Epithelial Hyperplasia in Human Papillomavirus-Associated IL-17 Suppresses Immune Effector Functions

IL-17 Suppresses Immune Effector Functions in Human Papillomavirus-Associated Epithelial Hyperplasia

This information is current as Christina Gosmann, Stephen R. Mattarollo, Jennifer A. of September 24, 2021. Bridge, Ian H. Frazer and Antje Blumenthal J Immunol 2014; 193:2248-2257; Prepublished online 25 July 2014; doi: 10.4049/jimmunol.1400216

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-17 Suppresses Immune Effector Functions in Human Papillomavirus-Associated Epithelial Hyperplasia

Christina Gosmann,* Stephen R. Mattarollo,* Jennifer A. Bridge,* Ian H. Frazer,*,† and Antje Blumenthal*,†

Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia that can progress to cancer and is thought to depend on immunosuppressive mechanisms that prevent viral clearance by the host. IL-17 is a cytokine with diverse functions in host defense and in the pathology of autoimmune disorders, chronic inflammatory diseases, and cancer. We analyzed biopsies from patients with HPV-associated cervical intraepithelial neoplasia grade 2/3 and murine skin displaying HPV16 E7 protein-induced epithelial hyperplasia, which closely models hyperplasia in chronic HPV lesions. Expression of IL-17 and IL- 23, a major inducer of IL-17, was elevated in both human HPV-infected and murine E7-expressing lesions. Using a skin- grafting model, we demonstrated that IL-17 in HPV16 E7 transgenic skin grafts inhibited effective host immune responses against Downloaded from the graft. IL-17 was produced by CD3+ T cells, predominantly CD4+ T cells in human, and CD4+ and gd T cells in mouse hyperplastic lesions. IL-23 and IL-1b, but not IL-18, induced IL-17 production in E7 transgenic skin. Together, these findings demonstrate an immunosuppressive role for IL-17 in HPV-associated epithelial hyperplasia and suggest that blocking IL-17 in persistent viral infection may promote antiviral immunity and prevent progression to cancer. The Journal of Immunology, 2014, 193: 2248–2257. http://www.jimmunol.org/ uman papillomaviruses (HPV) infect the epithelium of gd T cells, NK cells, NKT cells, lymphoid-tissue inducer-like the genital tract and the skin. Persistent infection with cells, and subsets of myeloid cells (7, 8). IL-17 induction in H high-risk HPV types can cause cancer, the most prevalent these cell types mostly requires signaling via the IL-23R, in ad- being cervical cancer with .0.5 million new cases and 0.2 million dition to other cytokines, including IL-1b and IL-18 (8, 9). IL-17 deaths per year (1, 2). Four major steps lead to the development of has a key role in host defense against infections and the patho- cervical cancer, as follows: 1) HPV transmission, 2) viral persis- genesis of some autoimmune and chronic inflammatory diseases tence, 3) progression of persistently infected epithelium to dys- (10–12). IL-17 has also been ascribed immune regulatory func- plastic epithelium, and 4) invasion through the epithelial basement tions in diseases such as asthma and colitis (13), as well as anti- membrane (3). Persistent infection with high-risk HPV is associ- and protumorigenic effects, depending on the disease context (14). by guest on September 24, 2021 ated with the expression of the HPV proteins E6 and E7 in epi- Recently, increased IL-17 production has been associated with thelial cells. E6 and E7 inhibit epithelial cell differentiation and persistent HPV16/18 infection and cervical epithelial neoplasia and apoptosis, thus promoting epithelial hyperplasia and transforma- transformation (15, 16). However, the function of IL-17 in viral tion (4). To date, there is no cure for chronic HPV infection. persistence and premalignant epithelial disease is unknown. Furthermore, little is known about the mechanisms that allow for In this study, we demonstrate