T-bet inhibits innate lymphoid cell–mediated eosinophilic airway inflammation by suppressing IL-9 production

Accepted Manuscript T-bet inhibits innate lymphoid cell-mediated eosinophilic airway inflammation by suppressing IL-9 production Ayako Matsuki, MD., Hiroaki Takatori, MD., PhD., Sohei Makita, MD., Masaya Yokota, MD., PhD., Tomohiro Tamachi, MD., PhD., Akira Suto, MD., PhD., Kotaro Suzuki, MD., PhD., Koichi Hirose, MD., PhD., Hiroshi Nakajima, MD., PhD. PII:

S0091-6749(16)31023-5

DOI:

10.1016/j.jaci.2016.08.022

Reference:

YMAI 12356

To appear in:

Journal of Allergy and Clinical Immunology

Received Date: 19 December 2015 Revised Date:

14 August 2016

Accepted Date: 23 August 2016

Please cite this article as: Matsuki A, Takatori H, Makita S, Yokota M, Tamachi T, Suto A, Suzuki K, Hirose K, Nakajima H, T-bet inhibits innate lymphoid cell-mediated eosinophilic airway inflammation by suppressing IL-9 production, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/ j.jaci.2016.08.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Full-length article

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T-bet inhibits innate lymphoid cell-mediated eosinophilic airway inflammation by

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suppressing IL-9 production

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Ayako Matsuki, MD. 1, #, Hiroaki Takatori, MD., PhD.1, #, *, Sohei Makita, MD.1,

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Masaya Yokota, MD., PhD.1, Tomohiro Tamachi, MD., PhD.1, Akira Suto, MD., PhD.1, Kotaro

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Suzuki, MD., PhD.1, Koichi Hirose, MD., PhD.1, and Hiroshi Nakajima, MD., PhD.1, *

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University, Chiba, Japan.

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Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba

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#

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*Corresponding author:

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Address: Department of Allergy and Clinical Immunology, Graduate School of Medicine,

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Chiba University, 1-8-1 Inohana, Chiba City, Chiba 260-8670, Japan.

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Phone number: +81 43 226 2198

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E-mail address: [email protected] or [email protected]

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Hiroaki Takatori or Hiroshi Nakajima

FAX number: +81 43 226 2199

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A.M. and H.T. contributed equally to this work.

Word count: 4264. Word count in Abstract: 250

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Number of Figures: 7

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Running head: Role of T-bet in ILC2-mediated airway inflammation

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The authors have no conflicting financial interests.

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Abstract

25 Background: Innate lymphoid cells (ILCs) are emerging subsets of immune cells that

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produce large amounts of cytokines upon cytokine and/or alarmin stimulation. Recent studies

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have shown that T-bet plays pivotal roles in the development of ILC3s and ILC1s; however,

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the roles of T-bet in lung ILC2s remain unknown.

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Objective: To determine the role of T-bet in ILC2-mediated airway inflammation.

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Methods: The expression of T-bet in lung ILCs (defined as Thy1.2+ Lin- cells) was examined.

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The roles of T-bet in the development of lung ILC2s and airway inflammation induced by

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IL-33 administration were examined by using T-bet-deficient (T-bet-/-) mice. Gene expression

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profiles of T-bet-/- lung ILCs were analyzed by RNA sequencing.

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Results: T-bet was expressed in lung ILC2s (defined as Thy1.2+ Lin- cells expressing ST2 or

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CD25) and IFN-γ enhanced its expression. While the development of lung ILC2s at

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steady-state conditions was normal in T-bet-/- mice, IL-33-induced accumulation of lung

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ILC2s and eosinophilic airway inflammation were exacerbated in T-bet-/- mice. The

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exacerbated accumulation of ILC2s and eosinophilic airway inflammation by the absence of

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T-bet was evident even in a RAG2-/- background, suggesting that T-bet expressed in

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non-T/non-B population is involved in the suppression of IL-33-induced eosinophilic airway

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inflammation. Transcriptome analysis revealed that IL-9 expression in IL-33-stimulated lung

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ILCs was up-regulated in T-bet-/- mice compared with that in wild-type mice. Importantly,

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neutralization of IL-9 markedly attenuated IL-33-induced accumulation of lung ILC2s and

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eosinophilic inflammation in T-bet-/- mice.

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Conclusion: T-bet suppresses IL-9 production from lung ILC2s and thereby inhibits

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IL-33-induced eosinophilic airway inflammation.

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Key messages

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T-bet expressed in lung ILC2s is involved in the counter-regulatory mechanisms of

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ILC2-mediated immune responses in the airways.

51 Capsule summary

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T-bet suppresses IL-9 production from lung ILC2s and thereby inhibits IL-33-induced

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accumulation of lung ILC2s and ILC2-mediated eosinophilic airway inflammation.

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55 Key words: T-bet, innate lymphoid cells, IL-9, eosinophils

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57 Abbreviations used in this paper:

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AhR; aryl hydrocarbon receptor

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BALF; bronchoalveolar lavage fluid

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ILCs; Innate lymphoid cells

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KLRG1; killer cell lectin like receptor G1

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Lin; lineage markers

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MIG; MSCV-IRES-GFP

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NCR; Natural cytotoxicity receptor

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T-bet; T-box expressed in T-cells

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Tregs; regulatory T cells

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WT; wild-type

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Introduction

71 Innate lymphoid cells (ILCs) are emerging subsets of immune cells that exist in mucosal

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and lymphoid tissues.1, 2, 3, 4 ILCs do not express antigen receptors or cell-lineage markers but

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do produce large amounts of cytokines upon cytokine and/or alarmin stimulation.1 ILCs have

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been classified into several subsets by the expression pattern of surface molecules, cytokines,

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and transcription factors analogous to helper T cells. Type 1 ILCs (ILC1s) consist of

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conventional NK cells and ILCs that express T-box expressed in T-cells (T-bet) and IFN-γ.2

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Type 2 ILCs (ILC2s), which express ST2 and CD25 and produce large amounts of IL-5 and

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IL-13 in response to epithelial cell-derived cytokines such as IL-25 and IL-33, have been

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shown to be required for anti-helminth responses and the development of allergic diseases

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such as asthma and chronic rhinosinusitis.3 Recent studies have shown that RORα, GATA3,

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and T cell factor 1 (Tcf1) are required for the development and the function of ILC2s.5, 6, 7, 8, 9

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Type 3 ILCs (ILC3s) are comprised of three subsets: lymphoid tissue inducer (LTi) cells,

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natural cytotoxicity receptor (NCR)+ ILC3, and NCR- ILC3, and are defined by the

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expression of RORγt and the production of IL-17A and/or IL-22.10, 11, 12 In addition to RORγt,

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GATA3 has been reported to be involved in the development of ILC3s.13

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T-bet is well known as the master regulator of Th1 cells.14, 15 In addition, T-bet has been

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shown to be important for the development and/or function of cytotoxic CD8+ T cells, NKT

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cells, DCs, and ILC1s.15 Moreover, it has recently been shown that T-bet is highly expressed

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in NCR+ ILC3s in the gut of mice16, 17, 18 and that T-bet-/- mice show a marked reduction of

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RORγt expression and a decrease in IL-22 production in ILC3s,16 suggesting that T-bet plays

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a pivotal role in gut NCR+ ILC3s. However, the roles of T-bet in the development of ILC2s

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remain to be determined.

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Allergic airway inflammation is an immunohistopathological feature of asthma.19 Not

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only Th2 cells induced by allergens but also ILC2s activated by epithelial cell-derived

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cytokines such as IL-25 and IL-33 are involved in the induction of allergic airway

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inflammation.20, 21 Regarding the role of T-bet in allergic airway inflammation, it has been 4

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shown that T-bet-/- mice on a C57BL/6 background develop eosinophilic airway inflammation

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spontaneously.22 By using ovalbumin-induced asthma models on a BALB/c background, we have shown that T-bet expressed in CD4+ T cells suppresses both Th2 cell-induced

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eosinophilic airway inflammation and Th17 cell-induced neutrophilic airway inflammation.23

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However, the roles of T-bet in ILC2-mediated airway inflammation remain unknown.

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In this study, we examined the role of T-bet in the development of lung ILC2s and

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eosinophilic airway inflammation induced by the administration of IL-33. While the

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development of lung ILC2s at steady-state conditions was normal in T-bet-/- mice,

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IL-33-induced eosinophilic airway inflammation and accumulation of lung ILC2s was

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exacerbated in T-bet-/- mice even in the absence of T cells. Importantly, IL-9 production was

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enhanced in T-bet-/- ILC2s and the neutralization of IL-9 markedly attenuated IL-33-induced

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eosinophilic airway inflammation in T-bet-/- mice, suggesting that T-bet inhibits the expression

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of IL-9 in lung ILC2s and thus suppresses IL-33-induced eosinophilic airway inflammation.

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Methods

112 Mice

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T-bet-/- mice22 on a BALB/c background (Jackson Laboratory, Bar Harbor, Me) were crossed

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with RAG2-/- mice to obtain T-bet-/- RAG2-/- mice. All mice were housed in microisolator

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cages under pathogen-free conditions. All experiments were performed according to the

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guidelines of Chiba University.

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118 Reagents

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Antibodies and cytokines used in this study are shown in Online Repository Methods.

