtprcaPepcb/BoyJ mice (2.5 × 106 cells) and Gfi1fl/−Rosa26ERT2-Cre mice (2.5 × 106 cells) mixed at a ratio of 1:1 (5 × 106 total cells). Approximately 7 weeks after injection, mice were given intraperitoneal injection of 90 mg tamoxifen (Sigma) per kg body weight once daily for 5 d. Two weeks after the first treatment with tamoxifen, mice were given intraperitoneal injection of 500 ng of IL-33 once daily for 4 d as described above. Bone marrow was col-lected at various time points (Supplementary Fig. 1b).

N. brasiliensis infection. Gfi1+/+, Gfi1+/− or Gfi1−/− mice were anesthetized and then infected subcutaneously on the flank with 500 N. brasiliensis L3 larvae in 200 µl saline. Control and infected mice were provided water con-taining polymixin b and neomycin for 5 d after infection. MLNs, BAL fluid, lungs and serum were collected 7 d after infection. Worm burden was assessed 10 d after infection. Adult worms were counted by being viewed with a dis-section microscope.

Papain administration. Gfi1+/+, Gfi1+/− or Gfi1−/− mice were anesthetized and then sensitized with papain (25 µg in 50 µl saline; Wako Chemicals) by intranasal instillation on days 0, 3 and 6. Mice were rechallenged on day 14. BAL fluid and lungs were collected on day 15.

Flow cytometry. Single-cell suspensions of MLNs, bone marrow or BAL fluid were washed in PBS containing 5 mM EDTA and 0.5% BSA. Cells were incu-bated for 15 min with Fc Receptor Block (2.4G2; BD Pharmingen) before being stained with antibody. The following antibodies were used for the identification and purification of ILC2 cells: biotinylated anti-ST2 (DJ8; MD Biosciences), eFluor 450–anti-KLRG1 (2F1; eBioscience) and V450–anti-CD45.2 (104; BD Pharmingen); phycoerythrin-conjugated antibodies to detect lineage markers (anti-CD8α (53-6.7; eBioscience), anti-CD49b (DX5; eBioscience), anti-Ter-119 (Ter-119; eBioscience), anti-CD19 (eBio1D3; eBioscience), anti-B220 (RA3-6B2; eBioscience), anti-CD5 (53-7.3; eBioscience), anti-TCRγδ (GL3; BD Pharmingen), anti-TCRβ (H57-597; eBioscience), anti-CD4 (RM4-5; BD Pharmingen), anti-F4/80 (BM8; Invitrogen), anti-FcεRI (MAR-1; eBioscience) and anti-Gr-1 (RB6-8C5; eBioscience)); peridinin chlorophyll protein–cyanine 5.5–anti-Sca-1 (D7; eBioscience); phycoerythrin- indotricarbocyanine–anti-CD127 (A7R34; eBioscience); allophycocyanin–anti-ICOS (C398.4A, BioLegend); and allophycocyanin–eFluor 780–anti-c-Kit (2B8; eBioscience) and allophycocyanin-indotricarbocyanine–anti-CD45.1 (A20; BD Pharmingen). The MLNs of IL-33-treated, IL-25-treated or N. brasiliensis– infected Gfi1+/+, Gfi1+/GFP or Gfi1GFP/GFP mice were first depleted of B cells with a CD19 microbead kit (130-052-201; Miltenyi Biotec) and an autoMACS Pro according to the manufacturer’s protocol (Miltenyi). Eosinophils were analyzed with the following antibodies: eFluor 450–anti-CD11b (M1/70; eBioscience), phycoerythrin–anti-Siglec-F (E50-2440; eBioscience), Alexa

Fluor 647–anti-F4/80 (BM8; eBioscience) and allophycocyanin–eFluor 780– anti-Gr-1 (RB6-8C5; eBioscience). V450–anti-CD4 (RM4-5; BD Biosciences) and allphycocyanin–anti-CD45 (104; eBioscience) were used for analysis of T cells and leukocytes, respectively. Ex vivo cytokine expression by ILC2 cells was analyzed with a Cytofix/Cytoperm kit (BD) and the following conjugated antibodies: Alexa Fluor 647–anti-IL-13 (eBio13A; eBioscience), peridinin chlorophyll protein–cyanine 5.5–anti-IL-17A (eBio17B7; eBio-science) and phycoerythrin-indotricarbocyanine–anti-IFN-γ (XMG1.2, eBioscience). Intracellular BrdU was detected with an APC BrdU Flow Kit (557892) according to the BD Biosciences protocol. Propidium iodide was used to discriminate between viable cells and dead cells. Cells were sorted on a FACSAria (BD) or were analyzed on a LSR Fortessa (Special Order Research products; BD Pharmingen). Flow cytometry data were analyzed with FlowJo software (TreeStar).