a locally immunosuppressive persistent infection. However, a local immunosuppressive envi- function of IL-17 in HPV-associated premalignant disease. We ronment that prevents viral clearance by the host has been deemed observed elevated IL-17 production in human cervical precan- critical for viral persistence (5). cerous lesions, as well as in skin in a mouse model of HPV16 E7 IL-17 is an inflammatory cytokine (6) produced by CD4+ Th17 protein-induced epithelial hyperplasia. We identified CD4+ and gd cells and CD8+ T cells as well as innate immune cells, including T cells in HPV16 E7 transgenic hyperplastic skin, and CD4+ T cells in human cervical neoplastic epithelium as the predominant cellular sources of IL-17. Using a skin transplantation model, *University of Queensland Diamantina Institute, Translational Research Institute, we further demonstrate that the presence of IL-17 in E7 transgenic Brisbane, Queensland 4102, Australia; and †Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Queensland 4102, Australia hyperplastic grafts suppresses graft rejection by an immunocompetent host. This identifies a local immunosuppressive role for IL- Received for publication January 23, 2014. Accepted for publication June 29, 2014. 17 in a mouse model of HPV-associated premalignant disease. This work was supported by National Health and Medical Research Council of Australia Program Grant 080214, National Cancer Institute Grant RFA-CA-08-018, and funding from the Australian Cancer Research Foundation. C.G. was in receipt Materials and Methods of University of Queensland International Research Tuition Award and University of Queensland Research Services postgraduate scholarships from the University of Human cervical intraepithelial neoplasia and control biopsies Queensland, and S.R.M. and A.B. were recipients of Balzan Fellowships. Biopsies from human cervical intraepithelial neoplasia (CIN)2 or CIN3 and Address correspondence and reprint requests to Dr. Ian H. Frazer, University of normal cervical tissue from the same patient were diagnosed and provided Queensland Diamantina Institute, Translational Research Institute, 37 Kent Street, by L. Perrin and S. Cattanach (Mater Medical Centre, Brisbane, QLD, Woolloongabba, Brisbane, QLD 4102, Australia. E-mail address: [email protected] Australia). Women participating in the study were over the age of 20. The online version of this article contains supplemental material. Women who were pregnant, had evident cancer or cervical infection with Abbreviations used in this article: CIN, cervical intraepithelial neoplasia; hIL, human another agent, or had had cervical treatment within the prior year were IL; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HPV, human papilloma- excluded from the study. All human subjects provided written informed virus; K14, keratin 14; MDSC, myeloid-derived suppressor cell. consent prior to the procedure, and the study was approved by the insti- tutional ethics committee (2009001060) and conducted in compliance with Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 the Helsinki guidelines. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400216 The Journal of Immunology 2249

Mice were purchased from BioLegend. Anti-human TCR-gd (IMMU510) and matched isotype control Abs were obtained from Beckman Coulter (Brea, C57BL/6J mice (designated wild type) and HPV16 E7 transgenic C57BL/6J CA), and anti-mouse mAbs against CD45.2 (104), CD3e (145-2C11), and mice, which express the HPV 16 E7 oncoprotein under control of the TCR-gd (GL3) were from BD Biosciences. a-GalCer–loaded CD1d tet- keratin 14 (K14) promoter (designated K14E7) (17), were obtained from ramer for detection of invariant NKT cells was provided by D. Godfrey the Animal Resources Centre (Perth, WA, Australia). Colonies of K14E7 (University of Melbourne). Cell suspensions were incubated