Prostaglandin I2 Promotes the Development of IL-17−Producing δγ T Cells That Associate with the Epithelium during Allergic Lung This information is current as of September 27, 2021. Zeina Jaffar, Maria E. Ferrini, Pamela K. Shaw, Garret A. FitzGerald and Kevan Roberts J Immunol published online 5 October 2011 http://www.jimmunol.org/content/early/2011/10/04/jimmun ol.1101261 Downloaded from

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 5, 2011, doi:10.4049/jimmunol.1101261 The Journal of Immunology

Prostaglandin I2 Promotes the Development of IL-17–Producing gd T Cells That Associate with the Epithelium during Allergic Lung Inflammation

Zeina Jaffar,* Maria E. Ferrini,* Pamela K. Shaw,* Garret A. FitzGerald,† and Kevan Roberts*

gd T cells rapidly produce and represent a first line of defense against microbes and other environmental insults at mucosal tissues and are thus thought to play a local immunoregulatory role. We show that allergic airway inflammation was associated with an increase in innate IL-17–producing gd T(gd-17) cells that expressed the aEb7 integrin and were closely

associated with the airway epithelium. Importantly, PGI2 and its receptor IP, which downregulated airway eosinophilic inflam-

mation, promoted the emergence of these intraepithelial gd-17 cells into the airways by enhancing IL-6 production by lung Downloaded from eosinophils and dendritic cells. Accordingly, a pronounced reduction of gd-17 cells was observed in the of naive mice

lacking the PGI2 receptor IP, as well as in the lungs during allergic inflammation, implying a critical role for PGI2 in the programming of “natural” gd-17 cells. Conversely, iloprost, a stable analog of PGI2, augmented IL-17 production by gd T cells but significantly reduced airway inflammation. Together, these findings suggest that PGI2 plays a key immunoregulatory role by promoting the development of innate intraepithelial gd-17 cells through an IL-6–dependent mechanism. By enhancing gd-17 cell

responses, stable analogs of PGI2 may be exploited in the development of new immunotherapeutic approaches. The Journal of http://www.jimmunol.org/ Immunology, 2011, 187: 000–000.

n response to an infection, , or environmental insult, other allergic diseases (2). Conversely, Th17 cells, which produce various cells of the innate and adaptive immune systems a distinctive set of cytokines, including IL-17 (also known as IL- participate in a coordinated fashion to mold an appropriate 17a), IL-17f, IL-21, and IL-22, play crucial roles during immune I + mucosal response. Three distinct subsets of CD4 Th cells—Th1, responses against diverse pathogens, including extracellular bac- Th2, and Th17 cells—play critical roles during adaptive immune teria and fungi (3, 4). The development of Th17 cells is critically responses. These effector T cells not only recruit and activate dependent on TGF-b and IL-6. Importantly, IL-6 is effective at by guest on September 27, 2021 CD8+ T cells, , eosinophils, , and other suppressing the TGF-b–induced generation of Foxp3+ regulatory effector cells, but also act directly on several cells in the mu- T cells (5, 6). IL-17 mediates several important effects in the cosal site, with the epithelial cells typically playing a key role. mucosal site by acting on epithelial cells to induce the expression The diverse functions of CD4+ T cells are typically mediated by of that recruit neutrophils (7), antimicrobial their secretion patterns. Th1 cells secrete IFN-g and are such as b-defensins (8), and the polymeric Ig receptor (9, 10). essential for controlling intracellular pathogens and promoting However, gd T cells can also produce IL-17, and these innate IL- IgG2a production, whereas Th2 cells, which produce IL-4, IL-5, 17–producing T cells are involved in sensing stress, injury, or and IL-13, are critical for IgE production, eosinophil recruitment, pathogens and serve an immunoregulatory role at epithelial sites and clearance of helminth infections (1). Th2 cells are also re- (11, 12). In the lung, the Vg4+ subset has been shown to be sponsible for driving the inflammatory response in asthma and induced during allergic lung inflammation and play an important role in limiting inflammation (13, 14). In marked contrast, the effector CD4+ T cells drive the mucosal inflammation and pro- *Center for Environmental Health Sciences, Biomedical and Pharmaceutical Scien- mote airway remodeling if left unregulated. † ces, University of Montana, Missoula, MT 59812; and Institute for Translational Emerging evidence suggests that prostanoids, such as PGI , Medicine and Therapeutics, University of Pennsylvania School of Medicine, Phila- 2 delphia, PA 19104 PGE2, and PGD2, play prominent roles in promoting and regu- Received for publication May 2, 2011. Accepted for publication September 5, 2011. lating immune responses by facilitating Th1 differentiation, am- plifying IL-23–mediated Th17 expansion (15–17), regulating This work was supported by grants from the National Institutes of Health, Heart, Lung and Institute (R01-HL079189) and Centers of Biomedical Research innate invariant NKT cell responses (18), and suppressing Th2- Excellence (P20RR017670). mediated responses (19–21). Prostanoids, a family of medi- Address correspondence and reprint requests to Dr. Kevan Roberts and Dr. Zeina ators consisting of the PGs and thromboxanes, are generated from Jaffar, Center for Environmental Health Sciences, 285B Skaggs Building, University arachidonic acid in response to various stimuli by the sequential of Montana, Missoula, MT 59812. E-mail addresses: [email protected] and [email protected] actions of cyclooxygenase 1 or 2, and the respective synthases. The online version of this article contains supplemental material. PGs are abundant at sites of inflammation and mediate their Abbreviations used in this article: gd-17, IL-17–producing gd T; AHR, airway hy- actions mainly through their respective G-protein–coupled re- perreactivity; ATTC, American Type Tissue Collection; BALF, bronchoalveolar la- ceptors that include the PGI2 receptor (IP), PGD2 receptor (DP), vage fluid; EPO, eosinophil peroxidase; LMC, lung mononuclear cell; MFI, mean and PGE receptor subtypes (EP1–EP4) (22). Each of these pros- fluorescence intensity; WT, wild-type. 2 tanoids, acting through its own receptor(s), differs in its effect on Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 cAMP (23) and regulation of pathophysiological processes. PGI2

