TCF-1 Controls ILC2 and NKp46+RORγt+ Innate Differentiation and Protection in Intestinal Inflammation

This information is current as Lisa A. Mielke, Joanna R. Groom, Lucille C. Rankin, Cyril of September 29, 2021. Seillet, Frederick Masson, Tracy Putoczki and Gabrielle T. Belz J Immunol 2013; 191:4383-4391; Prepublished online 13 September 2013;

doi: 10.4049/jimmunol.1301228 Downloaded from http://www.jimmunol.org/content/191/8/4383

Supplementary http://www.jimmunol.org/content/suppl/2013/09/13/jimmunol.130122 Material 8.DC1 http://www.jimmunol.org/ References This article cites 50 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/191/8/4383.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TCF-1 Controls ILC2 and NKp46+RORgt+ Innate Lymphocyte Differentiation and Protection in Intestinal Inflammation

Lisa A. Mielke,* Joanna R. Groom,* Lucille C. Rankin,* Cyril Seillet,* Frederick Masson,* Tracy Putoczki,† and Gabrielle T. Belz*,‡

Innate lymphocyte populations play a central role in conferring protective immunity at the mucosal frontier. In this study, we demonstrate that factor 1 (TCF-1; encoded by Tcf7), a transcription factor also important for NK and T cell differentiation, is expressed by multiple (ILC) subsets, including GATA3+ nuocytes (ILC2) and NKp46+ ILCs (ILC3), which confer protection against lung and intestinal inflammation. TCF-1 was intrinsically required for the differentiation of both ILC2 + and NKp46 ILC3. Loss of TCF-1 expression impaired the capacity of these ILC subsets to produce IL-5, IL-13, and IL-22 and Downloaded from resulted in crippled responses to intestinal infection with Citrobacter rodentium. Furthermore, a reduction in T-bet expression required for Notch-2–dependent development of NKp46+ ILC3 showed a dose-dependent reduction in TCF-1 expression. Collec- tively, our findings demonstrate an essential requirement for TCF-1 in ILC2 differentiation and reveal a link among Tcf7, Notch, and Tbx21 in NKp46+ ILC3 development. The Journal of Immunology, 2013, 191: 4383–4391.

nnate lymphoid cells (ILCs) are a developmentally related and (such as IL-5, IL-13, IL-17, and IL-22) produced by http://www.jimmunol.org/ family of cells that have emerged more recently as critical ILCs and Th cells to control cell fate and function, suggesting that I effectors of innate immunity, particularly at mucosal surfaces, a common program of differentiation is used by the innate and such as the intestine and the lung. ILCs can be activated by in- adaptive immune systems (5). flammatory signals, independent of Ag presentation, to rapidly Inhibitor of DNA binding 2 (Id2) is a helix–loop–helix tran- produce cytokines, suggesting that ILCs form part of the front line scription factor that lacks a DNA binding domain and hetero- of defense to infection and provide important cues for the devel- dimerizes with E-box proteins to prevent gene transcription (6). It opment of the adaptive immune response (1). is a key regulatory protein required for a wide range of develop- Although the classical members of the innate lymphocyte family— mental and cellular processes and is essential for the development NK cells and lymphoid tissue inducer (LTi) cells—have been of all ILC subsets (7, 8). Downstream of Id2, specific transcription by guest on September 29, 2021 known for .2 decades, newly described family members, such as factors and the cytokines that they produce appear to define in- the nuocyte (2), or natural helper cell associated with fat clusters dividual ILC lineages. Group 1 ILCs (ILC1) are composed of (3), and NKp46+ innate (NKp46+ ILC, also known as classical NK cells, which are distinct from other ILC subsets natural cytotoxicity receptor [NCR]+ ILC) have greatly expanded because they do not express CD127 and depend on IL-15, and an the family membership (4). Molecular analysis of these ILC innate lymphocyte subset, both of which express IFN-g (9, 10). subsets demonstrated that there is considerable similarity in the Group 2 ILCs (ILC2s) require GATA3 (11, 12) and RORa (13) for types of transcription factors (including Id2, Rorgt, and Tbx21) their development. They produce IL-5 and IL-13 and have been implicated in the development of airway hypersensitivity re- sponses (14). In addition, ILC2 are critical for defense against *Division of Molecular Immunology, Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3052, Australia; †Division of Inflammation, Walter intestinal parasites (2) and have both pro- and anti-inflammatory and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3052, Australia; properties in the skin (15). Group 3 ILCs (ILC3) are found pre- and ‡Department of Medical Biology, University of Melbourne, Melbourne, Victoria 3010, Australia dominantly in the GALT and require the transcription factor RORgt for their development (16, 17). This last subset can produce IL- Received for publication May 9, 2013. Accepted for publication August 15, 2013. 17 or IL-22, and both cytokines play a fundamental role in in- This work was supported by a project grant (APP1047903) from the National Health and Medical Research Council of Australia, the Victorian State Government Oper- testinal immune responses and tissue repair (18). IL-22 is thought ational Infrastructure Support, and an Australian Government National Health and to act exclusively on epithelial cells to promote proliferation and Medical Research Council (NHMRC) Independent Research Institutes Infrastructure barrier function in the intestine and, therefore, plays a protec- Support scheme. L.A.M. was supported by a Cancer Australia grant (APP1050241), J.R.G. was supported by an NHMRC postgraduate training fellowship, L.C.R. was tive role in inflammatory bowel disease and infections against supported by an NHMRC Dora Lush Biomedical postgraduate scholarship, and attaching and effacing bacterial pathogens (19, 20). ILC3 are G.T.B. was supported by an Australian Research Council Future Fellowship. made up of a number of subsets, including the classical LTi cells, Address correspondence and reprint requests to Dr. Gabrielle T. Belz, Division of which are required for initiation of lymphoid tissue development Molecular Immunology, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia. E-mail address: [email protected] during embryogenesis (21–25), and a second subset that expresses The online version of this article contains supplemental material. the NCR NKp46 [or NKp44 in humans (26)] and is known as + + + Abbreviations used in this article: Id2, inhibitor of DNA binding 2; ILC, innate NKp46 or NCR ILC3. NKp46 ILC3 produce IL-22, which is lymphoid cell; ILC1, group 1 innate lymphoid cell; ILC2, group 2 innate lymphoid further enhanced by stimulation with IL-23. In mice, these cells do cell; ILC3, group 3 innate lymphoid cell; LTi, lymphoid tissue inducer; MLN, mes- not produce IL-17 in response to IL-23, and this distinguishes enteric lymph node; NCR, natural cytotoxicity receptor; TCF-1, T cell factor 1. them from NCR2 or LTi ILC3, which can produce both IL-17 and Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 IL-22 (26). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301228 4384 TCF-1 AND INNATE LYMPHOID CELLS