Cell culture. MLNs from IL-25-, IL-33-treated and/or N. brasiliensis–infected Gfi1+/+, Gfi1+/−, Gfi1−/−, Gfi1fl/−Rosa26-ERT2-Cre− or Gfi1fl/−Rosa26-ERT2-Cre+ mice were depleted of B cells or T cells, then Lin−, Lin−GFP−, Lin−GFP+, Lin−IL-7rα+Sca-1+ICOS+c-Kit+ or Lin−IL-7rα+Sca-1+ICOS+c-Kit+GFP+ cells were sorted from the MLNs and cultured for up to 6 d in RPMI-1640 medium containing 10% FBS, glutamine, penicillin-streptomycin, 2-mercaptoethanol (55 µm; Gibco), recombinant human IL-2 (50 ng/ml; Peprotech), recombinant mouse IL-7 (10 ng/ml; R&D Systems), recombinant mouse IL-33 (100 ng/ml; prepared in-house) and/or recombinant mouse IL-25 (100 ng/ml; prepared in-house). Live cells were counted with the Violet AnnexinV/Dead Cell Apoptosis kit according to the manufacturer’s protocol (Invitrogen). Intracellular IL-5, IL-13, IL-17A and IL-17F were detected with a Cytofix/Cytoperm Kit according to the manufacturer’s protocol (BD Pharmingen) with the follow-ing antibodies: phycoerythrin-conjugated IgG1 (rat IgG1 isotype; eBRG1; eBioscience), phycoerythrin-conjugated IL-5 (TRFK5, eBioscience), perid-inin chlorophyll protein–cyanine 5.5–conjugated IgG2a (rat IgG2a isotype; eBR2a; eBioscience), peridinin chlorophyll protein–cyanine 5.5–conjugated anti-IL-17A (eBio17B7; eBioscience), Alexa Fluor 647–conjugated IgG1 (rat IgG1 isotype; eBRG1; eBioscience) and Alexa Fluor 647–conjugated anti-IL-13 (eBio13A; eBioscience). Propidium iodide was used to discrimi-nate between viable cells and dead cells. For analysis of IL-33 signaling, ILC2 cultures were allowed to ‘rest’ overnight in serum-containing medium but in the absence of cytokines. Cells were starved of serum for 90 min the following day and then were stimulated for 5 min with IL-33 (100 ng/ml). Intracellular phosphorylated p65 was detected with Alexa Fluor 647–anti-pp65 (93H1; Cell Signaling) by the BD Phosphoflow protocol. IL-25-elicited ILC2 cells from the MLNs of Gfi1−/− mice were spin-infected with retroviral super-natant of mouse stem cell virus expressing human CD4 or mouse stem cell virus expressing Gfi1 and human CD4 as described7. Cells were analyzed on an LSR Fortessa (BD). Flow cytometry data were analyzed with FlowJo software (TreeStar).

Histology. Tissues from Gfi1+/+ and Gfi1−/− mice were fixed in neutral buffer formalin (10%) and embedded in paraffin and then were sectioned (~5 µm in thickness), deparaffinized and stained with hematoxylin and eosin or Periodic acid–Schiff by standard procedures. Slides stained with periodic acid–Schiff were acquired by the Olympus Nanozoomer automated slide-scanning plat-form at a final magnification of ×200. Scanned slides were analyzed with the Matlab software package (MathWorks) as 24-bit RGB images. Areas spe-cific to the intestinal wall were identified by RGB thresholding and simple morphological filtering. Within those areas, a similar approach was used for the identification of goblet cells, with additional cutoffs applied to each area identified based on size and shape factor, which is a measure of round-ness, to reduce the influence of mucin, which has a similar appearance, in the intestinal lumen.