with Fc block heterozygous mice are maintained by breeding K14E7 males with (BD Biosciences) for 15 min. Live/Dead Fixable Aqua Dead Cell Stain C57BL/6J females, rendering C57BL/6J mice the appropriate control (Invitrogen) and Abs against cell surface markers were added for another strain. TCR-d2/2 (18) mice were purchased from The Jackson Laboratory 2/2 2/2 2/2 20 min of incubation. For intracellular cytokine staining, cells were fixed (Bar Harbor, ME). IL-17 (19), IL-12p40 (20), and IL-1R1 (21) and permeabilized using the BD Biosciences Cytofix/Cytoperm kit, mice were obtained from Y. Iwakura (University of Tokyo, Tokyo, Japan), according to the manufacturer’s instructions. Abs against cytokines and C. Farah (University of Queensland Centre for Clinical Research, Bris- matched isotype control Abs were added for 30 min. All steps were per- bane, QLD, Australia), and H. Thomas (University of Melbourne, Park- formed at 4˚C. Flow Count Fluorospheres (Beckman Coulter) were used ville, VIC, Austrailia), respectively. All knockout strains were on the for calculation of total cell numbers. A Gallios flow cytometer (Beckman C57BL/6J background. All mice were maintained locally under specific Coulter) was used to acquire samples. Data analysis was performed with pathogen-free conditions. To generate K14E7 mice deficient in TCR-d, IL- Kaluza software 1.2 (Beckman Coulter). Doublets and dead cells were 17, IL-12p40, or IL-1R1, hemizygous K14E7 mice were crossed with the excluded from the analysis. respective knockout mice and the E7-expressing progeny backcrossed with 2 2 2 2 2 2 2 2 homozygous TCR-d / , IL-17 / , IL-12p40 / , and IL-1R1 / mice. All Skin explant culture mice were used at 6–20 wk of age and were sex and age matched for all experiments. All animal procedures were approved by the University of Ears were split into halves and cultured dermis side down on DMEM Queensland Animal Ethics Committee (AEC 290/10/NHMRC/NIH). supplemented with FBS (10% v/v), penicillin/streptomycin/glutamine (1 mM), sodium pyruvate (1 mM) (Life Technologies), and HEPES Downloaded from Quantitative real-time PCR (10 mM) (Invitrogen) in 24-well plates (37˚C, 5% CO2). To remove any cytokine released as a result of tissue preparation, medium was exchanged For RNA extraction, frozen human biopsies and mouse ears were homog- after 1 h and another 2 h of culture. As determined by the CytoTox 96 enized in TRIzol (Invitrogen, Carlsbad, CA). RNA was purified using the NonRadioactive Cytotoxicity Assay (Promega, Fitchburg, WI), medium RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). RNA was reversely changes reduced the lactate dehydrogenase activity in the supernatant to transcribed using oligo d(T) (Applied Biosystems, Foster City, CA). Primers levels of control medium 2 h after the last medium exchange. Supernatants for SYBR Green quantitative real-time PCR were purchased from Integrated were collected 20 h after the last medium exchange for analysis by ELISA,

DNATechnologies (Coralville, IA) and were designed using the PrimerQuest and low lactate dehydrogenase activity was confirmed in supernatants of http://www.jimmunol.org/ PCR design tool (Integrated DNA Technologies). Primer sequences are as both wild-type and K14E7 skin explants (data not shown). follows: murine hypoxanthine-guanine phosphoribosyltransferase (HPRT) For cytokine neutralization in skin explant cultures, mAbs specific for the forward, 59- CCCCAAAATGGTTAAGGTTGC-39; murine HPRT reverse, cytokine or matched isotype control Abs were added to the medium. Anti- 59- AACAAAGTCTGGCCTGTATCC-39; human HPRT forward, 59-TC- mouse IL-17 and IL-23p19 neutralizing and control Abs were obtained from AGGCAGTATAATCCAAAGATGGT-39; human HPRT reverse, 59- AGT- Lilly Research Laboratories (Indianapolis, IN). IL-12/23p40 (C17.8), IL-1b CTGGCTTATATCCAACACTTCG-39; human IL (hIL)-17 forward, 