The molecular pathways that diversify these cell types into (104), CD117 (2B8), CD127 (A7R34), Sca1 (E13-161.7), and ST2 (DJ8). functionally distinct subsets are not well characterized. NKp46+ Intracellular staining was performed using the Transcription Factor ILC3 are intriguing because they appear to represent a terminally Staining Buffer Set (eBioscience) and Abs to GATA3 (TWAJ), Rorgt (AFKJS-9), IL-13 (eBio13A), IL-5 (TRFK5), and IL-22 (IL22JOP). In- differentiated effector subset that is greatly expanded in response tracellular staining for IL-22 was performed following stimula- to inflammation. Recently, we (27) and other investigators (28, 29) tion for 4 h with IL-23 (10 ng/ml) and IL-1b (10 ng/ml) and intracellular showed that T-bet (encoded by Tbx21) is essential for the devel- cytokine staining for IL-13 and IL-5 was performed following stimulation opment of NKp46+ ILC but not the NCR2 ILC3 or ILC2 pop- for 4 h with PMA (50 ng/ml) and ionomycin (100 ng/ml), in the presence 2 of brefeldin A (1 mg). Cells were analyzed using a FACSCanto II (BD ulations. T-bet is required to allow the transition of NCR ILC3 Biosciences), and FlowJo software was used for analysis. Flow cytometric + into NKp46 ILC3 through Notch2-dependent signaling (27). sorting was performed with a FACSAria (BD Biosciences). Following activation, the intracellular domain of Notch dissociates and interacts with the transcription factor RBP-Jk; mice lacking Histology + + this factor have fewer NKp46 ILC3 (30). Differentiation of NKp46 Tissues were fixed in 10% neutral buffered formalin and embedded in wax, ILC3 progenitors in an in vitro culture system required the expres- by standard protocols, prior to sectioning and staining with H&E. Images sion of Notch ligands (31), which also appear to be important for the were captured with an Aperio ImageScope. Colitis severity was assessed in vitro differentiation of ILC2 (13, 32). by a combined score of colon cellular infiltration and tissue disruption. The histological scoring was performed in a blinded fashion. To further define the molecular pathways and transcription + factors that give rise to functional NKp46 ILC3, we investigated Quantitative RT-PCR Notch target genes for their potential role in the differentiation Total RNA was prepared from purified lymphocyte populations with an of NKp46+ ILC3. T cell factor 1 (TCF-1; encoded by Tcf7) was Downloaded from RNeasy Mini kit (QIAGEN), and cDNAwas synthesized with oligo(dT) and identified as a direct target gene of Notch in T cells to promote Thermoscript reverse transcriptase (Invitrogen). Real-time PCR was done T cell development in the (33, 34). In line with this, we with the SensiMix SYBR no-Rox kit (Bioline). Primer sequences were as identified TCF-1 as a novel transcription factor required for the follows: Tcf7:59-CGCTGCATAACAAGCC-39 and 59-CCAGCTCACAGTA- development of NKp46+ ILC3, in addition to ILC2 (32). TCF-1 TGGG-39; Lef1:59-TGTTTATCCCATCACGGGTGG-39 and 59-CATGGA- AGTGTCGCCTGACAG-39; Gata3:59-CTCGGCCATTCGTACATGGAA-39 was required for IL-22 production and protection against the in- and 59-GGATACCTCTGCACCGTAGC-39;andRora:59-GACATCCGACC- testinal pathogen Citrobacter rodentium. Furthermore, expression AAACTTGACA-39 and 59-GTGGAGACAAATCGTCAGGAAT-39. http://www.jimmunol.org/ of a single allele of T-bet resulted in a significant reduction in Tcf7 + expression in NKp46 ILC3 but not other ILC3 subsets. These Results findings suggest that TCF-1 acts downstream of T-bet and Notch Tcf7 is highly expressed in ILCs and controls development of to facilitate NKp46+ ILC3 development. Id2+ innate immune cells ILCs exhibit a shared requirement for the transcriptional repressor Materials and Methods Id2, which acts to inhibit E protein transcription factor activity Mice and antagonize the development of B and T cell fates during ILC 2 2 Id2gfp mice (35), Rorc(gt)gfp mice (16), Prdm1gfp