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101261 2 PGI2 PROMOTES IL-17–PRODUCING gd T CELL DEVELOPMENT

is highly labile and rapidly hydrolyzed at physiological pH to RPMI media for 4 d in the presence of OVA323–339 (1 mg/ml; form the inactive 6-keto-PGF1a (24). Studies using mice deficient , San Diego, CA) and IL-4 (2 ng/ml, R&D Systems, Minne- in the PGI receptor IP (IP2/2 mice) provided strong evidence that apolis, MN) plus mAb anti–IFN-g (5 mg/ml R4-6A2; American Type Tis- 2 sue Collection [ATTC], Manassas, VA). After 4 d of incubation, cells were PGI2 (also known as prostacyclin) exerts multiple effects in pro- restimulated using culture conditions identical to those previously used, moting as well as suppressing immune and inflammatory but this time in the presence of exogenous IL-2 (10 ng/ml; R&D Systems) + responses (17, 19, 20, 25, 26). Specifically, PGI2 is known to sup- for a further 4 d. On day 8, the cells were depleted of class II cells by press allergic airway inflammation and remodeling in models of panning by incubating with anti-class II mAb (5 mg/ml M5/114; ATTC) for 30 min, then plate-bound mouse anti-rat IgG (10 mg/ml; Jackson Immu- asthma (19, 21, 27). noResearch, West Grove, PA) for 1 h. Nonadherent (Class II2 cells) CD4+ Many inflammatory characteristics of allergic asthma—namely, Th2 cells were collected for analysis. pulmonary eosinophilia, IgE production, mucus hypersecretion, + and airway hyperreactivity (AHR)—are mediated by CD4+ Th2 Transfer of polarized DO11.10 CD4 Th2 cells and OVA cells. It has become increasingly evident that airway epithelial challenge cells play a central role in the inflammatory response. Specifically, Eight-day polarized DO11.10 CD4+ Th2 cells (107 cells/mouse that are prolonged activation of epithelial cells promotes mucus produc- devoid of gd T cells) were adoptively transferred into BALB/c animals by tion and stimulates fibrogenic and remodeling processes (28–31). i.v. injection. Mice (four to six per group) were then challenged by ex- posure in a chamber to aerosolized solutions of OVA (0.5%, Grade V; However, the interaction of innate and adaptive immune cells with Sigma-Aldrich) for 20 min/day, over 7 consecutive days using a Wright’s the airway epithelium and the impact of the inflammatory re- nebulizer. Control mice were exposed to OVA aerosols but did not receive sponse on this collaboration are poorly understood. The aEb7 DO11.10 Th2 cells. integrin, which serves as a receptor for E-cadherin (32), is Downloaded from Intranasal administration of iloprost, anti–IL-6, or anti-Vg4 expressed by intestinal T cells and by T cells in the lung that associate with airway epithelial cells (33). TGF-b is known to play In certain experiments, mice were treated intranasally with iloprost, anti–IL- an important role in the induction of both IL-17 by T cells (4) and 6 mAb, or anti-Vg4 mAb (four instillations during the aerosol challenge, i.e., every 48 h) to examine the effect on Th2-mediated inflammation. the expression of the aEb7 integrin (34). In the current study, we Briefly, DO11.10 mice were given Th2 cells (6 3 106 cells/ml) and then demonstrated that an innate gd-17 cell response develops in challenged with aerosolized OVA for 7 consecutive days. On days 0, 2, 4,

tandem with the allergic lung inflammation and that these cells and 6 of OVA inhalation, Th2 recipient mice were lightly anesthetized with http://www.jimmunol.org/ expressed the aEb7 integrin and are closely associated with the isofluorane to allow intranasal administration of iloprost (2.5 mg; Cayman Chemical, Ann Arbor, MI), anti–IL-6 mAb (clone MP5-20F3, 15 mg; epithelium. Given that PGI2 plays an important immunoregulatory BioLegend), anti-Vg4 mAb (25 mg UC3; gift from Dr. Jeffrey A. Blue- role during allergic lung inflammation (20, 21), the aim was to stone, University of California) in 30 ml PBS or sterile PBS alone (vehi- examine the involvement of this prostanoid in molding the gd cle). T cell response. We found that PGI -IP signaling is essential in 2 OVA immunization and challenge of mice promoting the development of intraepithelial IL-17–producing gd 2 2 T(gd-17) cells. Our observation that naive IP2/2 mice lacked C57BL/6 wild-type (WT) and IP / mice were immunized with OVA (20 mg) using an alum adjuvant (100 mg) by intraperitoneal injection and after gd-17 cells in the thymus is a clear demonstration that PGI2 plays