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Induction of airway inflammation

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IL-33 (500 ng), IL-25 (1 µg), or papain (25 µg) was administered to mice (age 8-12 weeks)

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intranasally on days 0, 3, and 6 and airway inflammation was evaluated at 12 hs after the last

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administration. Where indicated, a neutralizing anti-IL-9 antibody (25 µg) or anti-IFN-γ

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antibody (500 µg) was administered to mice at 30 min before each of IL-33 administration.

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Histological analysis

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Lung sections (3 µm thick) were stained with H&E or PAS according to the standard

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protocols. Histological score on H&E-stained sections and goblet cell score on PAS stained

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sections were determined in a blinded manner as described previously.24

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Flow cytometric analysis

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After Fc receptors were blocked with anti-CD16/32 Ab (93; BioLegend), cells were stained

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and analyzed on a FACS Calibur (Becton Dickinson, San Jose, CA) using FlowJo software

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(Tree Star, Ashland, OR).

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Intracellular cytokine staining 6

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Cells were stimulated with PMA (20 ng/ml; Calbiochem, San Diego, CA) and ionomycin (1

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µg/ml; Calbiochem) for 4 hs, with monensin (2 µM; Sigma-Aldrich) added for final 3 hs.

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Intracellular staining of IL-4, IL-5, IL-9, IL-13, and IL-17A was performed using antibodies

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to IL-4 (11B11; BD Biosciences), IL-5 (TRFK5; BioLegend), IL-9 (RM9A4; BioLegend),

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IL-13 (eBio13A; eBioscience), and IL-17A (TC11-18H10.1; BioLegend).

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144 Isolation and culture of ILCs

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Lineage-negative cells were roughly purified from single-cell suspensions of lung cells by

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negative sorting with a Lineage cell depletion kit (Miltenyi Biotec, Auburn, CA) according to

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the manufacturer’s instruction. The resultant cells were stained with FITC-labeled antibodies

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to surface lineage markers (CD3ε, CD4, CD8α, CD19, CD5, B220, CD11b, CD11c, CD49b,

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FcεRI, TER119, TCRβ, TCRγδ, and GR-1), anti-ST2-APC, and anti-Thy1.2-PerCP. Thy1.2+

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Lin- cells and ST2+ Thy1.2+ Lin- cells were purified by a cell sorter SH800 (SONY, Tokyo,

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Japan). These cells (1.0 x 106 cells/ml) were cultured in MEMα medium (Thermo Fisher

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Scientific, Waltham, MA) supplemented with 20% heat-inactivated FCS and 2-ME (50 µM)

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(complete medium) containing IL-2 (10 ng/ml) and IL-7 (10 ng/ml). Where indicated, cells

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were cultured in the presence of IL-33 (10 ng/ml), IFN-γ (10 ng/ml), or anti-IL-9 antibody (50

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µg/ml).

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ELISA

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The levels of IL-4, IL-5 and IL-13 were measured by DuoSet ELISA kits (R&D Systems,

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Minneapolis, MN).

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qPCR analysis

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qPCR was performed as described previously.25 The expression levels of each gene were

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normalized to the levels of β-actin. The sequences of PCR primers are shown in Online

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Repository Methods.

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Retroviruses of MSCV-IRES-GFP (MIG) and MIG-T-bet (a kind gift from Dr. Steven L.

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Reiner) were produced as described previously.25 For retroviral transduction, isolated lung

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Thy1.2+ Lin- cells were cultured overnight in the presence of IL-2 (10 ng/ml), IL-7 (10 ng/ml),

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and IL-33 (10 ng/ml), then infected with retroviruses in the presence of IL-2, IL-7, and IL-33,

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and cultured for 4 days.

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173 RNA-Seq analysis

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RNA-Seq analysis was performed as described previously.25 Genes whose expression was

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enhanced by IL-33 stimulation and was higher in T-bet-/- Thy1.2+ Lin- cells than that in WT

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Thy1.2+ Lin- cells were selected with weighted average difference (WAD) method.26

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178 Statistical analysis

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Data are summarized as means ± SD. The statistical analysis of the results was performed by

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an unpaired t test. P values <0.05 were considered significant.

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Results

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IFN-γγ induces T-bet expression in ST2+ Thy1.2+ Lin- cells. A recent study has shown that IFN-γ inhibits IL-33-induced proliferation and cytokine production in ILC2s.27 More recently, it has been reported that IL-27 as well as IFN-γ

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suppresses the activation and function of ILC2s in a STAT1-dependent manner in

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IL-33-induced lung inflammation.28, 29 To examine the roles of T-bet, a representative

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IFN-γ-inducible gene in CD4+ T cells,30 in IFN-γ-mediated inhibition of ILC2 function, we

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first examined the expression of T-bet in Thy1.2+ Lin- cells isolated from lungs of wild-type

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(WT) mice. As shown in Fig. 1A, lung Thy1.2+ Lin- cells modestly expressed T-bet in neutral

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conditions and the expression was significantly increased by IFN-γ stimulation to the levels

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similar to Th1 cells. In addition, the expression of IL-12Rβ2, which is induced by T-bet in

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CD4+ T cells,31 was induced in Thy1.2+ Lin- cells by IFN-γ (Fig. 1A). On the other hand,

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IFN-γ itself was not significantly induced by IFN-γ in Thy1.2+ Lin- cells A,

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consistent with poor IFN-γ-producing ability of lung ILC2s.32

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We next examine protein expression of T-bet in Thy1.2+ Lin- cells in the lung by intracellular staining. As described elsewhere33, lung Thy1.2+ Lin- cells consist of two

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populations; ST2+ CD25+ cells and ST2- CD25- cells (Fig. 1B), which represent ILC2s and

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ILC1/3s, respectively. T-bet was expressed not only in ST2- CD25- population but also in

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ST2+ CD25+ population in steady-state conditions (B). The frequency of T-bet

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expressing cells in ST2+ Thy1.2+ Lin- cells was increased by IFN-γ but decreased by IL-33

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(Fig. 1C). IFN-γ partly antagonized the inhibitory effect of IL-33 on T-bet expression in ST2+

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Thy1.2+ Lin- cells (Fig. 1C). These results suggest that T-bet is expressed in lung ILC2s and

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IFN-γ enhances its expression.

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T-bet is dispensable for the development of lung ILC2s. Because T-bet has been shown to be indispensable for the development and function of a subset of NCR+ ILC3s,16, 17, 18 we next examined whether T-bet is essential for the 9

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development of lung ILC2s. As shown in Fig. E1A, the numbers of Thy1.2+ Lin- cells in the

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lung in T-bet-/- mice were comparable to those in WT mice. The number of CD25+ Thy1.2+

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Lin- cells in the lung was also similar between T-bet-/- mice and WT mice (Fig. E1B). Given

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that T-bet is required for the expression of IFN-γ in intestinal ILCs,34 we next investigated

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cytokine production from lung Thy1.2+ Lin- cells in T-bet-/- mice. In steady-state conditions,

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the frequencies of IL-4-, IL-5-, IL-13-, or IL-17A-producing Thy1.2+ Lin- cells were not

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significantly different between T-bet-/- mice and WT mice (Fig. E1C). The expression levels

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of GATA3 and RORγt in lung Thy1.2+ Lin- cells were also similar between T-bet-/- mice and

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WT mice (Fig. E1D). These results indicate that T-bet is dispensable for the development and

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cytokine production of lung ILC2s in steady-state conditions.

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IL-33-induced eosinophilic airway inflammation is exacerbated in T-bet-/- mice. It is well established that intranasal administration of IL-33 induces IL-5 and IL-13 production from ILC2s and causes eosinophilic airway inflammation.32 To determine the roles

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of T-bet in IL-33-induced airway inflammation, recombinant IL-33 was administered to WT

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mice and T-bet-/- mice intranasally at day 0, 3, and 6 and the numbers of eosinophils in

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bronchoalveolar lavage fluid (BALF) were evaluated at 12 hs after the last administration.

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The proportion and absolute numbers of eosinophils (Siglec-F+ CD11c- cells) in the BALF

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were significantly higher in IL-33-stimulated T-bet-/- mice than those in IL-33-stimulated WT

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mice (Fig. 2A). Consistently, histological analyses showed that peribronchial inflammation

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with eosinophil infiltration (Fig. 2B) as well as goblet cell hyperplasia (Fig. 2C) was more

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obvious in IL-33-stimulated T-bet-/- mice than that in IL-33-stimulated WT mice. In addition,

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the levels of IL-5 and IL-13 in the BALF were significantly higher in IL-33-stimulated T-bet-/-

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mice (Fig. 2D). Eosinophilic airway inflammation induced by IL-25, another epithelial

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cell-derived cytokine that induces IL-5 and IL-13 production from ILC2s,32 was also

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significantly exacerbated in T-bet-/- mice (Fig. E2). Furthermore, Papain-induced eosinophilic

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inflammation, which largely depends on ILC2s,35 was significantly exacerbated in T-bet-/-

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mice (Fig. E2). These results suggest that T-bet plays an inhibitory role in ILC2-mediated

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eosinophilic airway inflammation.