mice (36), Tbx21 / development (9, 23, 39). Therefore, Id2 expression marks he- by guest on September 29, 2021 mice (37), and Tcf72/2 mice (38) were described previously. Ly5.1+ 3 + matopoietic precursors that give rise to the different ILC subsets Ly5.2 mice were bred and maintained in-house. Mice were used at 8–12 (27). Using our Id2-GFP reporter mouse, in which endogenous wk of age, unless otherwise stated. All procedures involving animals were approved by the Animal Ethics Committee of the Walter and Eliza Hall expression of Id2 is tagged using GFP, we analyzed the devel- Institute of Medical Research. opment of ILC populations in the presence or absence of Tcf7. This showed that the overall expression of Id2-GFP+ lympho- Infection with C. rodentium cytes was substantially reduced in the absence of TCF-1 in the Mice were inoculated with 2 3 109 CFU of C. rodentium by oral gavage. lung, lamina propria of the small intestine and colon, and the Micewereanalyzed8dpostinfection,andbacterialdisseminationwas mesenteric lymph node (MLN) (Fig. 1A). Although Id2-GFP was determined by culture of livers and spleens from infected mice on agar + plates containing nalidixic acid, as previously described (27). reduced in T cell lineages (TCRb ) as expected, this loss of ex- pression was also found in lin2 (TCRb2) cells, suggesting the in- Papain-induced lung inflammation volvement of TCF-1 in the regulation of innate lymphocyte Mice were treated intranasally with 20 ml papain (5 mg/ml diluted in PBS) populations (Fig. 1B). To investigate which ILC subsets expressed or PBS alone on three consecutive days. Twenty-four hours following the Tcf7, quantitative RT-PCR analysis was performed on ILC subsets last treatment, lung cells were isolated and stained with Abs for CD45, isolated from the small intestinal lamina propria (Fig. 1C). This CD11c, and Siglec F to identify infiltrating eosinophils and then analyzed revealed that Tcf7 transcripts were highly expressed in all ILC by flow cytometry. subsets, including ILC1 (NK cells), ILC2 (nuocytes), and NCR2 Mixed fetal liver chimeras and NKp46+ ILC3 (Fig. 1C). LEF1, a closely related HMG tran- Ly5.1 mice were lethally irradiated and reconstituted with a 1:1 mixture of scription factor that functions redundantly with TCF-1 in promoting wild-type (Ly5.2) and Tcf72/2 (Ly5.2+Ly5.1+) fetal liver cells. Mice were maturation, was not expressed in lamina propria ILC allowed to reconstitute their hematopoietic system for 8 wk before use. subsets, but it was highly expressed in CD8+ T cells, as expected Isolation of lymphocytes and flow cytometry (data not shown). These results suggest that TCF-1 may play an important role in the development of Id2+ ILCs. Consistent with Intestinal lamina propria lymphocytes were isolated as previously described previous reports (40), we did not observe a developmental defect in by Rankin et al. (27). Mononuclear lung cells were isolated from the lung by generating single-cell suspensions following digestion with Collagenase NK cells (data not shown); thus, we proposed that TCF-1 may in- III (1 mg/ml; Worthington), DNase I (200 mg/ml; Roche), and dispase fluence the development of ILC2 and/or ILC3 innate subsets. (4 U/ml; Sigma-Aldrich) for 45 min at 37˚C. Cells were purified further by centrifugation on Histopaque density gradient (1.077 g/ml; Sigma-Aldrich) TCF-1 is required for development of ILC2 and ILC3 subsets for 18 min at 2000 rpm at room temperature. Single-cell suspensions were stained with the following Abs to cell surface receptors: CD19 (ID3), CD3 To delineate which ILC subsets were influenced by TCF-1, the (145-2C110), CD4 (GK1.4), TCRb (H57-597), CD8 (53-6.7), ICOS expression of lineage-specific markers (i.e, GATA3, Sca1, and (C398.4A), NKp46 (29A1.4), NK1.1 (PK136), CD45.1 (A20), CD45.2 ICOS for ILC2 and RORgt and NKp46 for ILC3) was analyzed in The Journal of Immunology 4385