10 d the mice were exposed to either OVA aerosols or PBS (control) for 20 by guest on September 27, 2021 an important role in the development of “natural” gd-17 cells. The min/day over 7 consecutive days. production of IL-17 by gd T cells was dependent on IL-6, whose Level of pulmonary inflammation expression was promoted by PGI2. Given the role of gd T cells in epithelial repair and , it is conceivable that promoting Following OVA inhalation for 7 d (i.e., on day 8), bronchoalveolar lavage responses by gd-17 cells using stable analogs of PGI2, such as was performed (3 3 0.5 ml PBS) to collect bronchoalveolar lavage fluid iloprost, may provide a novel approach to resolving some of the (BALF) for analysis. Eosinophil peroxidase (EPO) levels in the bron- epithelial changes that occur during allergic airway inflammation. choalveolar lavage cells were determined by colorimetric analysis. Cell differential percentages were determined by light microscopic evaluation of Hema3-stained cytospin preparations and expressed as absolute cell Materials and Methods numbers. Lung tissue was dispersed by collagenase (Type IV; Sigma- Mice Aldrich), and lung mononuclear cells (LMCs) were isolated by Percoll (Sigma-Aldrich) density gradient for functional analysis. All mice were maintained in microisolator cages and treated in accordance with National Institutes of Health guidelines and the American Association Flow cytometry and intracellular IL-17, IL-6, IL-4, and IFN-g of Laboratory Animal Care regulations. All animal experiments were ap- staining proved by the University of Montana Institutional Animal Care and Use Committee. Female or male C57BL/6, BALB/c (The Jackson Laboratory, FACSAria II (BD Biosciences, San Jose, CA) was used to enumerate the Bar Harbor, ME), IP2/2, and DO11.10 transgenic mice were used number of ab+ T cells, gd+ T cells, KJ1-26+ T cells (KJ-126 is an Ab that throughout (6–8 wk old). IP2/2 mice were developed and provided by Dr. recognizes I-Ad–restricted TCR specific for OVA), Gr-1+ (Gr-1 is a GPI- Garret A. FitzGerald’s Laboratory, Institute for Translational Medicine and linked myeloid differentiation marker also known as Ly-6G, expressed Therapeutics, Philadelphia, PA. DO11.10 transgenic mice (originally de- by granulocytes and ), CD11b+, CCR3+, CD11c+, and class II+ veloped by Dr. D.Y. Loh, Howard Hughes Medical Institute, St. Louis, cells in the LMCs, BALF, spleen, or thymus coexpressing the aEb7 in- MO) and provided by Dr. Ethan Shevach (National Institutes of Health, tegrin (a member of the integrin family expressed almost exclusively by Bethesda, MD) were bred under pathogen-free conditions in a barrier fa- cells of the T lineage in mucosal tissues); IL-17, IL-6, IL-4, cility. IFN-g, CD4, CD8, Vg4, Vg5, or Vg1, using specific mAbs that include anti-ab TCR (H57, FITC-conjugated; BD Biosciences), anti-gd TCR Media (GL3, allophycocyanin-conjugated; BD Biosciences), anti-mouse DO11.10 TCR (KJ1-26, allophycocyanin-conjugated; eBioscience, San Diego, CA), Cells were cultured in RPMI 1640 media supplemented with 10% FBS, anti-aE (anti-CD103, allophycocyanin-conjugated; BioLegend, San Diego, L-glutamine (Life Technologies, Carlsbad, CA), penicillin and strepto- CA) and anti-integrin b7 (M293, PE-conjugated; BD Biosciences), anti-CD4 mycin (Life Technologies), HEPES (Life Technologies), sodium pyruvate (GK1.5, allophycocyanin-Cy7–conjugated, BD Biosciences), anti-CD8 (53- (Life Technologies), and 2-ME (Sigma-Aldrich, St. Louis, MO). 6.7, PE-conjugated; BD Biosciences), anti-Vg4 (UC3, FITC-conjugated; gift Preparation of DO11.10 CD4+ Th2 cells from Dr. J.A. Bluestone), anti-Vg5 (F536, FITC-conjugated; gift from Dr. J.A. Bluestone), anti-Vg1 (2.11, FITC-conjugated; BioLegend), anti–Gr- To generate CD4+ Th2 effector cells, peripheral lymph nodes obtained 1 (Ly-6G/Ly-6C, RB6-8C5, allophycocyanin-Cy7–conjugated; BioLegend), from DO11.10 mice were first depleted of CD8+ cells using MACS beads anti-CD11b (M1/70, allophycocyanin-conjugated; BioLegend), anti-CCR3 (Miltenyi Biotech, Auburn, CA) and incubated (5 3 105/ml) in complete (CD193, TG14/CCR3, Alexa Fluor 647-conjugated; BioLegend), anti- The Journal of Immunology 3

CD11c (N418, allophycocyanin-conjugated; BioLegend), and anti-class II Lung histology (I-A/I-E, M5/114.15.2, allophycocyanin-conjugated; BioLegend). Spleen and thymus cells were first enriched by depletion of CD4+ and CD8+ cells Lung tissue was fixed in Histochoice (AMRESCO, Solon, OH) and em- (and also B220+ cells in the spleen cells), using MACS beads (Miltenyi bedded in paraffin using a Shandon Citadel tissue processor (Thermo Fisher Biotech). CD11b+CCR3+ cells were sorted using FACSAria II (BD Bio- Scientific, Pittsburgh, PA). Microtome sections were cut at 5-mm thickness sciences), and cytospin preparations were stained with Hema3 to confirm and stained with H&E using a Shandon Varistain 24–4 (Thermo Fisher eosinophil purity by light microscopic evaluation (.95%). Scientific). 6 For analysis of intracellular IL-17, IL-6, IL-4, or IFN-g, cells (1 3 10 ) Immunohistochemistry were stimulated with 50 ng/ml PMA plus 500 ng/ml ionomycin (Sigma- Aldrich) in the presence of 1 ml of the protein transport inhibitor BD For immunofluorescent staining, lung tissue was frozen in Sakura Tissue- GolgiPlug containing brefeldin A (BD Biosciences) for 4 h at 37˚C (this Tek O.C.T. Cryosections were mounted on glass slides, blocked, and dual step was omitted for IL-6 staining). Cells (0.5 3 106) were then blocked stained with 100 ml biotinylated anti–IL-17 mAb (10 mg/ml) followed by using 2.4G2 supernatant (ATCC) and stained with the appropriate conju- streptavidin Alexa Fluor 594 (Invitrogen) and 100 ml anti-gd TCR mAb gated mAb or control. Following treatment with fixation and (UC7-13D5 clone, 200 mg/ml; ATCC) followed by anti-hamster IgG permeabilization buffers (BioLegend), cells were intracellularly stained DyLight 488 (Jackson ImmunoResearch). Coverslips were mounted with using the following Abs (all from BioLegend): allophycocyanin- or PE- FluorSave (EMD, Gibbstown, NJ), and sections were examined using conjugated anti–IL-17 (clone TC11-18H10.1), PE-conjugated anti–IL-6 Olympus FV1000 IX81 inverted laser scanning confocal microscope (with (MP5-20F3), allophycocyanin-conjugated anti–IL-4 (clone 11B11), or spectral detection and TIRF Module). DyLight 488-labeled images (green) FITC-conjugated anti–IFN-g (clone XMG1.2) and analyzed by FAC- were acquired sequentially using the 488 laser line (8% power) and the SAria II. emission spectra collected at 500–549 nm. Alexa Fluor 594-labeled images (red) were acquired using the 559 laser line (10% power) and emission spectra collected at 589–695 nm. A lookup table linear adjustment was

Measurement of IL-17, IL-4, and IFN-g production made for the display. Downloaded from To measure IL-17 production, LMCs or spleen cells were stimulated with Pulmonary function measurement anti-CD3 (2 mg/ml 2C11; ATCC), anti-mouse TCR b-chain (5 mg/ml H57- 597), anti-mouse TCR Vg4(5mg/ml UC3), anti-mouse TCR Vg5(5mg/ Respiratory resistance (RL,cmH2O.s/ml) and dynamic compliance (CDyn, ml, F536), anti-mouse gd TCR (5 mg/ml UC7-13D5) (all Abs were kindly ml/cm H2O) were assessed in anesthetized and tracheotomized mice that provided by Dr. J.A. Bluestone), anti-mouse TCR Vg1(5mg/ml, 2.11; were mechanically ventilated in response to increasing concentration of BioLegend), or anti-CD40 Abs (2 mg/ml, IC10; BioLegend). Briefly, cells methacholine inhalation (1.5–24 mg/ml) using the pulmonary function 5 (1 3 10 per well) were cultured for 24 h in the presence of medium alone, equipment from Buxco Research Systems. http://www.jimmunol.org/ or plate-bound anti-CD3/TCR mAb or anti-CD40 mAb in 1.0 ml medium Effect of iloprost on IL-17 production in 96-well plates. After 24 h, the supernatants were harvested, and pro- duction of IL-4, IFN-g, IL-17 (R&D Systems), or IL-6 (R&D Systems) To examine the effect of iloprost on IL-17 production, spleen cells from was measured by ELISA. IL-4 and IFN-g were detected using capture C57BL/6 mice (2 3 106/ml) were stimulated with soluble anti-mouse gd (ATCC) and biotinylated Abs (BD Biosciences), followed by avidin-HRP, TCR (5 mg/ml UC7-13D5; ATTC) in the absence or presence of iloprost then TMB solution (Invitrogen, Carlsbad, CA). (0.5 and 1.0 mM; Cayman Chemical) at 37˚C for 4 d. Cell supernatants