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IL-33-induced accumulation of lung ILC2s is exacerbated in T-bet-/- mice. To address the mechanism underlying the enhanced IL-33-induced eosinophilic

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inflammation in T-bet-/- mice, we examined the numbers of ILC2s in the lung. As shown in

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Fig. 3A, the number of lung Thy1.2+ Lin- cells was significantly increased in IL-33-stimulated

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T-bet-/- mice as compared with that in IL-33-stimulated WT mice. In addition, while the

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frequency of CD25+ cells in lung Thy1.2+ Lin- cells (Fig. 3B, left panel) as well as the

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frequencies of CD25+ Thy1.2+ Lin- cells expressing ST2 and killer cell lectin like receptor G1

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(KLRG1) (Fig. 3C, left panels), markers of ILC2s,7, 32, 36 in IL-33-stimulated T-bet-/- mice was

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comparable to that in IL-33-stimulated WT mice, the absolute numbers of CD25+ Thy1.2+

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Lin- cells, ST2+ CD25+ Thy1.2+ Lin- cells, and KLRG1+ CD25+ Thy1.2+ Lin- cells were

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increased in IL-33-stimulated T-bet-/- mice (Fig. 3B and 3C, right panels). Consistently, the

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frequency of IL-5- or IL-13-producing cells in lung Thy1.2+ Lin- cells was similar between

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IL-33-stimulated T-bet-/- mice and WT mice (Fig. 3D). Even in IL-33-stimulated T-bet-/- mice,

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IL-4 production was negligible in lung Thy1.2+ Lin- cells, consistent with poor ability of IL-4

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production in ILC2s.32 Taken together, these results suggest that the accumulation of lung

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ILC2s is significantly enhanced but the ability of IL-5 and IL-13 production of ILC2s is not

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affected by the absence of T-bet. Meanwhile, the frequency of IL-17A-producing Thy1.2+ Lin-

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cells was increased in T-bet-/- mice (Fig. 3D), consistent with a previous report showing the

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increase of IL-17A-secreting ILCs in colon by the absence of T-bet.37 As expected, the

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frequency of CD4+ T cells was similar between IL-33-stimulated T-bet-/- mice and WT mice

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(Fig. E3A) and the production of IL-4, IL-5, and IL-13 from lung CD4+ T cells was negligible

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in IL-33-stimulated T-bet-/- mice and WT mice (Fig. E3B).

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T-bet expressed in non-T/non-B cells is involved in the suppression of IL-33-induced

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eosinophilic airway inflammation. 11

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It has been shown that ILC2s and Th2 cells form a positive feedback loop to mount type 2 immune responses.38 In addition, a recent study has shown that regulatory T cells (Tregs)

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inhibit ILC2-mediated allergic airway inflammation.39 To exclude the possibility that the lack

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of T-bet in adaptive immune cells including CD4+ T cells is primarily involved in the

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exacerbation of IL-33-induced airway inflammation in T-bet-/- mice, we compared

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IL-33-induced airway inflammation between RAG2-/- mice and T-bet-/- RAG2-/- mice.

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Consistent with the comparison between WT mice and T-bet-/- mice (Fig. 2), IL-33-induced

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eosinophil infiltration into the BALF was significantly enhanced in T-bet-/- RAG2-/- mice as

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compared with that in RAG2-/- mice (Fig. 4A). Moreover, the number of lung Thy1.2+ Lin-

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cells was significantly higher in IL-33-stimulated T-bet-/- RAG2-/- mice than that in

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IL-33-stimulated RAG2-/- mice (Fig. 4B). Interestingly, the frequency of CD25+ cells in lung

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Thy1.2+ Lin- cells and the expression levels of KLRG1 on CD25+ Thy1.2+ Lin- cells were

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significantly up-regulated in IL-33-stimulated T-bet-/- RAG2-/- mice as compared with those in

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IL-33-stimulated RAG2-/- mice (Fig. 4C and 4D). Furthermore, the production of IL-5, IL-13,

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and IL-17A in lung Thy1.2+ Lin- cells was significantly enhanced in IL-33-stimulated T-bet-/-

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RAG2-/- mice (Fig. 4E). Taken together with the finding that T-bet is expressed in ILCs (Fig.

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1), these results suggest that T-bet expressed in ILCs suppresses IL-33-induced accumulation

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of lung ILC2s and subsequent eosinophilic airway inflammation and that adaptive immune

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cells are involved in the suppression of cytokine production from lung ILCs in T-bet-/- mice.

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T-bet suppresses IL-9 production from lung Thy1.2+ Lin- cells. To determine the regulatory mechanism by which T-bet expressed in ILCs suppresses

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IL-33-induced airway inflammation, we searched for genes whose expression is up-regulated

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by the deficiency of T-bet in IL-33-stimulated lung Thy1.2+ Lin- cells. RNA-Seq analysis

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identified IL-9 as one of the genes up-regulated in IL-33-stimulated T-bet-/- Thy1.2+ Lin- cells

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as compared with IL-33-stimulated WT Thy1.2+ Lin- cells (Fig. 5A and Online Repository

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Table 1). qPCR analyses confirmed that mRNA levels of IL-9 but not of IL-4, IL-5, or IL-13

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were higher in IL-33-stimulated T-bet-/- Thy1.2+ Lin- cells than those in IL-33-stimulated WT 12

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Thy1.2+ Lin- cells (Fig. 5B). Moreover, flow cytometric analysis revealed that IL-9 production

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by IL-33-stimulated CD25+ Thy1.2+ Lin- cells was enhanced in T-bet-/- mice than that in WT

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mice, whereas the production of IL-5 as well as the expression of CD25 and ST2 in

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IL-33-stimulated Thy1.2+ Lin- cells was similar between T-bet-/- mice and WT mice (Fig. 5C).

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To elucidate the molecular mechanisms of T-bet-mediated suppression of IL-9 production

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in ILC2s, we investigated the expression of transcription factors that are involved in IL-9

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production in CD4+ T cells such as GATA3, IRF4, BATF, PU.1, and aryl hydrocarbon

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receptor (AhR).40, 41, 42, 43 As shown in Fig. E4, the expression levels of GATA3, IRF4, BATF,

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and AhR were not significantly different between IL-33-stimulated WT and T-bet-/- Thy1.2+

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Lin- cells, while PU.1 was not detectable in both IL-33-stimulated WT and T-bet-/- Thy1.2+

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Lin- cells, suggesting that T-bet down-regulates IL-9 production in ILC2s in GATA3-, IRF4-,

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BATF-, AhR-, or PU.1-independent mechanisms. On the other hand, the expression of IL-9

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receptor (IL-9R) tended to be up-regulated in IL-33-stimulated T-bet-/- Thy1.2+ Lin- cells (Fig.

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E4). In addition, the number of surviving ST2+ Thy1.2+ Lin- cells in culture was increased in

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T-bet-/- mice as compared with that in WT mice and was more strongly reduced by the

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neutralization of IL-9 in T-bet-/- mice (Fig. 5D).

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We further examined the effect of enforced T-bet expression on IL-9 production in ILCs. Lung Thy1.2+ Lin- cells isolated from WT mice were infected with retrovirus of

311

MSCV-IRES-GFP (MIG)-T-bet or MIG (as a control) and cultured for an additional 4 days in

312

the presence of IL-2, IL-7, and IL-33. As shown in Fig. 5E, IL-9 production was markedly

313

reduced by the enforced expression of T-bet. On the other hand, the expression of ST2 and

314

KLRG1 as well as the production of IL-5 and IL-13 was not significantly affected by the

315

enforced expression of T-bet (Fig. 5E), although the enforced expression of T-bet

316

reproducibility resulted in the reduction in the numbers of surviving infected Thy1.2+ Lin-

317

cells (data not shown). Taken together, these results suggest that T-bet preferentially

318

down-regulates IL-9 production in ILC2s and may suppress an autocrine and/or paracrine

319

loop of IL-9-IL-9R signaling in ILC2s.

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To determine the role of IFN-γ-T-bet axis in the regulation of IL-33-induced IL-9

322

production from ILC2s in vivo, we next examined the effect of in vivo administration of

323

anti-IFN-γ antibody on IL-9 production from CD25+ Thy1.2+ Lin- cells in IL-33-stimulated

324

WT mice and T-bet-/- mice. Without anti-IFN-γ antibody administration, IL-9 production from

325

CD25+ Thy1.2+ Lin- cells was significantly exacerbated in IL-33-stimulated T-bet-/- mice as

326

compared with that in IL-33-stimulated WT mice (Fig. 6A), consistent with in vitro data

327

shown in Fig. 5. Importantly, anti-IFN-γ antibody significantly enhanced IL-9 production

328

from CD25+ Thy1.2+ Lin- cells in IL-33-stimulated WT mice but not in IL-33-stimulated

329

T-bet-/- mice (Fig. 6A). These results suggest that endogenously produced IFN-γ suppresses

330

IL-33-induced IL-9 production from ILC2s in a T-bet-dependent manner. Meanwhile, the

331

expression of T-bet in CD25+ Thy1.2+ Lin- cells was undetectable in IL-33-treated mice (Fig.

332

6B), consistent with the inhibitory effect of IL-33 on T-bet expression in ILC2s (Fig. 1C).

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Neutralization of IL-9 cancels the enhanced IL-33-induced airway inflammation in

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T-bet-/- mice.