FIGURE 1. TCF-1 is critical for development of Id2- expressing cells. (A) Expression of Id2-GFP in total lymphocytes (CD45+) isolated from the lung, small intestine or colon lamina propria, or MLN of Id2 re- porter mice (Id2gfp/gfpTcf7+/+) or TCF-1–deficient mice (Id2gfp/gfpTcf72/2). (B) Id2-GFP expression in lin2 2

(TCRb ) cells isolated from the indicated tissues. Results Downloaded from in (A)and(B) show one representative experiment of two (n = 4 mice analyzed/genotype). (C) Quantitative PCR analysis of Tcf7 in purified CD8+ or CD19+ splenocytes of wild-type mice or ILC subsets from the small intestine of Rorc(gt)+/gfp mice. Innate lymphocyte subsets were defined as NK cells (CD45+CD32CD192RORgt-GFP2 + + 2 2 2

NKp46 ), ILC2 (CD45 CD3 CD19 RORgt-GFP http://www.jimmunol.org/ NKp462KLRG1+), NCR2 ILC3 (CD45+CD32CD192 RORgt-GFP-NKp462), NKp46+ ILC3 (CD45+CD32 CD192RORgt-GFP+NKp46high), and NKp46int ILC3 (CD45+CD32CD192RORgt2GFP+NKp46int). Results are representative of three independent experiments (n =6 mice/group). by guest on September 29, 2021

Tcf7+/+ and Tcf72/2 mice. Strikingly, both the frequency and mice (data not shown). Interestingly, we observed that TCF-1 was 2 total number of lin GATA3+Sca1+ ILC2 (Fig. 2A, 2B) were required for the development of RORgt+NKp46+ ILC3 but not markedly reduced compared with wild-type control animals. Of NCR2 ILC3, including the CD4+ or CD42 subset (Fig. 2C, 2D). A the remaining GATA3+ ILC2 in TCF-1–deficient mice, expres- significant proportion of Rorgt+NKp46+ ILC3 expressed KLRG-1, sion of the IL-33R subunit, ST2, was consistently reduced, whereas and the frequency of these cells was reduced .10-fold in the other cell surface molecules commonly expressed on ILC2 re- absence of TCF-1 (Fig. 2C, right panels). Previously, we showed mained unchanged, including KLRG1 and ICOS (Supplemental that NKp46+ ILC3, but not NCR2 ILC3, express the transcrip- Fig. 1A) and IL-17RB (IL-25R; Supplemental Fig. 1B). Consis- tional repressor Blimp-1 (encoded by Prdm1) (27). NKp46+ ILC3 2 2 tent with the decreased expression of ST2, ILC2s from Tcf7 / do not require Blimp-1 for their differentiation (27); therefore, we mice did not proliferate in response to IL-33, and we could not were able to use Blimp-1 reporter mice to provide a means by 2 2 detect the proliferation of either wild-type or Tcf7 / ILC2 in which to determine whether TCF-1 might also regulate NKp46 response to IL-25 (Supplemental Fig. 1C). These data are in line expression. Blimp1-GFP expression in ILC3 populations remained with recent reports suggesting that IL-33 is more potent than IL- unchanged in the presence or absence of TCF-1, indicating that 25 in promoting ILC2 (41). These results highlight the importance loss of the NKp46+ ILC3 subset was a TCF-1–dependent devel- of TCF-1 in the development of ILC2 and suggest that TCF-1 may opmental defect rather than the result of modulation of cell surface regulate the expression of some, but not all, cell surface markers receptor expression (Supplemental Fig. 1D). The development of on ILC2. Analysis of the small intestine revealed that TCF-1 is NCR2 ILC3 were not dependent on TCF-1, suggesting that TCF-1 required for normal Peyer’s patch development: Peyer’s patches plays a specific role in the generation of ILC3 cells by controlling develop but the size of each is reduced compared with wild-type the differentiation of NKp46+ ILC3. 4386 TCF-1 AND INNATE LYMPHOID CELLS