FIGURE 1. Allergic airway in- by guest on September 27, 2021 flammation is associated with an in- creased number of gd-17 cells in the lungs. DO11.10 CD4+ Th2 cells were adoptively transferred into BALB/c mice that were then exposed to aero- solized OVA for 7 d. Control mice did not receive Th2 cells but were exposed to OVA aerosols for 7 d. A, Cell dif- ferential counts in the BALF were determined by light microscopic eval- uation of stained cytospin prepara- tions. Results are expressed as absolute numbers (per mouse) of (Lym), macrophages (Mac), eosino- phils (Eos), and neutrophils (Neu). EPO levels in the BALF from Th2 recipient or control mice were assessed by colorimetric analysis. B, The pro- portion of gd T cells, ab T cells, and clonotypic KJ1-26+ cells coexpressing IL-17 or IL-4 in LMCs from Th2 re- cipient or control mice was determined by FACS analysis using intracellular staining. C, LMCs were stimulated with anti-gd TCR (5 mg/ml) or anti- CD3 (2 mg/ml) for 24 h and superna- tant analyzed for IL-17 or IL-4 pro- duction by ELISA. Results are mean 6 SE of four to six individual mice ana- lyzed per group in triplicates and rep- resent four independent experiments. *p , 0.05. 4 PGI2 PROMOTES IL-17–PRODUCING gd T CELL DEVELOPMENT were then harvested and IL-17 production measured by ELISA (R&D Ab KJ1-26 (Supplemental Fig. 1A), or anti-CD4 (Supplemental Systems). Fig. 1B) and were thus not of donor origin. Further analysis Statistical analysis revealed that the majority of the IL-17–expressing cells were in- deed gd T cells (Fig. 1B) that bore a CD42CD82 phenotype Data are expressed as means 6 SE. Comparisons were analyzed for sta- tistical significance by the Mann–Whitney U test, with p values ,0.05 (Supplemental Fig. 1B). In sharp contrast, the IL-4–expressing considered significant. Analysis was performed with Prism software T cells were predominantly ab T cells and were OVA specific (GraphPad, La Jolla, CA). (KJ1-26+;Fig.1B). Moreover, LMC from OVA-challenged Th2 recipients produced high levels of IL-17 and IL-4 in response to Results stimulation with anti-gd TCR Ab and anti-CD3, respectively (Fig. Allergic lung inflammation is associated with an increase in 1C). Control mice (no Th2 transfer) did not develop any airway intraepithelial gd-17 cell numbers inflammation following OVA inhalation and had low numbers of To resolve the cellular events involved in allergic lung in- IL-4– and IL-17–producing T cells present in the lungs (Fig. 1A–C). flammation that impact on CD4+ T cell responses, we used both Because gd-17 cells require TGF-b for their development (35) the OVA immunization and the passive CD4+ T cell transfer and play an important role in orchestrating epithelial barrier models of asthma. Immunization with OVA is the commonly used function during health and disease (36, 37), we evaluated whether method that reproduces many key features of asthma (e.g., airway these cells expressed the TGF-b–inducible mucosal integrin, inflammation, IgE production, and AHR), whereas the adoptive aEb7 (34), during allergic lung inflammation. Of interest, the transfer of transgenic Th2 cells approach has the advantage that majority of IL-17–producing T cells from Th2 recipient mice it allows the tracking of OVA-specific T cells, using the anti- expressed the aE and b7 integrin chains when characterizing Downloaded from clonotypic TCR Ab KJ1-26, during the inflammatory response. T cells in both the LMCs (Fig. 2A) and the BALF (Fig. 2B). With the adoptive transfer model of allergic airway inflammation, Conversely, this high level of aEb7 expression was not evident in DO11.10 CD4+ Th2 cells were generated in vitro and transferred the control group. Moreover, immunohistological examination of into BALB/c mice that subsequently inhaled aerosolized OVA for lung tissue revealed expression of IL-17+TCR+ cells in the air- 7 d consecutively. Following OVA exposure, Th2 recipients, but ways of Th2 recipients, but not control mice (Fig. 2C). Together,

not control mice, developed pronounced airway inflammation, these data show that in tandem with Th2-mediated inflammation http://www.jimmunol.org/ characterized by a marked increase in the number of lymphocytes intraepithelial gd-17 cells in the airways show a marked increase. and eosinophils and the level of EPO in the BALF (Fig. 1A). The eosinophilic inflammation was invariably associated with an in- gd-17 cell response in the lung during allergic airway crease in IL-17–expressing T cells, as well as IL-4–expressing inflammation is dependent on PGI2 T cells in the lungs (Fig. 1B). The IL-17–expressing T cells in the It has been proposed that innate intraepithelial gd-17 cells serve LMCs failed to stain with anti-ab TCR (Fig. 1B), anti-clonotypic as the sentinels of epithelial surfaces and play a central role in by guest on September 27, 2021