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It has been shown that the main producers of IL-9 in the lung during papain-induced airway inflammation44 and infection with Nippostrongylus brasiliensis45 were ILC2s. With respect to

338

the function, IL-9 has been reported to protect ILC2s from apoptosis.45 To determine whether

339

the excessive production of IL-9 is involved in the enhanced IL-33-induced airway

340

inflammation in T-bet-/- mice, we finally examined the effect of neutralizing anti-IL-9

341

antibody on IL-33-induced airway inflammation in T-bet-/- mice and WT mice. Whereas

342

anti-IL-9 antibody did not significantly inhibit eosinophilic airway inflammation in

343

IL-33-stimulated WT mice, anti-IL-9 antibody significantly suppressed eosinophilic airway

344

inflammation in IL-33-stimulated T-bet-/- mice (Fig. 7A). Anti-IL-9 antibody also suppressed

345

the accumulation of CD25+ Thy1.2+ Lin- cells in IL-33-stimulated T-bet-/- mice but not in

346

IL-33-stimulated WT mice (Fig. 7B). These results suggest that the excessive production of

347

IL-9 from lung ILC2s is involved in the exacerbation of IL-33-induced, ILC2-mediated

348

eosinophilic airway inflammation in T-bet-/- mice.

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Discussion

351 In this study, we show that ILC2-mediated eosinophilic airway inflammation is

353

exacerbated by the absence of T-bet in mice. Regarding the involvement of T-bet in the

354

pathogenesis of asthma, various studies have shown that single nucleotide polymorphisms

355

(SNPs) or variants in TBX21 gene, encoding T-bet, are associated with the development of

356

asthma in humans.46, 47, 48, 49 With respect to T-bet-mediated regulation of asthma, we have

357

previously shown that T-bet expressed in CD4+ T cells is crucial for the inhibition of Th2

358

cell-mediated eosinophilic airway inflammation in ovalbumin-induced asthma models.23 In

359

this study, we show that IL-33-induced eosinophilic airway inflammation, which is known to

360

be dependent on ILC2s but not on T cells,50 is exacerbated in T-bet-/- mice (Fig. 2). These

361

results suggest that T-bet is pleiotropically involved in the suppression of eosinophilic airway

362

inflammation.

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We show that IL-33-induced accumulation of ST2+ CD25+ Thy1.2+ Lin- cells, which represent ILC2s,33 in the lung is exacerbated in T-bet-/- mice (Fig. 3). It is well established that

365

T-bet is the most critical transcriptional factor for Th1 cell differentiation.15 On the other hand,

366

it has been shown that T-bet, together with runt-related transcription factor 3 (Runx3), is

367

required for silencing of Il4 gene in Th1 cells.51 Moreover, it has been reported that T-bet

368

interacts with GATA3 and dissociates GATA3 from the promoters of Il5 and Il13.22 These

369

findings suggest that T-bet directly suppresses Th2 cytokine production in CD4+ T cells. By

370

contrast, we found that IL-33-induced production of IL-5 and IL-13 in Thy1.2+ Lin- cells was

371

not affected by the absence of T-bet (Fig. 3D). In addition, we showed that the enforced

372

expression of T-bet did not suppress IL-5 and IL-13 production in Thy1.2+ Lin- cells (Fig. 5E),

373

suggesting that T-bet does not directly regulate the production of IL-5 and IL-13 in

374

IL-33-stimulated ILC2s. On the other hand, we found that IL-33-induced accumulation of

375

lung ST2+ CD25+ Thy1.2+ Lin- cells was enhanced in T-bet-/- mice (Fig. 3). The enhanced

376

accumulation of lung ST2+ CD25+ Thy1.2+ Lin- cells by the absence of T-bet was observed

377

even in a RAG2-/- background (Fig. 4). Taken together, these results suggest that T-bet

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expressed in non-T/non-B population plays an inhibitory role in IL-33-induced accumulation

379

of lung ILC2s. Regarding the mechanism underlying the enhanced IL-33-induced accumulation of lung

381

ILC2s and eosinophilic airway inflammation in T-bet-/- mice, we found that IL-9 production

382

from IL-33-stimulated CD25+ Thy1.2+ Lin- cells was up-regulated by the absence of T-bet

383

(Fig. 5B). We also found that the enforced expression of T-bet in lung Thy1.2+ Lin- cells

384

inhibited IL-9 production (Fig. 5E), suggesting that T-bet intrinsically inhibits IL-9

385

production in IL-33-stimulated ILC2s. In vivo, we showed that IL-9 neutralization by

386

anti-IL-9 antibody attenuated IL-33-induced accumulation of lung CD25+ Thy1.2+ Lin- cells

387

and eosinophilic airway inflammation in T-bet-/- mice (Fig. 7). Taken together, these results

388

suggest that the enhanced IL-9 production of T-bet-/- ILC2s seems responsible for the

389

enhanced IL-33-induced eosinophilic airway inflammation in T-bet-/- mice.

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It has recently been shown that ILC2s represent a potentially important source of IL-9

391

during papain-induced lung inflammation and helminth lung infection by using IL-9 reporter

392

mice and fate-mapping studies.44, 45 Importantly, one of the functions of IL-9 is to act on

393

ILC2s in an autocrine manner to enhance their survival in a helminth infection model,45 which

394

is consistent with the expression of IL-9R on Thy1.2+ Lin- cells (Fig. E4) and the reduction of

395

surviving ST2+ Thy1.2+ Lin- cells by IL-9 neutralization (Fig. 5D). Moreover, it has been

396

shown that IL-9 expression during helminth infection precedes the expression of IL-5 and

397

IL-13 and that the production of IL-5 and IL-13 is abrogated in IL-9R-/- mice,45 suggesting

398

that IL-9-IL-9R signaling acts in the upstream of IL-5 and IL-13. Interestingly, it has been

399

reported that the expression of IL-9 is more transient than that of IL-5 or IL-13 in ILC2s,44

400

suggesting that IL-9 production is more tightly regulated than that of IL-13 or IL-5 in ILC2s.

401

Importantly, we showed here that the expression of IL-9 but not of IL-13 or IL-5 in Thy1.2+

402

Lin- cells was significantly enhanced by the absence of T-bet (Fig. 5B). In addition, we

403

showed that IL-33-induced IL-9 production from CD25+ Thy1.2+ Lin- cells was significantly

404

enhanced by the neutralization of IFN-γ in WT mice but not in T-bet-/- mice (Fig. 6). Taken

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together, these results suggest that T-bet might be involved in the tight regulation of IL-9

406

expression in ILC2s as a downstream target of IFN-γ.

407

It has been reported that various transcription factors such as STAT5, STAT6, GATA3, IRF4, BATF, and PU.1 are involved in the differentiation of Th9 cells.41, 42, 43, 44, 52 More

409

recently, analogous to the findings in Th9 cells, IRF4 has been shown to be essential for IL-9

410

production from ILC2s in response to IL-33 and thymic stromal lymphopoietin (TSLP).53 On

411

the other hand, we found that the expression of IRF4 was not significantly affected by the

412

absence of T-bet in Thy1.2+ Lin- cells (Fig. E4), suggesting that IRF4 might not be involved

413

in T-bet-mediated suppression of IL-9 production in ILC2s. Our results also suggest that

414

GATA3, BATF, AhR, and PU.1 are not responsible for the enhanced IL-9 production in

415

T-bet-/- ILC2s (Fig. E4). Further studies including comprehensive analyses of DNA binding of

416

T-bet in ILC2s are needed to reveal the mechanisms of T-bet-mediated suppression of IL-9

417

expression in ILC2s.

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Our findings suggest that T-bet expressed in lung ILC2s plays an inhibitory role in ILC2s-mediated immune responses. Recently, several groups have investigated the

420

counter-regulatory mechanisms of ILC2s and have shown that IFN-β, IFN-γ, and IL-27

421

regulate the activation and function of ILC2s during lung inflammation in a

422

STAT1-dependent manner.27, 28, 29, 54 Importantly, Kudo et al. have shown that IFN-γ

423

produced by activated NKT cells is important for the suppression of IL-5 and IL-13

424

production from lung ILC2s.54 In this regard, we found that IFN-γ induced the expression of

425

T-bet in lung ST2+ Thy1.2+ Lin- cells (Fig. 1). Moreover, we found that the enforced

426

expression of T-bet suppressed IL-33-induced IL-9 but not IL-5 or IL-13 production in lung

427

Thy1.2+ Lin- cells (Fig. 5D), consistent with a previous finding that IFN-γ inhibits

428

IL-33-induced IL-5 production in a manner dependent on STAT1 but not on T-bet.28 Therefore,

429

it is likely that IFN-γ suppresses the function of ILC2s via both T-bet-dependent and

430

-independent mechanisms.

431 432

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In conclusion, our findings indicate that T-bet intrinsically suppresses the expression of IL-9 in lung ILC2s during IL-33-induced lung inflammation and thereby suppresses the 17

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accumulation of lung ILC2s and subsequent eosinophilic airway inflammation. Although

434

further studies are needed, our results should add a new insight into the counter-regulatory

435

mechanisms for ILC2s in asthma and suggest that the induction of T-bet expression in ILC2s

436

could be a therapeutic strategy in the treatment of asthma.