FIGURE 2. TCF-1 is essential for development of ILC2 and NKp46+ ILC3. (A) Analysis of GATA3- and Sca1-expressing ILC2 in CD32CD192 lymphocytes isolated from the lung, lamina propria of small intes- tine or colon, and MLN of wild-type (Tcf7+/+) or TCF- 1–deficient mice (Tcf72/2). (B) Total number of ILC2 (CD45+CD32CD192GATA3+Sca1+) isolated from lung, Downloaded from lamina propria of small intestine or colon, and MLN of wild-type (Tcf7+/+) or TCF-1–deficient mice (Tcf72/2). Data show mean 6 SE pooled from three indepen- dent experiments (n =6).(C) Flow cytometry of CD45+CD32CD192 gated lymphocytes isolated from the small intestine of wild-type (Tcf7+/+)orTCF-1– http://www.jimmunol.org/ deficient (Tcf72/2)mice(left panels) stained for intra- cellular expression of RORgt. Expression of NKp46, CD4, and KLRG1 in RORgt+ ILC3 isolated from the small intestine of wild-type (Tcf7+/+)orTCF-1–deficient (Tcf72/2)mice(middle and right panels). Results show one representative experiment of three (n =6mice analyzed/genotype). (D)TotalcellnumberofILC3 (CD45+CD32CD192RORgt+) subsets isolated from the small intestine of wild-type (Tcf7+/+) or TCF-1–deficient 2 2 (Tcf7 / ) mice. Data are mean 6 SE pooled from three by guest on September 29, 2021 independent experiments (n =6).*p , 0.05, **p , 0.01.