FIGURE 2. Allergic airway inflammation is associ- ated with an increased number of intraepithelial gd-17 cells. DO11.10 Th2 cells were injected into BALB/c mice that were then exposed to aerosolized OVA for 7 d. Control mice inhaled OVA but did not receive Th2 cells. The proportion of IL-17–expressing cells that coexpress gd TCR, aE integrin, or b7 integrin in LMCs (A) or BALF (B) from Th2 recipients or con- trol mice was determined by FACS analysis using in- tracellular staining. C, Expression of IL-17+ and gd-TCR+ cells by lung tissue from Th2 recipient mice or control animals was determined by immunohisto- chemical staining (203) and acquired using laser scan- ning confocal microscopy, as described in Materials and Methods. Data represent four independent experi- ments with similar results. The Journal of Immunology 5 maintaining mucosal barrier integrity. These gd T cells rapidly per se were essentially the same in the lungs of WT and IP2/2 produce IL-17 and regulate pathogen clearance, inflammation, and mice (5.8 and 5.1%, respectively). It was observed that IL-17– epithelial homeostasis in response to tissue stress (12, 38). Given expressing ab T cells were also present in the lungs of OVA- that high levels of PGI2 are produced during allergic lung in- challenged C57BL/6 (Fig. 3A), and, to a lesser extent, in BALB/c flammation and serve to inhibit the Th2-mediated inflammatory mice (Fig. 1B). This finding reflects a slightly higher prevalence of response and remodeling (20, 21, 27), we examined whether this “natural” IL-17–expressing ab T cells in the lungs of naive prostanoid exerted any immunoregulatory action on gd T cell C57BL/6 compared with BALB/c mice (Supplemental Fig. 2). 2/2 2 2 response. IP mice lacking the PGI2 receptor IP were used, and These cells were found to be CD4 CD8 invariant NKT cells the animals were OVA immunized and exposed to OVA aerosols typically present in both the lungs and the spleen (data not shown), for 7 d to induce allergic inflammation. The IP2/2 mice had en- and have been described previously (39). hanced peribronchial inflammation (Supplemental Fig. 3A) with Consistently, a pronounced loss of IL-17 production by gd augmented eosinophil numbers and EPO levels in the airways T cells was present in both LMCs and spleens (stimulated with (Supplemental Fig. 3B), when compared with OVA-challenged anti-gd TCR or anti-Vg4 Ab) of IP2/2 mice compared with WT WT C57BL/6 mice. Control IP2/2 and WT mice that inhaled mice (Fig. 4A). This loss of IL-17 production by gd T cells PBS did not develop any pulmonary inflammation. In marked appears to consist mainly of Vg4+ cells (50–60% of IL-17 pro- contrast to the augmented allergic pulmonary inflammation, duction by WT gd T cells was by Vg4+ cells; Fig. 4A), which is in a dramatic loss in the proportion (Fig. 3A) and the absolute accordance with the report of Murdoch et al. (40). Consistent with number of gd-17 cells (Fig. 3B) was observed in the lungs of these data, typically, 30% of gd T cells present in the LMCs of 2 2 2 2 OVA-challenged IP / mice compared with WT mice. Particu- OVA-challenged WT and IP / mice were Vg4+ T cells (Fig. 4B). Downloaded from larly noteworthy was a loss of gd T cells expressing aE integrin Stimulation of LMC with plate-bound anti-Vg5 or anti-Vg1 Abs in the LMC of IP2/2 mice (Fig. 3A). The number of IL-17– showed that the remaining IL-17 production was not by Vg5+ or expressing ab T cells was unaffected. Control mice that inhaled Vg1+ T cells (data not shown). In keeping with these findings, PBS had negligible numbers of IL-17–expressing T cells in the mice receiving Th2 cells and treated daily with indomethacin, lungs (Fig. 3A). It is important to note that only the gd-17 cells a nonselective cyclooxygenase 1 and 2 inhibitor, elicited a reduced 2/2 were affected in IP mice, because the numbers of gd T cells gd-17 cell response in the lungs (Fig. 4C). Collectively, these http://www.jimmunol.org/ results demonstrate that prostanoids, PGI2 in particular, play a key role in promoting gd-17 T cell response in the lung. Loss of programming of gd-17 cells in the thymus and spleen of naive IP2/2 mice: enhancement by iloprost We next investigated whether this loss of gd T cells was a con- sequence of a preexisting defect in the generation of natural gd-17 cells in the thymus and spleen of IP2/2 mice. We discovered a dramatic reduction in the proportion (Fig. 5A) and total number by guest on September 27, 2021 (Fig. 5B) of natural gd-17 cells in both the thymus and the spleen from naive IP2/2 mice. Similar to lung gd T cells, the majority of natural gd T cells in the thymus expressed aEb7 integrin (Fig. 5A). As depicted in Fig. 5B, the absolute number of gd-17 cells per mouse was 8.5 3 105 in spleen of naive WT mice, compared with 4.1 3 105 in IP2/2 mice. In marked contrast, the number and proportion of IL-172 gd TCR+ cells were similar in both IP2/2 and WT tissues, suggesting that the absence of PGI2-IP signaling affects IL-17 expression rather than favoring the expression of particular gd TCR gene rearrangements. Conversely, iloprost (1.0 mM), a stable analog of PGI2, signif- icantly enhanced IL-17 production by splenic gd T cells stimu- lated with anti-gd TCR Ab (5 mg/ml) (Fig. 5C). Relatively low levels of IFN-g and IL-4 were produced by the gd T cells (data not shown). In summary, a pronounced reduction in the number of gd-17 cells (but not gd T cell numbers) was observed in the thymus and spleen of naive IP2/2 mice, implying a critical role for PGI2 in the programming of natural gd-17 cells. IL-6 production by eosinophils and dendritic cells during allergic lung inflammation is dependent on PGI2 and promotes FIGURE 3. Loss of gd-17 cells in the lungs of IP2/2 mice during al- gd-17 cell development 2/2 lergic inflammation. C57BL/6 WT or IP (IPKO) mice were immunized Given that IL-6 is required for promoting the development of with OVA, and after 10 d the mice were exposed to either aerosolized OVA IL-17–producing ab-TCR–expressing T cells (5), we examined or PBS (control) for 7 d. A, The proportion of gd T cells and ab T cells whether PGI facilitated IL-6 production and subsequent gd-17 coexpressing IL-17 or aE integrin in LMCs obtained from OVA-chal- 2 lenged or control IP2/2 and WT mice, determined by FACS analysis using cell development. In the first instance, we examined the role of IL- intracellular staining. B, The absolute number/mouse of gd T cells 6 in generating the gd-17 response. Using the Th2 transfer model expressing IL-17 in LMCs of OVA-challenged or control WT and IP2/2 of asthma, treatment of Th2 recipient mice with intranasal anti– mice. Data are mean 6 SE (n = 3). FACS data represent four independent IL-6 mAb (0.6 mg/kg, every 48 h during the 7-d OVA inhalation experiments. *p , 0.05, compared with OVA-challenged WT. period) caused a reduction in the number of gd-17 cells and 6 PGI2 PROMOTES IL-17–PRODUCING gd T CELL DEVELOPMENT Downloaded from FIGURE 4. Loss of gd-17 cells in the lungs of IP2/2 and indomethacin-treated mice: role for Vg4 gd T cells. A, C57BL/6 WT or IP2/2 (IPKO) mice were immunized with OVA, and after 10 d the mice were exposed to either aerosolized OVA or PBS for 7 d. LMCs or splenic cells (SPLN) were harvested and stimulated for 24 h with anti-gd TCR or anti-Vg4(5mg/ml), and IL-17 production in the supernatants was then measured by ELISA. B, The proportion of gd T cells expressing Vg4 in LMCs from OVA-challenged WT and IPKO mice analyzed by FACS. C, DO11.10 CD4+ Th2 cells were adoptively transferred into BALB/c mice that were then either treated daily by intraperitoneal injection of 5 mg/kg of indomethacin (Th2 + INDO group) or PBS vehicle (Th2 group). Control mice did not receive Th2 cells (Con). All mice were exposed to OVA aerosols for 7 d. Results are mean 6 SE of four to six individual mice analyzed per group in triplicates and represent three independent experiments. *p , 0.05 compared with WT or Th2 group. http://www.jimmunol.org/