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Acknowledgements

438 439

We thank Drs. Ken Nonaka and Osamu Ohara (Kazusa DNA Research Institute, Japan) for RNA-Seq analysis and Dr. Steven L. Reiner (University of Pennsylvania) for the

441

MIG-T-bet. We also thank Ms. Juri Iwata, Kazumi Nemoto, and Yumiko Hanabuchi (Chiba

442

University, Chiba, Japan) for technical support. This work was supported in part by

443

Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports,

444

Science and Technology (MEXT, WG24390207, G26461486), and LGS (Leading Graduate

445

School at Chiba University) Program, MEXT, Japan, and the Takeda Science Foundation,

446

Japan.

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Figure Legends

605 Figure 1. IFN-γγ induces T-bet expression in lung ILC2s.

607

(A) Thy1.2+ Lin- cells were isolated from the lung of WT mice and cultured with IL-2 and

608

IL-7 in the presence or absence of IFN-γ for 6 days. mRNA levels of T-bet, IL-12Rβ2, and

609

IFN-γ were analyzed by qPCR analysis. Naïve CD4+ T cells, Th0 cells, and Th1 cells were

610

used as controls. Data are means ± SD of three independent experiments. **p<0.01. (B) The

611

expression of T-bet and GATA3 in lung CD25+ ST2+ Thy1.2+ Lin- cells or CD25- ST2-

612

Thy1.2+ Lin- cells was examined by intracellular staining in WT mice. T-bet-/- mice were used

613

as controls. Data are representative of three independent experiments. (C) ST2+ Thy1.2+ Lin-

614

cells were isolated from the lung of WT mice and cultured with IL-2 and IL-7 in the presence

615

or absence of IFN-γ and/or IL-33 for 6 days. Representative data of T-bet staining and means

616

± SD of the frequency of T-bet-expressing cells are shown. *p<0.05. Data are representative

617

of three independent experiments.

TE D

618

M AN U

SC

RI PT

606

Figure 2. IL-33-induced eosinophilic airway inflammation is exacerbated in T-bet-/- mice.

620

(A) Representative CD11c vs. Siglec-F staining, and means ± SD of the frequency and

621

absolute numbers of Siglec-F+ CD11c- cells (eosinophils) in the BALF are shown (n=5, each).

622

*p<0.05. (B) Representative photomicrographs (H&E staining) and means ± SD of

623

histological score of the lung are shown. *p<0.05. n=4, each. Scale bars, 100 µm. (C)

624

Representative photomicrographs (PAS staining) and means ± SD of goblet cell score are

625

shown. *p<0.05. n=4, each. Scale bars, 50 µm. (D) Means ± SD of the levels of IL-4, IL-5,

626

and IL-13 in the BALF are shown (n=3, each). *p<0.05. NS=not significant. ND=not

627

detectable.

AC C

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619

628 629

Figure 3. IL-33-induced accumulation of lung ILC2s is exacerbated in T-bet-/- mice.

630

(A) Representative Lin vs. Thy1.2 staining of isolated lung cells, and means ± SD of the

631

frequency and absolute numbers of Thy1.2+ Lin- cells in the lung are shown (n=5, each). 25

Matsuki et al.

ACCEPTED MANUSCRIPT

*p<0.05. (B) Means ± SD of the frequency of CD25+ cells in Thy1.2+ Lin- cells and absolute

633

numbers of CD25+ Thy1.2+ Lin- cells in the lung are shown (n=3, each). *p<0.05. NS=not

634

significant. (C) Means ± SD of the frequency of ST2+ cells and KLRG1+ cells in CD25+

635

Thy1.2+ Lin- cells and absolute numbers of ST2+ CD25+ Thy1.2+ Lin- cells and KLRG1+

636

CD25+ Thy1.2+ Lin- cells are shown (n=3, each). (D) Isolated lung cells were stimulated with

637

PMA and ionomycin for 4 hs, and subjected to intracellular staining for IL-4, IL-5, IL-13, and

638

IL-17A. Shown are representative Thy1.2 vs. IL-4, IL-5, IL-13, or IL-17A staining of Thy1.2+

639

Lin- cells, and means ± SD of the frequency of the indicated cells (n=3, each). *p<0.05. Data

640

are representative of four independent experiments.

M AN U

641

SC

RI PT

632

Figure 4. T-bet expressed in non-T/non-B cells is involved in the suppression of

643

IL-33-induced eosinophilic airway inflammation.

644

(A) Representative CD11c vs. Siglec-F staining of BALF cells and means ± SD of the

645

absolute numbers of Siglec-F+ CD11c- cells in the BALF are shown (n=3, each). (B)

646

Representative Lin vs. Thy1.2 staining of isolated lung cells and means ± SD of the absolute

647

numbers of Thy1.2+ Lin- in the lung are shown (n=3, each). *p<0.05. (C) Representative

648

histogram of CD25 staining gating on Thy1.2+ Lin- cells and means ± SD of the frequency of

649

CD25+ cells are shown (n=3, each). *p<0.05. (D) Representative histogram of ST2 or KLRG1

650

staining gating on CD25+ Thy1.2+ Lin- cells and means ± SD of the frequency of ST2+ or

651

KLRG1+ cells are shown (n=3, each). **p<0.01. NS=not significant. (E) Isolated lung cells

652

were stimulated with PMA and ionomycin for 4 hs, and subjected to intracellular staining for

653

IL-4, IL-5, IL-13, and IL-17A. Shown are representative CD25 vs. IL-4, IL-5, IL-13, or

654

IL-17A staining of Thy1.2+ Lin- cells and means ± SD of the frequency of cytokine-producing

655

cells (n=3, each). *p<0.05. Data are representative of three independent experiments.

AC C

EP

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642

656 657

Figure 5. T-bet suppresses IL-9 production in lung ILC2s.

658

(A) Isolated lung Thy1.2+ Lin- cells from WT mice and T-bet-/- mice were cultured with IL-2

659

and IL-7 in the presence or absence of IL-33 for 6 days, and RNA-Seq analysis was 26

Matsuki et al.

ACCEPTED MANUSCRIPT

performed. Shown is a heatmap of top 10 genes whose expression is up-regulated in

661

IL-33-stimulated T-bet-/- Thy1.2+ Lin- cells. (B) Isolated lung Thy1.2+ Lin- cells from

662

IL-33-stimulated WT mice and T-bet-/- mice were cultured with IL-2, IL-7, and IL-33 for 6

663

days, and mRNA levels for indicated cytokines were analyzed by qPCR. Data are means ±

664

SD of 3 independent experiments. *p<0.05. (C) Isolated lung Thy1.2+ Lin- cells from

665

IL-33-stimulated WT mice and T-bet-/- mice were cultured with IL-2, IL-7, and IL-33 for 6

666

days and then subjected to flow-cytometric analysis. Representative CD25 vs. ST2 staining

667

(upper panels) and IL-5 vs. IL-9 staining gating on CD25+ population (lower panels) are

668

shown. Data are representative of three independent experiments. (D) Isolated lung ST2+

669

Thy1.2+ Lin- cells from WT mice and T-bet-/- mice were cultured with IL-2 and IL-7 in the

670

presence or absence of anti-IL-9 antibody for 6 days, and the number of surviving cells was

671

analyzed. Data are means ± SD of surviving cells. *p<0.05, **p<0.01. (E) Isolated lung

672

Thy1.2+ Lin- cells from IL-33-stimulated WT mice were stimulated with IL-2, IL-7, and IL-33

673

for 24 hs. These cells were infected with retroviruses of MSCV-IRES-GFP (MIG) (as a

674

control) or MIG-T-bet, cultured for 4 days in the presence of IL-2, IL-7, and IL-33, and

675

subjected to flow cytometric analyses. Shown are representative FACS profiles of ST2 vs.

676

KLRG1, IL-5 vs. IL-13, and IL-5 vs. IL-9 gating on GFP+ CD25+ cells. Data are

677

representative of three independent experiments.

EP

TE D

M AN U

SC

RI PT

660

678

Figure 6. Anti-IFN-γγ antibody enhances IL-33-induced IL-9 production from ILC2s in

680

WT mice but not in T-bet-/- mice.

681

Representative CD25 vs. IL-9 staining (A) and CD25 vs. T-bet staining (B) of Thy1.2+ Lin-

682

cells in the lung and means ± SD of the absolute numbers of IL-9-producing CD25+ Thy1.2+

683

Lin- cells in the lung are shown (n=3, each). *p<0.05. **p<0.01. NS=not significant. Results

684

are representative of two independent experiments.

AC C

679

685 686

Figure 7. Anti-IL-9 antibody cancels the enhanced IL-33-induced airway inflammation

687

in T-bet-/- mice. 27

Matsuki et al.

ACCEPTED MANUSCRIPT

(A) Representative CD11c vs. Siglec-F staining of BALF cells and means ± SD of the

689

absolute numbers of Siglec-F+ CD11c- cells in the BALF are shown (n=3, each). *p<0.05.

690

**p<0.01. NS=not significant. (B) Representative Lin vs. Thy1.2 staining of isolated lung

691

cells and means ± SD of the absolute numbers of Thy1.2+ Lin- cells and CD25+ Thy1.2+ Lin-

692

cells in the lung are shown (n=3, each). Results are representative of two independent

693

experiments.