ILC2 and ILC3 subsets are regulated by an intrinsic TCF-1 intestine was analyzed. Ly5.2+/+ wild-type cells reconstituted ILC2 pathway compartments normally in the lung and intestine (Fig. 3A, 3B) and 2/2 ILC3 compartments in the intestine (Fig. 3C, 3D). In contrast, Tcf7 mice lack expression of TCF-1 in both hematopoietic and + + 2/2 nonhematopoietic (stromal) compartments, and TCF-1 has multiple Ly5.1 Ly5.2 Tcf7 fetal liver cells completely failed to repo- roles in the development of the hematopoietic cells and tissues (33, pulate ILC2 (Fig. 3A, 3B). The frequency and total number of ILC3 were reduced (Fig. 3C, 3D). Analysis of the ILC3 subsets revealed 38, 40, 42), both of which could influence the development of ILC that Tcf72/2 fetal liver cells did not fully repopulate NKp46+ ILC3, populations. To determine whether loss of ILC2 and ILC3 cells was 2 2 whereas in NCR ILC3 repopulations, CD4 and CD4+ were not a TCF-1–driven cell-intrinsic effect, we generated mixed fetal liver affected (Fig. 3C, 3D). Thus, the loss of ILC2 and NKp46+ ILC3 chimeras by combining congenically marked Tcf72/2 (Ly5.1+ + +/+ +/+ populations in both intact mice and fetal liver chimeras showed that Ly5.2 ) and Tcf7 (Ly5.2 ) fetal liver cells at a 1:1 ratio and TCF-1 is required in a cell-intrinsic manner. adoptively transferred them into lethally irradiated Ly5.1+/+ recipi- 2/2 ent mice. This approach allowed us to trace the hematopoietic or- Tcf7 mice lack functional ILC2 and ILC3 subsets igin of ILC2 and ILC3 subsets. Eight weeks after transplantation, ILC2 characteristically secrete the cytokines IL-5 and IL-13, which reconstitution of ILC2 and ILC3 populations in the lung and small are critical in type 2 immune responses to helminth infection and the The Journal of Immunology 4387 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. TCF-1 controls ILC2 and ILC3 development by a cell-intrinsic mechanism. Reconstitution of innate cells in Ly5.1+/+ mice reconstituted with a 1:1 mixture of Tcf72/2 (Ly5.1+Ly5.2+) and Tcf7+/+ (Ly5.2+) fetal liver cells. Frequency (A) and total cell number (B) of ILC2 in the lung and small intestinal lamina propria expressing GATA3 and ICOS. Frequency (C) and total cell number (D) of ILC3, CD42NKp462 ILC3, CD4+ ILC3, or NKp46+ ILC3 expressing RORgt and CD4 in CD32CD192 lymphocytes isolated from the small intestinal lamina propria. All cells were gated on CD45+CD32 CD192 lymphocytes. Data show one of two similar independent experiments (n = 6 mice/group). **p , 0.01. development of allergic responses (11, 13, 14), whereas ILC3 can inflammatory responses. To determine whether loss of TCF-1 also produce IL-17 and IL-22 (1). To determine whether loss of TCF-1 impaired the production of cytokines characteristic for ILC3, cells resultedinimpairedcytokineproduction, ILC from various tissues were stimulated ex vivo to induce production of IL-17 and IL-22. (lung, small intestine, colon, and MLNs) were stimulated in vitro to Analysis of IL-22 production by ILC3 from the small intestine induce IL-5 and IL-13. We observed large numbers of IL-5- and IL- revealed that the frequency and total number of IL-22–producing 13–producing cells in the lungs of wild-type mice (Fig. 4A, Supple- NKp46+ ILC3 were significantly reduced in Tcf72/2 mice (Fig. 4B, mental Fig. 2A), and these cells were almost completely absent in Supplemental Fig. 2C), although IL-22 production by NCR2 ILC3 Tcf72/2 mice (Fig. 4A). We also observed decreased IL-5 and IL-13 was not influenced by the loss of TCF-1 (Fig. 4B). Similar to previous production from the small intestine, colon, and MLN (Fig. 4A, Sup- reports showing that TCF-1 acts to suppress the Il17 promoter (43–45), plemental Fig. 2A). To determine whether loss of Tcf7 influences we observed a significant increase in IL-17 production by NCR2 ILC3 ILC2-mediated lung inflammation in vivo, we used a model in which from Tcf72/2 mice. Although IL-17 production by NKp46+ ILC3 was mice are treated intranasally with papain; this induces ILC2 to pro- slightly increased in the absence of Tcf7, the total number of NKp46+ duce IL-5 and IL-13 to enhance lung eosinophil infiltration. Tcf7- ILC3 producing IL-17 was extremely small, and the differences were deficient mice showed reduced eosinophil migration to the lung not statistically significant (Fig. 4B, Supplemental Fig. 2C). Loss of following papain treatment (Supplemental Fig. 2B). These results functional NKp46+ ILC3 and increased IL-17 production indicated that confirm the importance of TCF-1 in innate immune-mediated TCF-1 may play a critical role in maintaining intestinal homeostasis. 4388 TCF-1 AND INNATE LYMPHOID CELLS

of TCF-1 in regulating ILC populations, Tcf7+/2 ILC2 and T-bet+/2 NKp46+ ILC3 were analyzed by quantitative RT-PCR for their expression of Gata3 and Tcf7, respectively, in the different sub- sets. In addition, Tcf7, Lef1, and Rora were analyzed in ILC2; as expected, Tcf7 expression decreased in Tcf7+/2 ILC2, whereas Lef1 expression was increased in Tcf7 heterozygous mice, con- sistent with previous reports that Lef1 is regulated by Tcf7 and can partially compensate for the loss of Tcf7 (Fig.4C)(44).Both Gata3 and Rora expression were decreased in Tcf7+/2 ILC2, suggesting that TCF-1 is a critical transcription factor required for expression of GATA3 and RORa, two transcription factors known to be required for the differentiation and function of ILC2 (Fig. 4C) (12, 13). NCR2 ILC3 did not require TCF-1 for their de- velopment (Fig. 2C, 2D); however, Tcf7 expression was higher in NCR2 ILC3 cells compared with NKp46+ ILC3 (Fig. 1C), sug- gesting that Tcf7 might be important in the transition to NKp46+ ILC3, similar to that of T-bet (27). Therefore, we analyzed ILC3 isolated from Tbx21+/2Rorc(gt)+/gfp heterozygote mice and found

that the expression of Tcf7 in the NKp46+ ILC3 subset was re- Downloaded from duced by approximately half (Fig. 4D). These data, combined with our previous findings that T-bet is required to induce Notch2 in NCR2 ILC3 for the differentiation of NKp46+ ILC3, imply that Tcf7 plays an important role in optimal signaling through the T-bet/Notch pathway.