59- (B122) neutralizing, and matched control Abs were purchased from AGACTACCTCAACCGTTCCA-39; hIL-17 reverse, 59-GAGCTTCCCA- eBioscience (San Diego, CA), and neutralizing Ab against mouse IL-18 GATCACAGAG- 39;hIL-23forward,59-CCCCAAAATGGTTAAGGTTGC- (93-10C) and the corresponding isotype control Ab were obtained from 39; hIL-23 reverse, 59- AACAAAGTCTGGCCTGTATCC-39. Predesigned MBL (Woburn, MA). The dorsal (or ventral) half from one ear was treated TaqMan gene expression assays (Integrated DNA Technologies) were used with isotype control Ab and compared with the dorsal (or ventral) half for the quantification of mouse IL-23A mRNA expression. Quantitative from the other ear of the same mouse, treated with neutralizing Ab. by guest on September 24, 2021 real-time PCR was performed on a 7900HT Fast Real-Time PCR System Supernatants were replaced after 20 h with Ab-supplemented medium and (Applied Biosystems). Data were analyzed using SDS Software 2.4 (Ap- collected after another 20 h for analysis by ELISA. plied Biosystems). mRNA expression relative to the housekeeping gene HPRT was calculated as 2^(cycle threshold [HPRT] 2 cycle threshold Cytokine ELISA [gene of interest]). ELISA to measure the concentrations of mouse IL-17, IL-23 (p40/p19), and Isolation and stimulation of cells IFN-g (eBioscience) was performed according to the manufacturer’s instructions. Samples undetectable within the range of the assay were To isolate cells from CIN and control biopsies, the tissue samples were plotted as the detection limit. Cytokine concentrations in supernatants were minced in 0.25% trypsin, 0.1% EDTA (Life Technologies, Mulgrave, VIC, normalized to weight of the skin explant and are shown as pg/mg tissue. Australia), 0.3 mg/ml collagenase, and 5 mg/ml DNase (Roche, Basel, Switzerland) in PBS and incubated for 20 min at 37˚C. Ears from two mice Skin grafting per group were pooled to obtain sufficient cell numbers for analysis. To obtain cells from mouse dermis and epidermis, using forceps, ears were Donor ear skin was grafted onto recipient flanks, as previously described split into halves and incubated dermis side down in 2 mg/ml dispase in (22, 23). Briefly, skin from dorsal and ventral ear halves of donor mice PBS (Roche) for 1 h at 37˚C. Following incubation, dermis and epidermis (∼1.5 cm2) was placed onto the flank region of an anesthetized recipient. were carefully separated and incubated in PBS containing 0.3 mg/ml Grafts were held in place with antibiotic-permeated gauze (Bactigras; collagenase/5 mg/ml DNase (Roche) and 0.25% trypsin/0.1% EDTA/5 Smith and Nephew, London, U.K.) and bandaged with micropore tape and mg/ml DNase, respectively, for 30 min at 37˚C. The obtained human and Flex-wrap (Lyppard, QLD, Australia). Bandages were removed 7 d fol- mouse tissue pieces were vigorously pipetted to facilitate the release of lowing grafting, and grafts were monitored three times weekly. Grafts were cells from the tissue, and the cell suspension was filtered through a 40-mm considered rejected if there was a loss of distinct border and necrosis to cell strainer (BD Biosciences, San Jose, CA). ˃80% of the graft area. Up to 1 3 106 of total cells/mL were stimulated with 1 mg/ml PMA and 0.5 mg/ml ionomycin (Calbiochem/EMD4 Biosciences, San Diego, CA) in Western blot RPMI 1640 or DMEM supplemented with FBS (10% v/v), penicillin/ Mouse ears were snap frozen and homogenized in 500 ml freshly prepared streptomycin/glutamine (1 mM), sodium pyruvate (1 mM) (Life Tech- radioimmunoprecipitation assay buffer (1% Triton X-100, 20 mM Tris [pH nologies), and HEPES (10 mM, final concentrations) (Invitrogen) for 4 h at 7.5], 150 mM NaCl, protease inhibitor [Roche], in Milli-Q H2O) on ice, 37˚C. After 30 min, monensin (BioLegend, San Diego, CA) and GolgiPlug using a T 10 basic ULTRA-TURRAX homogenizer (IKA). After 20 min of (BD Biosciences) were