AC C

EP

TE D

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SC

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688

28

ACCEPTED MANUSCRIPT

Fig. 1

**

Thy1.2+ Lin-

T cells

Thy1.2+ Lin-

2.5

10 5

10

ST2+

Thy1.2

10 3

2

CD25+ Thy1.2+ Lin-

10 1

10 1

104

10 2

Lin

10 3

10 4

10 3

10 2

10 2

10 5

ST2-

102 101

14.1

100 100

1

2

10

10

10

ST2

10 3

10 4

9.7

0

10 5

6.7

10 5

10

10 3

10 2

10 2

0

10 2

47.8 10 3

10 4

0

10 2

81.3 10 3

10 4

0.0

10 5

0.5

4

10 3

35.8

18.7

0

10 5

78.3 0

10 2

21.2 10 3

10 4

10 5

4

10

IFN-γ 11.9

AC C

103

102

102

1

1

10

(%) 15

* *

10

100

0

200

400

600

4

10

IL-33

10

0.0

GATA3

PBS 6.6

4

10

103

PBS

4

0.0

EP

C

10

CD25Thy1.2+ Lin-

10 2

91.2

4

0

3

5.5

TE D

CD25

103

10 5

10 3

0

82.8

3.3

10 4

100 800 1000 0

1.8

103

103

102

102

101

101

100 0

200

SSC

400

600

200

400

600

800 1000

5

4

10

100 800 1000 0

*

10 9.0

T-bet

10 0

0.0

10 4

0

10 0

T-bet-/-

SC

4

Thy1.2+ Lin-

T cells

WT 10

IFN-γ

0.1 0.08 0.06 0.04 0.02 0

200

400

600

800 1000

0 IL-33

-

-

+

+

IFN-γ

-

+

-

+

T-bet

**

T cells

B

IL-12Rβ2

0.5 0.4 0.3 0.2 0.1 0

RI PT

T-bet 0.01 0.008 0.006 0.004 0.002 0

M AN U

Relative expression

A

ACCEPTED MANUSCRIPT

WT 10

0.0

103 102

102

101

101

100 100

101

102

103

101

102

103

(%)

104

102

1

1

10

10

0 0

10

1

10

2

3

10

10

100 104 100

1

2

10

10

3

10

2

40

1

20 0

0

WT T-bet-/-

SC

2

*

60

103

IL-33 10

4

10

CD11c

B

T-bet-/-

M AN U

WT

(X106) 3

**

80

52.3

104

103

10

Siglec-F+ CD11c- cells

100 0 104 10

14.6

104

14.6

0.0

103

Siglec-F

PBS

T-bet-/-

104

4

RI PT

A

Fig. 2

Histological score

*

4

PBS

3 2 1

C

AC C

PBS

IL-4 (pg/ml) NS 50 40 30 20 10 ND ND 0 IL-33 - + - +

WT T-bet-/-

+

-

+

T-bet-/-

WT

Goblet cell score 6

2 ND

0 IL-33 -

ND

+

-

IL-5

*

1000

500

500 ND

-

ND

+

-

IL-13

(pg/ml) 1500

1000

0 + IL-33

WT T-bet-/-

+

T-bet-/-

WT

(pg/ml) 1500

0 IL-33

*

4

IL-33

D

ND

T-bet-/-

EP

WT

TE D

IL-33

ND

0 IL-33 -

*

ND

-

ND

+

-

+

WT T-bet-/-

WT T-bet-/-

ACCEPTED MANUSCRIPT

101

101

100 100

101

102

103

104

100 100

101

102

103

104

Lin Thy1.2+ Lin- cells (%)

* 1

5 0

B

WT T-bet-/-

(X106) 1.5

0 WT T-bet-/-

C

*

1.0 0.5

WT T-bet-/-

EP

(X106) 1.0 0.8 0.6 0.4 0.2 0

*

AC C

NS

WT T-bet-/-

WT T-bet-/-

KLRG1+ CD25+ Thy1.2+ Lin- cells (%) 100 80 60 40 20 0

NS

WT T-bet-/-

(X106) 1.0 0.8 0.6 0.4 0.2 0

102

103

100 104 100

*

WT T-bet-/-

101

4

10

54.7

3

10

102

102

101

101

1

10

2

10

3

10

100 0 10

4

10

104

103

0

104

54.2

1

10

103 102

101

101

100 100

101

102

103

100 104 100

101

14.5

2

10

3

10

102

102

101

101

0

WT T-bet-/-

IL-13+ cells

103

104

1

10

2

10

Thy1.2

3

10

10

4

10

0

10

1

10

2

10

(%) 80 60 40 20 0

NS

WT T-bet-/-

IL-17A+ cells

0

0

10

NS

40

0

4

10

50.5

102

IL-5+ cells

20

23.4

103

NS

WT T-bet-/-

(%) 60

104

103

IL-4+ cells

1

104

102

10

102

54.2

10

104

ST2+ CD25+ Thy1.2+ Lin- cells

(%) 100 80 60 40 20 0

101

TE D

100 80 60 40 20 0

NS

100 100

103

CD25+ Thy1.2+ Lin- cells (%)

101

100 0 10

0 WT T-bet-/-

101

3

*

10

1.0 102

4

2

15

1.6 102

10

(X106)