TCF-1 plays a critical role in host defense against http://www.jimmunol.org/ C. rodentium infection Previous reports suggest that NKp46+ ILC3 play a crucial role in protection against C. rodentium infection (27). In this model, NKp46+ ILC3 produce IL-22 to enhance epithelial cell repair and prevent dissemination of C. rodentium (19). To assess innate im- mune responses, we analyzed infected mice on day 8 following infection before they start to clear the bacteria. Intestinal inflam-

mation caused by C. rodentium infection induces colon shorten- by guest on September 29, 2021 ing, which was exacerbated in the absence of TCF-1 (Fig. 5A). Histological analysis revealed increased mucosal hyperplasia and crypt damage in Tcf72/2 mice (Fig. 5B, 5C). Furthermore, infected Tcf72/2 mice displayed increased bacterial dissemination to the liver and spleen compared with wild-type controls (Fig. 5D). We observed that ILC3 make up .95% of IL-22–producing cells at this time point, and the overall IL-22 production was FIGURE 4. TCF-1 controls factors critical for functional ILC2. (A) decreased in the absence of TCF-1 (data not shown, Supplemental Total number of IL-5–producing (upper panels) or IL-13–producing (lower Fig. 3). Enhanced susceptibility to C. rodentium in Tcf72/2 mice 2 panels) ILC2 following stimulation with PMA and ionomycin in lin correlated with reduced IL-22 production by total ILC3 (Fig. 5E) lymphocytes isolated from lung, small intestine, and colon lamina propria and loss of IL-22–producing NKp46+ ILC3 (Fig. 5F, 5G). and MLN of wild-type (Tcf7+/+) or TCF-1–deficient (Tcf72/2) mice. (B) 2 Therefore, TCF-1 plays a crucial role in the innate immune re- Total number of IL-22–producing NKp46+ ILC3 (left panel), NCR ILC3 + 2 C. rodentium. (left middle panel), or IL-17–producing NKp46 or NCR ILC3 (right sponse to infection with middle and right panels) isolated from the small intestine of wild-type (Tcf7+/+) or TCF-1–deficient (Tcf72/2) mice following short-term in vitro Discussion stimulation with IL-23 (10 ng/ml). Data in (A) and (B) show mean 6 SE The mucosal immune system constantly balances the response to pooled from three independent experiments (n = 6). (C) Quantitative PCR environmental Ags, such as virus, bacteria, and nutrients. This +/+ analysis of Tcf7, Lef1, Gata3, and Rora in purified wild-type (Tcf7 )or sentinel role is orchestrated by the interplay of a network of +/2 TCF-1 heterozygous (Tcf7 ) ILC2 isolated from small intestine. Data innate lymphocytes, such as NK cells (ILC1), nuocytes (ILC2), and show mean 6 SE of data pooled from three independent experiments. (D) + 2 2 NKp46 and NCR innate lymphocytes (ILC3), and their envi- Quantitative PCR analysis for Tcf7 from NK cells, NCR ILC3, or + +/+ +/gfp ronment. In this study, we identify an essential requirement for NKp46 ILC3 isolated from the small intestine of Tbx21 Rorc(gt) or + Tbx21+/2Rorc(gt)+/gfp mice. Data are mean 6 SD of samples pooled from TCF-1 in the generation of the ILC2 and NKp46 ILC3 subsets. two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. TCF-1 (encoded by Tcf7) was identified as a critical regulator of T cell specification and is highly expressed in early thymic