Cytokine expression profiling. Peritoneal lavage fluid and/or BAL fluid was extracted from Gfi1+/+ and Gfi1−/− mice infected with N. brasiliensis or treated with PBS, IL-25, or IL-33. The concentration of IL-5 and/or IL-13 in the peritoneal lavage fluid or BAL fluid was determined with a mouse Bio-Plex Pro Assay (Bio-Rad Group I and II) on a Luminex 100 cytokine analysis system (Luminex).

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Real-time quantitative PCR. Total RNA was extracted from cells with the use of an RNeasy Mini kit according to the manufacturer’s guidelines (Qiagen) and was reverse-transcribed with a first-strand cDNA synthesis kit according to the manufacturer’s instructions (Invitrogen). Taqman probe sets for mouse 18S RNA, Gfi1, Il1rl1, Il1rap, Il17ra, Il17rb, Gata3, Rora, Rorc, Ahr, Il17a, Il17f and Il23r (Applied Biosystems) were used for quantitative gene-expression analysis on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Expression was normalized relative to the expression of 18S RNA.

Microarray gene-expression profiling. Total RNA was extracted from cells with the use of an RNeasy Mini i kit according to the manufacturer’s guide-lines (Qiagen). RNA samples were quantified with a Nanodrop ND-1000 UV-spectrophotometer (Thermo Scientific) and RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies). The quantity of total RNA used in a two-round amplification protocol ranged from 10 ng to 50 ng per sample. First-round amplification and second-round cDNA synthesis was done with the Message Amp II aRNA Amplification Kit (Applied Biosystems). Indodicarbocyanine dye was incorporated with the Quick Amp Labeling kit (Agilent Technologies). Each indodicarbocyanine-labeled test sample (750 ng) was pooled with indocarbocyanine-labeled Universal Mouse Reference RNA (Stratagene) and hybridized onto Whole Mouse Genome 4 × 44K arrays as described in the manufacturer’s protocol (Agilent). Arrays were washed and dried and then were scanned on an Agilent scanner accord-ing to the manufacturer’s protocol. Microarray image files were analyzed with Feature Extraction software, version 10.7.3.1, with the default settings, including linear and Lowess normalization (Agilent Technologies). R Project software package, version 2.15.2, was used for statistical calculations for analysis of gene expression microarray data. Limma package of Bioconductor was used for linear modeling. Control probes, probes with mostly missing values and probes without Entrez Gene annotation were excluded. Nominal P values were adjusted to compensate for multiple-hypothesis testing with the

eBayes function. IPA software (Ingenuity System) was used for pathway analysis with P values and change in expression (‘log fold’) for all probes not excluded as described above.

ChIP-seq. ILC2 cells were sorted from the MLNs of IL-25-treated mice as described above and were cultured for 6 d in RPMI-1640 medium contain-ing 10% FBS, glutamine, penicillin-streptomycin, 2-mercaptoethanol (55 µm; Gibco), recombinant human IL-2 (50 ng/ml; Peprotech), recombinant mouse IL-7 (, 10 ng/ml; R&D Systems), recombinant mouse IL-33 (100 ng/ml; prepared in-house) and recombinant mouse IL-25 (100 ng/ml; prepared in-house). ~3 × 107 cells were fixed, washed and snap-frozen according to the Cell Fixation protocol from Active Motif (http://www.activemotif.com/documents/1848.pdf). Chromatin fragments bound by Gfi1 were immuno-precipitated with rabbit polyclonal antibody to Gfi1 raised against amino- and carboxy-terminal peptides (made in-house). Immunoprecipitation and DNA sequencing was done by Active Motif. Computational and statistical analyses of ChIP-seq data was done as described37. Gfi1-binding peaks were identified by quantitative enrichment of Sequence Tags (QuEST) version 2.4 with input chromatin as control. The MEME algorithm (version 4.8.1) was used for the identification of motifs within 200 base pairs (in either direction) of the peak maxima. Genomic location and distance of binding sites from the nearest tran-scriptional start site were analyzed with the PeakAnnotator package (version 1.4) and derived from the mm10 genome (National Center for Biotechnology Information assembly of the mouse genome).

36. Wilhelm, C. et al. An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation. Nat. Immunol. 12, 1071–1077 (2011).

37. Glasmacher, E. et al. A genomic regulatory element that directs assembly and function of immune-specific AP-1-IRF complexes. Science 338, 975–980 (2012).