(%) 2

103

IL-4

102

103

IL-5

102

Thy1.2

3

10

T-bet-/-

104

IL-13

3

10

WT

104

11.1

IL-17A

7.6

T-bet-/-

RI PT

104

gating on Thy1.2+ Lin-

SC

WT

104

D

M AN U

A

Fig. 3

3

10

4

10

(%) 40 30 20 10 0

*

WT T-bet-/-

ACCEPTED MANUSCRIPT

10 1

10 0

10 0 10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

RAG2-/-

CD11c

103

103

2

2

10

10

101

101

100 100

T-bet-/-

101

102

100 104 100

103

Lin

RAG2-/-

E

gating on Thy1.2+ Lin-

150

100 80

60.5

100

60 40

50

20 0 100

101

102

CD25+ cells

T-bet-/- RAG2-/-

103

104

0 100

101

102

(%) 100 86.9 80 60 40 20 0 103 104

0.2

100 100

400 250 200

81.7

150

300

100 100

50 0 100

101

102

103

0 100

101

102

250

80

200

68.9

60

150

40

100

20

50

0 100

101

(%) 100 86.3 80 60 40 20 0 3 4 10

10

AC C

ST2

104

102

KLRG1

103

104

0 100

101

102

(%) 100 85.6 80 60 40 20 0 3 4 10 10

102

103

62.9

100 104 100

4

10

28.7

103

103

102

102

101

ST2+ cells NS

100 100

101

102

1.2

1

10

102

103

104

2.9

0

0

0

1

10

2

10

2.6

3

4

10

48.2

10

10

6.2

0

10

2

10

2.2

104

103

1

10

3

4

10

47.8

2

10

12.8

1

10

1

0

0

0

10

1

10

2

10

CD25

3

10

(%) 50 40 30 20 10 0

4

10

56.5

10

0

10

1

10

2

10

3

10

60.9

IL-5

*

(%) 20 15 10 5 0

IL-13

*

RAG2-/- T-bet-/RAG2-/-

(%) IL-17A 15

2

10

NS

RAG2-/- T-bet-/RAG2-/-

10

103

10

IL-4

RAG2-/- T-bet-/RAG2-/-

21.4

1

104

RAG2-/-

104

2

10

10

103

(%) 1.0 0.8 0.6 0.4 0.2 0

73.1 46.5

101

104

9.9

10

cells

T-bet-/-

RAG2-/- T-bet-/RAG2-/-

31.7

2

10

RAG2-/-

3.8

103

10

**

102

33.1

10

KLRG1+

101

100 104 100

103

103

RAG2-/- T-bet-/RAG2-/-

0.6

101

104

EP

200

101

1.0

TE D

T-bet-/- RAG2-/-

0.3

101

4

RAG2-/-

2 0

104

102

10

gating on CD25+ Thy1.2+ Lin-

103

103

101

D

102

T-bet-/- RAG2-/-

104

0.5

103 102

RAG2-/- T-bet-/RAG2-/-

CD25

RAG2-/104

*

*

4

gating on Thy1.2+ Lin-

M AN U

RAG2-/-

101

SC

C

Thy1.2

10

2

T-bet-/- RAG2-/(x106) 23.1 6

Thy1.2+ Lin- cells

104

IL-4

10 1

3

RAG2-/12.7

IL-5

10

2

10

104

IL-13

3

T-bet-/- RAG2-/41.2

IL-17A

10

10 4

Siglec-F

RAG2-/7.6

10 4

B

Siglec-F+ CD11c- cells 6 (x10 ) 8 * 6 4 2 0

RI PT

A

Fig. 4

4

10

10

*

5 0 RAG2-/- T-bet-/RAG2-/-

ACCEPTED MANUSCRIPT

Il9 Oaz1-ps Pla2g7 Dgat2 Tpsab1 Prss23 Iltifb Il33 Pth2r Tpsb2

High WT 10 4

96.4

10 2

10 1

2.4 10 0

10 1

10 2

10 3

10 10 4

CD25 10 4

11.9

10

0

10

2

10

1

10

10 0

10 1

10 2

10 3

Cell numbers

(x103) 10

**

8

Relative expression 10 4

10 4

10 0

*

*

0.5 0 WT T-bet-/-

T-bet-/-

4

10

3

MIG

7.3

10

4

10

3

86.2

10 2

10 2

10 1

10 1

5.6

10 0 10

0

10

1

10

2

10

3

10

MIG-T-bet 15.3 80.4

2.6

10 0 4

10

0

10

1

10

2

10

3

10 1

10 3

10 4

74.6

71.3

2

10 3

10

10 1

73.5

2

10 1

10 0 10 0

10 1

10 2

10 3

10 4

11.5

10 3

T-bet-/-

10 0

10 1

10 2

10 3

10 4

10 3

10 4

2.8

10 3

10 2

10 2

10 1

10 1

10 0 10 1

IL-5

0

10 4 10 4

10 0

2

5.9

10.0 10 0

Control anti-IL-9

4

10 4

10 3

10

10 2

10

ST2 10 4

10 0

WT

1.0

0

6 4

10 3

10

17.9

76.2

IL-5

D

10 2

EP

10

1

10 1

10 3

AC C

10

2

10 0

IL-13

1.5

gating on GFP+ CD25+

10 4

10 3

gating on CD25+

6.4

0

IL-9

10

10 1

WT T-bet-/-

IL-5

E

ST2

10 3

10 2

T-bet-/-

WT

91.3

10 4

10 3

0

0

T-bet-/-

TE D

C

0.4 0.2

M AN U

Low

*

WT 1.2 1.0 0.8 0.6 0.4 0.2 0

IL-4

0.6

KLRG1

WT

1.0 0.8 0.6 0.4 0.2 0

IL-13

T-bet-/-

(x10-2)

IL-9

T-bet-/-

IL-9

RI PT

WT

B

IL-2+IL-7+IL-33

SC

IL-2+IL-7

Relative expression

A

Fig. 5

10 2

10 3

75.2

10 4

10 0

10 1

10 2

85.3

ACCEPTED MANUSCRIPT

A 4

0.5

10

3.4

8.6

10 1

10 1

10 0

10 0 10

10 4

0

10

1

10

2

10

3

1.0

10

4

10 10

6.0

(x103) 30

2

4

0

10

1

IL-33 + anti-IFN-γ

10 2

10 2

10 1

10 1

10

0

10

1

B

10

2

10

3

10

4

10

4

10

0

10

1

10

2

10

3

10

4

T-bet-/10

4

0.1

0.1

0.0

10 3

10 2

TE D

10 3

10 2

10 1

10 1

10 0

10 0

10 1

10 2

10 3

10 4

10 0

10 1

10 2

10 3

EP

10 0

0.4

0.0

0.0

10 4

0.2

10 2

10 2

10 1

10 1

10 0

10 0

10 1

10 2

CD25

10 3

10 4

T-bet

10 3

AC C

10 3

10 4

**

*

10

WT 4

0.1

IL-33 + anti-IFN-γ

3

9.1

CD25

10 4

10

0.0

10 0

10 0

IL-33

2

10 3

10 3

10

10

20

NS

10 0 10 0

10 1

10 2

10 3

10 4

IL-33 IL-33 + anti-IFN-γ

RI PT

10

SC

2

IL-9

10

0.0

10 3

10 3

IL-33

4

0

M AN U

10

IL-9+ CD25+ Thy1.2+ Lin- cells

T-bet-/-

WT

Fig. 6

WT

T-bet-/-

ACCEPTED MANUSCRIPT WT 24.4

3

10

2

(x106) 3

2

10

10

101

101

100 0 10 4

1

10

2

10

3

10

100 0 10

4

10

4

25.8

10

2

10

3

10

10

102

102

101

101

1

10

2

10

3

10

100 0 10 10 4

4

NS

1 0

1

10

2

10

3

10

WT IL-33

4

10

WT 5.6

3

10

10

102

102

1

1

(x106) 2

10

100 100

101

102

103

104

4

100 100 4

10

3.1

103

103

IL-33 + 102 anti-IL-9

2

101

102

103

101

100 100

101

103

104

100 100

AC C

Lin

102

101

CD25+ Thy1.2+ Lin- cells

102

103

104

*

**

(x106) 1.0

NS

104

2.9

EP

10

101

IL-33 + anti-IL-9

Thy1.2+ Lin- cells

Thy1.2

10

10

10.9

3

TE D

IL-33

T-bet-/-

104

T-bet-/-

M AN U

CD11c

104

*

10

3

10

100 0 10

B

*

2 1

10

18.2

10

3

IL-33 + anti-IL-9

60.8

RI PT

3

10

IL-33

T-bet-/-

4

10

SC

4

10

Siglec-F

A

Fig. 7

0.5

1

*

**

NS

0

0

WT IL-33

T-bet-/IL-33 + anti-IL-9

WT IL-33

T-bet-/IL-33 + anti-IL-9

ACCEPTED MANUSCRIPT

A

Thy1.2+ Lin- cells 1.8

104

(%)

4

103

102

102

1

1

10

Thy1.2

103

T-bet-/-

1

2

10

10

3

10

100 10 100 4

10

2

10

100

10

4

10

0

Lin

0

WT T-bet-/-

CD25+ Thy1.2+ Lin- cells

gating on Thy1.2+ LinWT T-bet-/78.2

60

100 80 60 40 20 0

30 40

10

0 10 1

10 2

CD25

10 3

10 0

10 4

10 1

10 2

10 3

C

NS

10 4

NS

200

M AN U

20 20

0

(X103) 300

(%)

80.7

40

WT T-bet-/-

SC

B

10 0

200

1

3

NS

2

Lin 1

NS

3

10

100 100

(X103) 300

RI PT

WT

1.9

104

WT

100

0

T-bet-/-

WT T-bet-/-

gating on Thy1.2+ LinIL-4

104 103

101

102

103

104

22.5±4.1

102

101

101

101

100 100

101

102

103

104

20.8±6.6

EP 103

3

10

0.2±0.1

102

AC C

101 100 100

101

102

103

104

100 100

101

102

104

100 100

8.6±0.9

3

10

101

101

101

0

102

103

104

10

NS

(MFI) 10

1

10

RORγt NS

5

0

0 T-bet-/-

104

3.6±0.1

0

0

10

10 5

103

3

102

101

102

10

102

100 100

101

104

102

GATA3

WT

103

104

Thy1.2

(MFI) 15

103

102

10

10

9.1±2.9

103

IL-17A 4.2±0.5

104

102

4

4

T-bet-/-

103

IL-13

104

TE D

101 100 100

IL-5

104

0.4±0.2

102

WT

D

Fig. E1

WT T-bet-/-

2

10

3

10

4

10

10

0

10

1

10

2

10

3

10

4

10

ACCEPTED MANUSCRIPT

11.6

3

3

10

10

102

102

1

1

10 5

10

100 0 10 4

15

1

10

2

10

3

10

4

10

100 0 10 4

10

31.0

3

3

10

10

Papain 102

102

101

101

100 0 10

1

10

2

10

3

10

4

10

2

10

EP AC C

3

10

4

10

57.4

100 0 10

1

10

2

10

TE D

CD11c

1

10

3

10

4

10

**

SC

10

10

(X103) 20

32.5

0

-/(X104) WT T-bet 30 * 20

Siglec-F

IL-25

T-bet-/-

4

10

RI PT

WT

M AN U

4

10

Fig. E2

10

0

WT T-bet-/-

ACCEPTED MANUSCRIPT

15.0

10.6

103

103

102

102

101

101

32.4 101

102

103

104

37.9 101

102

103

2

1

10

101

102

0.2 100

103

0.1

104 100

101

102

103

102

103

104

104

24.7

4

10

3

10

gating 102 on CD3+

102

101

101

68.5 102

103

104

100 100

68.0

101

102

103

104

3

10

10

2

10

101

101

0

10

100

101

IL-5

EP

TE D

CD4

AC C

4

10

3.4

102

3.0

3

10

CD8

10

M AN U

IL-5

3

101

0.2

10

100 100

104

T-bet-/-

3

2

1

104 10

10 10

100 100

26.2

100 100

0.5

3

10

CD3ε 104

WT

IL-4

100 100

104

IL-13

10

RI PT

10

B

T-bet-/-

4

SC

WT

4

CD19

A

Fig. E3

2

0.1 100

103

104 100

0.4 101

104

ACCEPTED MANUSCRIPT

1.0 0.5 0

WT T-bet-/-

(x10-2)

BATF

2.0

WT T-bet-/AhR

1.0

TE D

Relative expression

1.5

0.5

WT T-bet-/-

EP AC C

WT T-bet-/PU.1

0.5

(x10-4)

0

1.5 1.0

1.0 0

(x10-4)

IRF4

M AN U

Relative expression

3.0

1.0 0.8 0.6 0.4 0.2 0

RI PT

Relative expression

2.0 1.5

(x10-2)

SC

GATA3

(x10-2)

Fig. E4

0

(x10-3)

5.0 4.0 3.0 2.0 1.0 0

ND

ND

WT T-bet-/IL-9R

WT T-bet-/-

Matsuki et al.