+ progenitors (34); however, its expression pattern and role in TCF-1 controls NKp46 ILC3 via the T-bet/Notch–signaling other lymphocyte populations are less clear. We found that TCF-1 pathway was expressed by all ILC subsets, but it was essential for the GATA3 is essential for ILC2 development and controls IL-5 and IL- development of ILC2 cells and also was required for the devel- 13 production (11), whereas T-bet influences the differentiation of opment of NKp46+ ILC3 cells. Loss of TCF-1 did not impair the NKp46+ ILC3 by inducing Notch (27). To further explore the role development of NK cells in the steady-state or NCR2 ILC3. The Journal of Immunology 4389 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. TCF-1–deficient mice lack NKp46+ ILC3–mediated immune responses to C. rodentium. Colon length (A), histological analysis (B), and H&E staining of colon (C) from wild-type and Tcf72/2 mice at 8 d after oral infection with C. rodentium. Areas of normal mucosa (arrows), extensive mucosal damage (arrowhead), and inflammation (*) are indicated. Scale bar, left panels, 500 mm; middle and right panels, 100 mm. Images are repre- sentative of eight animals from two independent experiments. (D) Dissemination of C. rodentium to the liver and spleen of mice following C. rodentium infection. (E) Frequency and total number of IL-22–producing ILC3 cells in the small intestine of Tcf7+/+ or Tcf72/2 mice following infection with C. rodentium.(F) Frequency and total cell number of NKp46+ ILC3 (CD32NKp46+RORgt+) in the small intestine of wild-type (Tcf7+/+) or TCF-1–deficient mice (Tcf72/2) following infection with C. rodentium.(G) Representative dot plots of IL-22–producing NKp46+ ILC3 (CD32NKp46+RORgt+) from the small intestine of wild-type (Tcf7+/+) or TCF-1–deficient mice (Tcf72/2) following infection with C. rodentium.Datain(D–F) are mean 6 SE pooled from two independent experiments (n =8).(H) Schematic diagram of ILC3 differentiation. CD4+ and NCR2 ILC3 develop from a common precursor that expresses Id2 and Rorgt, and these cells can produce IL-17 and IL-22. T-bet, Notch, and TCF-1 cooperate to induce the differentiation of NKp46+ ILC3 from NCR2 ILC3, and these cells lose their ability to produce IL-17.

Despite this, Tcf7 expression in NCR2 cells was high, suggesting Notch expression in the absence of T-bet (27). This loss was as- that induction of expression might play an important role in the sociated with a significant reduction in IL-22 production and im- transition of NCR2 cells into NKp46+ ILC3. Recently, we (27) and paired protection during C. rodentium infection. In , other investigators (28, 29) showed that the transcription factor T- Notch signals induce TCF-1, which, in turn, imprints T cell fate by bet was critical for the development of NKp46+ ILC3 and that this switching on the expression of T cell essential genes (34). Collec- depended on T-bet activation of Notch2. We found in our analyses tively, these findings suggest that a key action of Notch is to induce of T-bet heterozygous ILC that loss of a single allele of T-bet di- TCF-1 generally in lymphocytes; in addition, it acts to integrate the minished Tcf7 expression in a dose-specific manner in NKp46+ signals between T-bet and TCF-1 in the transcriptional network to ILC3, but not other ILC3, subsets. We similarly found a loss of determine the fate outcome of different ILC subsets (Fig. 5H). 4390 TCF-1 AND INNATE LYMPHOID CELLS

Although the major focus of TCF-1 has been in fate decisions of T-bet, Eomes, and Gata3, neither ILC2 nor ILC3 express Eomes thymocytes and in the development of memory CD8+ T cells, which, at least in Th2 cells, controls IL-5 production through a more significant role for TCF-1 was recently implicated in the Gata3, a signature cytokine of ILC2 (51). Thus, it appears that development of innate lymphocytes (32, 43). Yang et al. (32) unique, but overlapping, combinations of factors enable differen- observed a significant reduction in the production of IL-5 and IL- tiation and cytokine production. Our work emphasizes the need to 13 in the lungs of Tcf72/2 mice, impairing their capacity to mount further delineate differences between the molecular circuitry that ILC2-mediated innate type 2 immune responses. This was at- guides the development and cytokine expression of innate lymphocyte tributable to the requirement for TCF-1 in promoting ILC2 gen- subsets from other lymphocytes, such as T cells. eration, which appeared to act through both GATA3-dependent and -independent pathways. TCF-1 also appeared to be an im- Acknowledgments portant factor for the expression of IL-33R by ILC2. IL-33 plays We thank Rebecca Cole and Jayne Vella for maintaining and caring for the a critical role in inducing ILC2 effector functions; however, be- mice and the Walter and Eliza Hall Institute of Medical Research flow cause we did not find TCF-1 consensus binding sites in the pro- cytometry core facility for technical assistance. moter or enhancer regions of ST2, we hypothesize that it may indirectly regulate IL-33R/ST2 expression. The molecular regu- Disclosures lation of ST2 has not been studied in depth. 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