added to the unstimulated and stimulated cell incubation (4˚C), the homogenate was centrifuged (18,000 relative cen- suspensions to block cytokine release from the Golgi/endoplasmic retic- trifugal force, 10 min, 4˚C), and the supernatant was collected and filtered ulum complex. through a 70-mm cell strainer (BD Biosciences, San Jose, CA) to remove Flow cytometry aggregates. Total protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). mAbs against human CD45 (H130) and CD3 (OKT3), and anti-mouse CD8a Denatured skin lysates were separated on a 15% polyacrylamide gel and (53-6.7) and CD4 (RM4-5) Abs were from eBioscience. Anti-human CD4 transferred onto a 0.45-mm polyvinylidene fluoride membrane (EMD (RPA-T4) and IL-17A (BL168), anti-mouse IL-17A (TC11-18H10.1), Millipore, Billerica, MA). Blots were blocked with 5% skim milk in TBS CD11b (M1/70), Gr-1 (RB6-8C5), and matched isotype control Abs containing 0.1% Tween 20 (1 h, room temperature) and incubated with 2250 IMMUNOREGULATION BY IL-17 IN SKIN primary Abs against mouse IL-1b (R&D Systems, Minneapolis, MN) mRNA expression in CIN2/3 lesions when compared with mor- or b-actin (Sigma-Aldrich, St. Louis, MO) (1:2000 dilution in blocking phologically normal cervical control tissue from the same patients buffer, overnight, 4˚C), followed by HRP-conjugated secondary Ab (Fig. 1A). Furthermore, mRNA expression of the p19 subunit of (1:5000 dilution in blocking buffer, 1 h, room temperature). Blots were developed using SuperSignal West Dura Chemiluminescent Substrate IL-23 (IL-23A), a major initiator of IL-17 production, was higher (Thermo Fisher Scientific, Waltham, MA). Images were acquired with in precancerous compared with control specimens in 8 of 11 ChemiDoc (Bio-Rad, Hercules, CA). patients (Fig. 1B). Consistent with these findings, a higher per- Statistics centage of cells isolated from the CIN2/3 specimens produced IL- 17, with or without in vitro stimulation with PMA and ionomycin Statistical analysis was performed using Prism (GraphPad Software, La (Fig. 1C, 1D). Jolla, CA). Skin graft survival was plotted as Kaplan–Meier plots. Paired Student’s t test was used to assess differences between data sets from We next analyzed in a mouse model whether HPV16 E7-driven skin explant cytokine neutralization experiments. Wilcoxon matched-pairs hyperplasia of skin epithelium (K14E7 mice) (17) is similarly signed rank test was applied to human data sets. For all other data, un- associated with higher production of IL-17. Indeed, IL-17 con- paired Student’s t test was performed. Differences were considered sig- centrations were significantly elevated in culture supernatants of p , p , p , p , nificant at 0.05 and are indicated as * 0.05, ** 0.01, *** K14E7 compared with wild-type skin explants (Fig. 2A). In 0.001, and n.s., not significant. support of higher IL-17 production in K14E7 skin, we also detected significantly elevated IL-23A mRNA expression and Results significantly higher concentrations of IL-23 protein in super- Elevated IL-17 expression in human cervical precancerous natants of K14E7 compared with wild-type skin explants lesions and in a mouse model of HPV16 E7-induced epithelial (Fig. 2B). Furthermore, significantly more cells in the dermis and Downloaded from hyperplasia epidermis of K14E7 mice produced IL-17 compared with wild- Recent studies showed an increase in IL-17 production in human type dermis and epidermis (Fig. 2C, 2D). Whereas numbers of IL- CIN (15, 16). We similarly observed significantly elevated IL-17 17–producing cells were similar between dermis and epidermis of http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Increased production of IL-17 in human CIN2/3 compared with control tissue. (A and B) IL-17 and IL-23A mRNA expression in human CIN2/3 and control cervical tissue from the same patient, measured by quantitative real-time PCR (n = 12 patients). (C and D) Flow cytometric analysis of cells isolated from CIN2/3 lesions and control tissue, unstimulated or stimulated with PMA and ionomycin. Numbers shown in (C) indicate the percentage of CD45+IL-17+ cells, or of cells treated with isotype Ab, of total live cells for one patient (representative analysis). The results obtained for four to five patients are summarized in (D). (In one of five patients, unstimulated cell suspensions contained too few IL-17+ cells to perform a meaningful analysis. Data not shown.) Groups were compared by Wilcoxon test; ***p , 0.001. n.s., not significant. The Journal of Immunology 2251 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. Higher production of IL-17 and IL-23 in K14E7 compared with wild-type mouse skin. (A) IL-17 concentration in supernatants of C57BL/6 and K14E7 ear skin explants following 20 h of culture, assessed by ELISA (n = 4 mice per group). In supernatants from wild-type skin explant culture, IL-17 concentrations were below the detection limit of the assay (n.d., not detectable). Results shown are representative of two independent experiments. (B) IL-23A mRNA expression in C57BL/6 and K14E7 ear skin as determined by quantitative real-time PCR (n = 4 mice per group). IL-23 concentration in supernatants of C57BL/6 and K14E7 ear skin explants following 20 h of culture, assessed by ELISA (n = 4 mice per group). Results shown are representative of two independent experiments. (C and D) Flow cytometric analysis of cells isolated from dermis and epidermis of C57BL/6 and K14E7 mice, unstimulated or stimulated with PMA and ionomycin. Total live cells were gated for CD45.2+IL-17+ cells. Numbers in (C)indicate the numbers of CD45.2+IL-17+ cells per plot. Plots in (C) are representative of four independent experiments. The total numbers of CD45.2+IL-17+ cells in wild-type and K14E7 dermis and epidermis obtained from four independent experiments (n = 2 mice per strain per experiment) were calculated using Flow Count Fluorospheres and are summarized in (D). Bars indicate the mean 6 SEM. Groups were compared by unpaired t test. *p , 0.05, **p , 0.01, ***p , 0.001. wild-type mice, signiﬁcantly more IL-17–producing cells were Together, these results demonstrate elevated production of IL-17 detected in K14E7 transgenic epidermis compared with dermis in HPV-associated premalignant disease in humans and a mouse from K14E7 mice. model of HPV-induced hyperplasia. 2252 IMMUNOREGULATION BY IL-17 IN SKIN

IL-17 expression in K14E7 skin grafts suppresses an effective skin. To this end, we neutralized IL-17 in K14E7 skin explant E7-directed immune response cultures with an anti–IL-17 mAb and observed no reduction in + 2/2 + Despite expressing a non-self Ag, skin transgenic for the HPV16 E7 IFN-g secretion (Fig. 3C). Furthermore, E7 IL-17 and E7 IL- 17+/2 control skin explants secreted similar amounts of IFN-g protein is not rejected when transplanted onto otherwise syngeneic, + 2/2 + +/2 nontransgenic recipient mice (24), most likely due to a local im- (Fig. 3D), and E7 IFN-g and E7 IFN-g control skin explants munosuppressive environment. To functionally address the role of produced similar amounts of IL-17 (Fig. 3E). These findings IL-17 production by K14E7 skin in K14E7 graft survival, we demonstrate that IL-17 and IFN-g production in K14E7 skin are grafted E7 transgenic IL-17–deficient (E7+IL-172/2) skin onto not directly linked, although coregulation of their production is not C57BL/6 recipients. As a control, skin from E7-negative IL-172/2 excluded, as absence of either is permissive for graft rejection. (E7-IL-172/2) and E7-positive IL-17+/2 (E7+IL-17+/2) littermates was placed next to the E7+IL-172/2 grafts. Ten of 13 E7+IL-172/2 CD4 and gd T cells in K14E7 skin, and CD4 T cells in human grafts were rejected with a median time to rejection of 15 d, CIN are the