CD103+ cells (aE-expressing), but not ab T cells, compared with expression by CD11b+, CD11c+, class II+, Gr-1+, or CCR3+ cells vehicle-treated Th2 recipient mice (Fig. 6A). In sharp contrast, in LMCs from OVA-challenged or control animals was de- OVA-challenged control animals had negligible numbers of IL- termined in this way. By gating on forward and side-scatter, the 17–expressing T cells in the lungs. majority of IL-6–expressing cells in the lungs during allergic in- To examine the cellular source of IL-6 in the lung during allergic flammation were found to consist of CD11b+CCR3+ eosinophils inflammation, C57BL/6 WT or IP2/2 mice were immunized with and, to a lesser extent, CD11c+ class II+ dendritic cells (Fig. 6B). + + OVA/Alum prior to exposure to either aerosolized OVA or PBS Moreover, a marked increase in the number of CD11b CCR3 by guest on September 27, 2021 (control) for 7 consecutive days. The cellular source of IL-6 eosinophils was found in the lungs of both OVA-challenged WT production during allergic airway inflammation was identified (15.1%) and OVA-challenged IP2/2 (18.5%) mice compared with by intracellular staining using three-color flow cytometry. IL-6 control animals (3.1%) (Fig. 6B). Importantly, the level of IL-6

FIGURE 5. Loss of gd-17 cells in the spleen and thymus of naive IP2/2 mice: enhancement by iloprost. The proportion (A) and the absolute number per mouse (B)ofgd T cells coexpressing IL-17, aE integrin, or b7 integrin in the spleen (de- pleted of CD4+, CD8+, and B220+ cells) or thymus (depleted of CD4+ and CD8+ cells) from naive C57BL/6 WT mice or IP2/2 mice were determined by FACS analysis using intracellular staining. C, IL-17 production by splenic gd T cells stimulated with anti-gd TCR in the ab- sence or presence of iloprost, a stable 6 analog of PGI2. Spleen cells (2 3 10 /ml) from naive C57BL/6 mice were stimu- lated with soluble 5 mg/ml anti-gd TCR 6 iloprost (0, 0.5, and 1.0 mM) for 4 d, and IL-17 production was measured by ELISA. Data are mean 6 SE (n = 3). FACS results represent three to four in- dependent experiments. *p , 0.05. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 6. IL-6 production by eosinophils during allergic pulmonary inflammation is dependent on PGI2-IP signaling and promotes gd-17 cell de- velopment. A, DO11.10 CD4+ Th2 cells were transferred into BALB/c mice that were either treated with intranasal anti–IL-6 mAb (Th2 + anti–IL-6, 0.6 mg/kg in 30 ml PBS) or PBS vehicle (Th2) four times every 48 h during the 7-d OVA inhalation period. Control mice were vehicle treated but did not receive Th2 cells. All mice were exposed to aerosolized OVA for 7 d. IL-17–expressing ab Tcells,gd T cells, and CD103+ (aE+) cells in the LMCs were determined by FACS analysis and expressed as absolute cell number per mouse. B, C57BL/6 WT or IP2/2 (IPKO) mice were immunized with OVA, and after 10 d the mice were exposed to either aerosolized OVA or PBS (control) for 7 d. The number of lung CD11b+CCR3+ eosinophils coexpressing IL-6 (expressed as MFI) and the expression of IL-6 by whole LMCs from challenged IPKO and WT or control mice were determined by FACS analysis using intracellular staining. Results are mean 6 SE (n = 4) and represent three to four independent experiments. *p , 0.05 compared with vehicle-treated Th2 group. by guest on September 27, 2021 expression by the total LMC population and, in particular, the in the airways (Fig. 7A,7B) but caused a 2-fold increase in IL-6 CD11b+CCR3+ eosinophils, was markedly less in IP2/2 than in production by LMCs (Fig. 7C). Moreover, ∼50% inhibition was WT mice (mean fluorescence intensity [MFI] of 800 versus 1403, noted in the proportion (Fig. 7D) and total number (Fig. 7E)of respectively) (Fig. 6B). It is important to note that the CD11b+ OVA-specific CD4+KJ1-26+ T cells in the lungs of mice treated CCR3+ cells were initially sorted using FACSAria II and cytospin with iloprost. Control animals had negligible levels of CD4+KJ1- preparations were stained with Hema3 to confirm eosinophil pu- 26+ T cells in the lungs (Fig. 7D,7E). Consistently, an augmented rity by light microscopic evaluation, which was .98%. IL-6 ex- level of AHR, indicated by increased resistance and decreased pression by lung CD11c+ class II+ dendritic cells was also less in compliance, was observed in the Th2 recipient compared with OVA-challenged IP2/2 compared with WT mice (MFI of 1106 control mice and was reduced by iloprost treatment (Fig. 7F). versus 1330, respectively). Depletion of Vg4 gd T cells in vivo augments airway Collectively, these results suggest that PGI -dependent IL-6 2 Th2-mediated inflammation production by eosinophils and dendritic cells promotes the de- velopment of intraepithelial gd-17 cells. To investigate the contribution of Vg4 gd T cells in the allergic inflammatory process, anti-Vg4 Ab (1 mg/kg) was given in- Iloprost treatment reduces allergic airway inflammation and tranasally to mice to deplete Vg4 gd T cells that had accumulated AHR but enhances IL-6 production in the airways. BALB/c mice were first adoptively transferred with + To examine the immunomodulatory effects of PGI2 in vivo, mice DO11.10 CD4 Th2 cells and the mice were treated intranasally were treated with the stable analog of PGI2, iloprost, and the ef- with either anti-Vg4 Ab (Th2 + anti-Vg4) or vehicle (Th2) every fect on allergic airway inflammation and AHR was determined. 48 h during the OVA inhalation period. Control mice were vehicle According to the Th2 transfer model of asthma, Th2 recipient treated but did not receive any Th2 cells (no transfer). All mice mice were intranasally administered either iloprost (0.1 mg/kg) or inhaled aerosolized OVA for 7 d consecutively. Treatment of Th2 vehicle every 48 h during the OVA inhalation period. Control mice recipient mice with the anti-Vg4 Ab caused a marked decrease did not receive any Th2 cells but were vehicle treated. All mice in the proportion (Fig. 8A) and number (Fig. 8B) of CD103+gd+ were exposed to aerosolized OVA for 7 consecutive days. Fol- T cells in the lungs, compared with Th2 recipients untreated with lowing OVA inhalation, a pronounced influx of lymphocytes and the Ab. This depletion of intraepithelial Vg4 gd T cells resulted in eosinophils into the airways of Th2 recipients took place (Fig. an augmented number of Ag-specific T cells (CD4+KJ1-26+ cells) 7A), with increased EPO levels in their BALF, compared with that and an increase in the number of eosinophils and the level of EPO in control animals (Fig. 7B). Treatment with iloprost significantly activity in the airways (Fig. 8C–E). Control mice (no Th2 transfer) reduced the volume of eosinophil and lymphocyte accumulation did not develop any airway inflammation. Together, these results 8 PGI2 PROMOTES IL-17–PRODUCING gd T CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/