ACCEPTED MANUSCRIPT

1

Online Repository

2 T-bet inhibits innate lymphoid cell-mediated eosinophilic airway inflammation by

4

suppressing IL-9 production

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5

Ayako Matsuki, MD. 1, #, Hiroaki Takatori, MD., PhD.1, #, *, Sohei Makita, MD.1,

7

Masaya Yokota, MD., PhD.1, Tomohiro Tamachi, MD., PhD.1, Akira Suto, MD., PhD.1,

8

Kotaro Suzuki, MD., PhD.1, Koichi Hirose, MD., PhD.1, and Hiroshi Nakajima, MD.,

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PhD.1, *

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1

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University, Chiba, Japan.

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Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba

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#

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*Corresponding author:

16

Address: Department of Allergy and Clinical Immunology, Graduate School of

17

Medicine, Chiba University, 1-8-1 Inohana, Chiba City, Chiba 260-8670, Japan.

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Phone number: +81 43 226 2198

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E-mail address: [email protected] or [email protected]

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Hiroaki Takatori or Hiroshi Nakajima

FAX number: +81 43 226 2199

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A.M. and H.T. contributed equally to this work.

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Matsuki et al.

ACCEPTED MANUSCRIPT

21

Online Repository Methods

22 Reagents

24

Antibodies to murine CD3ε (145-2C11), CD4 (RM4-5), CD8α (53-6.7), CD19 (6D5),

25

CD5 (53-7.3), B220 (RA3-6B2), CD11b (M1/70), CD11c (N418), CD49b (Dx5), FcεRI

26

(MAR-1), TER119, TCRβ (H57-597), TCRγδ (UC7-13D5), GR-1 (RB6-8C5), Thy1.2

27

(30-H12), CD25 (PC61), IL-33Rα (ST2) (DIH9), and KLRG1 (2F1/KLRG1) were

28

purchased from BioLegend (San Diego, CA). Anti-Siglec-F antibody (E50-2440) was

29

purchased from BD Biosciences (San Jose, CA). Anti-IL-9 antibody (MM9C1) and

30

anti-IFN-γ antibody (XMG1.2) were purchased from Bio X Cell (West Lebanon, NH).

31

Anti-GATA3 antibody (TWAJ), anti-T-bet antibody (eBio4B10), and anti-RORγt

32

antibody (B2D) were purchased from eBioscience (San Diego, CA). Recombinant

33

murine IL-2, IL-7, IL-25, and IL-33 were purchased from BioLegend. Recombinant

34

murine IFN-γ was purchased from PeproTech (Rocky Hill, NJ). Papain was purchased

35

from Sigma-Aldrich (St. Louis, MO).

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The sequences of PCR primers:

38

IFN-γ, forward, 5”-TCAAGTGGCATAGATGTGGAAGAA-3”,

39

reverse, 5”-TGGCTCTGCAGGATTTTCATG-3”;

40

IL-4, forward, 5”- GGCATTTTGAACGAGGTCACA -3”,

41

reverse, 5”-GACGTTTGGCACATCCATCTC-3”;

42

IL-5, forward, 5”-ACGGAGGACGAGGCACTTC-3”,

43

reverse, 5”-TCCATTGCCCACTCTGTACTCA-3”;

44

IL-9, forward, 5”-CGTCCCCAGGAGACTCTTCA-3”,

45

reverse, 5”-CGAAAAGCCATGCAACCAG-3”;

46

IL-13, forward, 5”-GGTCCTGTAGATGGCATTGCA-3”,

47

reverse, 5”-GGAGCTGAGCAACATCACACA-3”;

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Matsuki et al.

IL-9R, forward, 5”-GGCAGCAGCGACTATTGCAT-3”,

49

reverse, 5”-ACACAGGAAGGGCCACAGG-3”;

50

IL-12Rβ2, forward, 5”-TTCATAGTCCGTGTTACTGCC-3”,

51

reverse, 5”-TCATCTTCCCACTGCAGTGTA-3”;

52

T-bet, forward, 5”-CAACAACCCCTTTGCCAAAG-3”,

53

reverse, 5”-TCCCCCAAGCAGTTGACAGT-3”;

54

IRF4, forward, 5”-TCCGACAGTGGTTGATCGAC-3”,

55

reverse, 5”-CCTCACGATTGTAGTCCTGCTT-3”;

56

GATA3, forward, 5”-AGAACCGGCCCCTTATCAA-3”,

57

reverse, 5”-AGTTCGCGCAGGATGTCC-3”;

58

BATF, forward, 5”-CTGGCAAACAGGACTCATCTG-3”,

59

reverse, 5”- GGGTGTCGGCTTTCTGTGTC-3”;

60

AhR, forward, 5”-TTCTATGCTTCCTCCACTATCCA-3”,

61

reverse, 5”-GGCTTCGTCCACTCCTTGT-3”;

62

PU.1, forward, 5”-AGAGCTATACCAACGTCCAATGC-3”,

63

reverse, 5”-TTCTCAAACTCGTTGTTGTGGAC-3” ;

64

β-actin, forward, 5”-TGTTACCAACTGGGACGACA-3”,

65

reverse, 5”-CCATCACAATGCCTGTGGTA-3”.

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66

Online Repository Table

67

Online Repository Table 1. Top 10 genes whose expression is up-regulated in

68

IL-33-stimulated T-bet-/- Thy1.2+ Lin- cells ranking

FC

Gene symbol (name)

Function

RI PT

Absolute

4.25

Il9 (interleukin 9)

cytokine

2

2.49

Oaz1-ps (ornithine decarboxylase antizyme 1)

enzyme

3

2.22

Pla2g7 (phospholipase A2, group VII)

enzyme

4

2.07

Dgat2 (diacylglycerol O-acyltransferase 2)

5

2.31

Tpsab1 (tryptase alpha/beta 1)

6

2.05

Prss23 (protease, serine 23)

7

3.33

Iltifb (interleukin 10-related T cell-derived inducible factor beta)

cytokine

8

2.23

Il33 (interleukin 33)

cytokine

9

4.17

Pth2r (parathyroid hormone 2 receptor)

receptor

10

3.92

Tpsb2 (tryptase beta 2)

enzyme

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1

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WAD: Weighted average difference, FC: fold change

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WAD

4

enzyme enzyme enzyme

Matsuki et al.

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70

Online Repository Figure Legends

71 Figure E1. T-bet is dispensable for the development of ILCs in the lung.

73

(A-D) Single-cell suspensions of lung cells were isolated from WT mice and T-bet-/-

74

mice at steady-state conditions and subjected to flow-cytometric analysis. (A)

75

Representative lineage markers (Lin) vs. Thy1.2 staining of isolated lung cells, and

76

means ± SD of the frequency and absolute numbers of Thy1.2+ Lin- cells in the lung are

77

shown (n=3, each). NS=not significant. (B) Representative histograms of CD25 staining

78

gating on Thy1.2+ Lin- cells, and means ± SD of the frequency of CD25+ cells and

79

absolute numbers of CD25+ Thy1.2+ Lin- cells are shown (n=3, each). (C) Isolated lung

80

cells were stimulated with PMA and ionomycin for 4 hs, and subjected to intracellular

81

staining for IL-4, IL-5, IL-13, and IL-17A. Representative Thy1.2 vs. IL-4, IL-5, IL-13,

82

or IL-17A staining of Thy1.2+ Lin- cells, and means ± SD of the frequencies of

83

cytokine-producing cells are shown (n=3, each). (D) Means ± SD of mean fluorescent

84

intensity (MFI) for GATA3 and RORγt staining of lung Thy1.2+ Lin- cells are shown

85

(n=3, each). NS=not significant.

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72

Figure E2. Eosinophilic inflammation is exacerbated in T-bet-/- mice upon IL-25 or

88

papain administration.

89

IL-25 (1 µg) or papain (25 µg) was administered to WT mice and T-bet-/- mice

90

intranasally at days 0, 3, and 6. Twelve hours after the last administration, cells

91

harvested from the BALF were subjected to flow-cytometric analysis. Representative

92

CD11c vs. Siglec-F staining of BALF cells and means ± SD of the absolute numbers of

93

Siglec-F+ CD11c- cells in the BALF are shown (n=3, each). Data are representative of

94

two independent experiments.

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Matsuki et al.

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Figure E3. The frequency and cytokine production of CD4+ T cells in

97

IL-33-stimulated T-bet-/- mice.

98

(A-B) IL-33 was administered to WT mice and T-bet-/- mice intranasally as described in

99

the Methods. (A) Twelve hours after the last administration, isolated lung cells were

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96

subjected to flow-cytometric analysis. Representative CD3ε vs. CD19 staining of

101

single-cell suspensions of lung cells and CD4 vs. CD8 staining of CD3ε+ cells are

102

shown. (B) Isolated lung cells were stimulated with PMA and ionomycin for 4 hs, and

103

subjected to intracellular staining for IL-4, IL-5, and IL-13. Shown are representative

104

IL-5 vs. IL-4 and IL-5 vs. IL-13 staining of CD4+ cells. Data are representative of 3

105

independent experiments.

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Figure E4. The expression levels of Th9 cell-related genes in Thy1.2+ Lin- cells.

108

Isolated lung Thy1.2+ Lin- cells from IL-33-stimulated WT mice and T-bet-/- mice were

109

cultured with IL-2, IL-7, and IL-33 for 6 days, and mRNA levels for indicated

110

molecules were analyzed by qPCR. Data are means ± SD of 3 independent experiments.

111

*p<0.05.

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