FIGURE 7. Iloprost treatment downregulates allergic airway inflammation and AHR but enhances IL-6 production. DO11.10 CD4+ Th2 cells (6 3 106/ mouse) were adoptively transferred into BALB/c mice and were treated with either intranasal iloprost (Th2 + ILOP, 0.1 mg/kg in 30 ml PBS) or PBS vehicle (Th2) every 48 h during the 7-d OVA inhalation period (i.e., four instillations). Control mice were vehicle treated but did not receive Th2 cells. All mice were exposed to aerosolized OVA for 7 d. A, Cell differential counts in the BALF were determined by light microscopic evaluation of stained cytospin preparations. Results are expressed as absolute numbers (per mouse) of lymphocytes (Lym), macrophages (Mac), eosinophils (Eos), and neutrophils (Neu). B, The level of EPO in the BALF was assessed by colorimetric analysis. C, IL-6 production by LMC from the Th2 or Th2 + ILOP group was determined by ELISA. The proportion (D) and absolute number (E) of clonotypic CD4+KJ1-26+ T cells in LMCs was determined by FACS analysis. F, Lung resistance

(RL) and dynamic compliance (CDyn) was assessed in anesthetized and tracheotomized mice that were mechanically ventilated in response to increasing

concentration of methacholine inhalation. Data are mean 6 SE of four to six individual mice analyzed per group in triplicates and represent three to four by guest on September 27, 2021 independent experiments. *p , 0.05 compared with vehicle-treated Th2 group. suggest that Vg4 gd T cells play an important immunoregulatory development of these cells in the thymus and periphery, the nature role during allergic pulmonary inflammation. of Ag recognized, and their role in mucosal inflammation. Cer- tainly, the juxtaposition of these cells to the epithelium strongly Discussion suggests a role for them in modulating the behavior of airway Using a Th2 adoptive transfer model of allergic lung inflamma- epithelial cells during the inflammatory phase. + tion, we have previously examined the CD4 Th2 response and its Our previous studies have revealed that PGI2 limits the pro- + regulation by the prostanoid PGI2 generated during the inflam- gression of CD4 Th2 cell responses (20, 21), primarily because matory response (20, 21). Curiously, we observed that during the the IP receptor for PGI2 is upregulated by IL-4 generated during allergic inflammation, IL-17–producing T cells accumulated in the allergic lung inflammation. As a result of this, the immunoregu- airways. In the current study, we sought to use this model to latory properties of this prostanoid are most evident during Th2- characterize these IL-17–expressing T cells. Surprisingly, the IL- mediated inflammatory responses. Consequently, we examined 17–producing T cells in the inflamed lungs were predominantly gd whether the gd T cell response was also influenced by PGI2, using T cells. Although only low numbers of gd T cells were found to mice lacking the IP receptor. Our data revealed that allergic lung be resident in the lung tissue of naive mice, following the onset inflammation was augmented in IP2/2 mice [as previously shown of Th2-mediated airway eosinophilic inflammation, a marked in- (19)] but, in stark contrast, the appearance of gd-17 cells in the crease in the number of host intraepithelial CD42CD82 gd T cells lungs of these animals was attenuated. This finding was surprising in the lungs was noted. Moreover, the vast majority of gd T cells because the emergence of gd-17 cells closely paralleled the level in this inflammatory site produced IL-17. The accumulation of of allergic inflammation. Consequently, this observation strongly gd-17 cells in the lung during mucosal inflammation induced by suggested that PGI2 is an essential component, underpinning the inhaled allergen was intriguing and prompted speculation that lung gd-17 cell response. This effect stemmed from a markedly they may play a role in the inflammatory process or its regulation. reduced number of “natural” innate gd-17 cells in the IP-null Strikingly, the gd T cells in the inflamed lung tissue uniformly mice. This defect was also evident in the thymus, where a failure expressed the aEb7 integrin that promotes adhesion to E-cadherin to generate gd-17 cells expressing the aEb7 integrin was noted in 2/2 (32), and, expectedly, these cells were largely associated with the naive IP mice. Conversely, the stable analog of PGI2, iloprost, airway epithelium. Such “priming” of the airway epithelium with markedly increased IL-17 production by splenic gd T cells but gd-17 cells during allergic inflammation is consistent with the significantly reduced airway inflammation. proposed key function of these cells as sentinels of epithelial sur- The pronounced reduction in IL-17 production by gd T cells faces (12). However, many questions remain that pertain to the evident in IP2/2 mice was surprising and strongly implied that The Journal of Immunology 9

FIGURE 8. Vg4 depletion in vivo augments Th2-mediated pulmonary inflammation. DO11.10 CD4+ Th2 cells were adoptively transferred into BALB/c mice and were administered intranasally (every 48 h during 7-d OVA inhalation period) with either anti-Vg4 mAb (Th2 + anti-Vg4, 25 mgin30mlPBS)orPBSvehicle(Th2).Control mice were vehicle treated but did not receive Th2 cells (no transfer). All mice were exposed to aerosolized OVA for 7 days. The proportion (A) and absolute number (B)ofgd T cells coex- pressing aE integrin (CD103) in LMCs from Th2, Th2 + anti-Vg4, or no transfer groups were determined by FACS analysis. C, The number of CD4+KJ1-26+ T cells was determined by

FACS analysis, and results were expressed as cell Downloaded from number per mouse. D, Cell differential counts in the BALF were determined by light micro- scopic evaluation of stained cytospin prepara- tions. Results are expressed as absolute numbers (per mouse) of lymphocytes (Lym), macrophages (Mac), eosinophils (Eos), and neutrophils (Neu).

E, EPO levels in the BALF were assessed by http://www.jimmunol.org/ colorimetric analysis. FACS results are represen- tative of two independent experiments. Data are mean 6 SE of four to six individual mice ana- lyzed per group in triplicates and represent three independent experiments. *p , 0.05 compared with Th2 group. by guest on September 27, 2021

PGI2 played a critical role in the programming of IL-17 production gd T cells differ markedly from ab T cells in their TCR receptor by these cells in the thymus, and possibly in the periphery. This diversity and a propensity to localize to epithelial sites (50). The defect in the IP2/2 mouse could not be a consequence of altered gd preferential homing of gd T cells to epithelial tissues is an intrinsic TCR expression per se because their total numbers and Vg usage function of this cell type, exemplified by the observation that ap- were similar to those in WT littermates. To date, both TGF-b and proximately one third of intestinal intraepithelial cells express a gd RORgt have been shown to be essential for the generation of nat- TCR (50). The use of a particular Vg segment by the gd TCR is ural gd-17 cells (35, 41). Furthermore, a role for IL-23 in promoting highly relevant because in early life the diversity of the gd TCR is IL-17 release by gd T cells has been proposed (42, 43), and the a function of the embryonic stage of development, with T cells, augmentation of IL-17 production by ab T cells by PGE2 via an IL- generated early and expressing a canonical gd TCR using Vg5and 23–dependent mechanism has been well documented (44–46). In Vg6, emigrating from the thymus to the skin and female re- contrast, PGI2 and its receptor had an important part in augmenting productive tract, respectively (51, 52). Subsequently, T cells leaving IL-6 production by eosinophils, as well as by dendritic cells, which the thymus display higher levels of diversity and seed into peripheral have been shown to express IP (47). That eosinophils are responsive sites. Interestingly, in the current study a significant amount of IL-17 + to PGI2 might be expected from our previous finding that IL-4 is an production in the lung was found by Vg4 gd T cells, but not Vg5 important cytokine for inducing expression of the receptor (21) and cells or Vg1. Vg4-expressing cells in the lung have been observed from reports that eosinophils are an important source of this cyto- previously during OVA-induced lung inflammation (13, 14, 53) or kine, which was clearly illustrated in mouse eosinophils, using IL-4 respiratory syncytial virus infection (54). It has been demonstrated GFP reporter mice (48). Certainly, human eosinophils impact on the that both short- and long-term OVA inhalation induced Vg4- inflammatory process by releasing a range of cytokines that include expressing suppressor cells that inhibited AHR (53, 55, 56) and IL-4, IL-13, IL-6, TGF-b, and IL-10 (49). Conceivably, during al- reduced the IgE response (57). This Vg4 response required CD8+ lergic inflammation the programming of cytokine expression is dendritic cells to develop (55) and did not affect the inflammatory + strongly influenced by PGI2 in an environment where IL-4 has response (14). Vg6 cells have been shown to be present in the lungs a central role. The observation that PGI2 had an important function of mice following infection with Bacillus subtilis (58), whereas in the development of both natural and inflammatory gd-17 cells is Vg1+ cells promote AHR in a model of allergic inflammation (59). indicative of this mechanism being operative when these cells are More recent work has shown that gd-17 cells are involved in the generated both in the thymus and in the periphery during allergic resolution of allergic airway inflammation and AHR (40). inflammation, although the relative importance of eosinophils and The priming of the airways with intraepithelial gd-17 cells raises dendritic cells may differ in these two scenarios. the issue of whether this contributes to the inflammatory process. 10 PGI2 PROMOTES IL-17–PRODUCING gd T CELL DEVELOPMENT

A notable property of gd T cells is the rapid cytokine release on nisms that are likely to be operative at mucosal sites and point to encountering Ag, a characteristic attributed to the prior pro- a role for prostanoids in molding the developing immune re- gramming of these T cells in the thymus and possible polarization sponse. Conceivably, modifying intraepithelial gd T cell function in the periphery (38). As such, these cells are regarded as “first using stable PGI2 analogs, such as iloprost, may provide a novel responders” and provide an immediate response to environmental approach capable of regulating some changes to the airway epi- insult or infection. gd T cells are able to recognize nonpeptidic thelium that occur in chronic lung inflammatory diseases such as Ags expressed by stressed cells that are recognized by pattern asthma. recognition receptors. However, innate responses elicited by gd T cells can also ensue following engagement of the pattern rec- Acknowledgments ognition receptors Dectin-1 and TLR 2 (42, 43). gd T cells that We thank the Fluorescence Cytometry Facility Core and Lou Herritt, produce IL-17 have been implicated in a series of immune Diane Brooks (Molecular Histology and Fluorescence Imaging Core), and responses that include the clearance of tubercu- Mary Buford (Inhalation and Pulmonary Physiology Core) for technical losis (60), Escherichia coli (61, 62), and Candida albicans (63). assistance. IL-17 production in the mucosal site is of particular importance because this cytokine exerts a wide range of effects at epithelial Disclosures surfaces that include release of b-defensins (64), promoting the The authors have no financial conflicts of interest. recruitment of neutrophils (65), inducing the expression of the polymeric Ig receptor, and trans-epithelial transport of IgA (9,

10). Conceivably, gd-17 cells play a role in the recruitment of References Downloaded from 1. Mosmann, T. R., and R. L. Coffman. 1989. TH1 and TH2 cells: different patterns neutrophils to the lung during allergic lung inflammation. Al- of lymphokine secretion lead to different functional properties. Annu. Rev. though the most striking aspect of allergic lung inflammation is Immunol. 7: 145–173. the pronounced infiltration of eosinophils into the airways, several 2. Robinson, D. S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, C. Corrigan, S. R. Durham, and A. B. Kay. 1992. Predominant TH2-like bron- reports have documented the involvement of neutrophils in severe choalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326: human asthma (66–68). Given the pronounced immunoregulatory 298–304. 3. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. 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