predominant source of IL-17 whereas none of the E7+IL-172/+ and other control grafts were To identify the cellular sources of IL-17 in K14E7 skin, IL-17– rejected (Fig. 3A). These data show that IL-17 expression in the expressing cells detected by intracellular flow cytometry were E7 transgenic graft contributes to local suppression of an effective, analyzed for expression of hematopoietic non-T cell (CD32)and E7-directed immune response. In contrast, the ability of the re- Tcell(CD3+) markers (Fig. 4A, 4B). The majority of IL-17– cipient to produce IL-17 was not required for K14E7 graft sur- producing cells in K14E7 skin were CD3+ T cells (epidermis, vival, as K14E7 grafts were not rejected by IL-17–deficient 85.8% 6 7.1%; dermis, 93.5% 6 2.4%). Among these, the recipients (Fig. 3B), confirming that local IL-17 production is key largest populations of IL-17–producing cells were gd T cells, Downloaded from to regulating local immune effector function. with intermediate expression levels of CD3 and gd-TCR (re- We have previously demonstrated an immunosuppressive role ferred to as CD3intgd-TCRint; epidermis, 32.6% 6 5.4%; dermis, for IFN-g in K14E7 skin grafts (25). As we obtained similar 49.1% 6 4%) and CD4 T cells (epidermis, 19.9% 6 2.2%; results for IL-17 in the current study, we sought to investigate dermis, 20% 6 3.1%). Low numbers of epidermal gd T cells, a possible link between IFN-g and IL-17 production in K14E7 a T cell population exclusively found in mouse epidermis and http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. IL-17 in K14E7 grafts suppresses graft rejection. (A) Kaplan–Meier survival curves of E7+IL-172/2 (median graft survival: 15 d) and littermate control E7+IL-17+/2 and E72IL-172/2 grafts on C57BL/6 recipients. (B) Kaplan–Meier survival curves of C57BL/6 and K14E7 grafts on IL-172/2 recipients. Numbers of donor and recipient mice per group are indicated. Pooled results from three independent experiments. (C)IFN-g concentrations in the supernatants of K14E7 skin explants cultured in the presence of neutralizing anti–IL-17 or isotype (ISO) control Ab (10 mg/ml) for 40 h, measured by ELISA. Data points are from six individual mice analyzed in two independent experiments. Groups were compared by paired t test. (D)IFN-g concentration in the supernatants of K14E7 transgenic IL-17+/2 (n = 9 mice), IL-172/2 (n = 11 mice), and IFN-g2/2 (n = 3 mice; n.d., not detectable) skin explants, measured by ELISA. Pooled results from four independent experiments. Bars indicate the mean 6 SEM. (E) IL-17 concentration in the supernatants of K14E7 transgenic IFN-g+/2 (n = 3 mice) and IFN-g2/2 (n = 4 mice) skin explants, measured by ELISA. Pooled results from two independent experiments. Bars indicate the mean 6 SEM. Groups were compared by unpaired t test. n.s., not signiﬁcant (p $ 0.05). The Journal of Immunology 2253 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. gd and CD4+ T cells are the predominant producers of IL-17 in K14E7 skin, and in human CIN2/3 lesions. (A and B) Flow cytometric analysis of cells isolated from the dermis and epidermis of K14E7 skin, stimulated with PMA and ionomycin. CD45.2+IL-17+ cells were gated for CD3+,CD3+ gd-TCR+ (CD3intgd-TCRint and CD3highgd-TCRhigh in epidermis, and CD3intgd-TCRint in dermis), CD3+CD4+, and CD3+CD8+ T cells and invariant NKT cells. Numbers shown in (A) indicate the percentages of these cell subsets in the CD45.2+IL-17+ cell fraction. Plots in (A) are representative of four independent experiments (n = 2 mice per experiment), the results of which are shown in (B). Bars indicate the mean 6 SEM. (C and D) Flow cytometric analysis of cells isolated from human CIN2/3 lesions and control tissue, stimulated with PMA and ionomycin. Numbers shown in (C) indicate the percentages of CD3+,CD3+CD4+, and CD3+gd-TCR+ T cells of CD45+IL-17+ cells. Plots in (C) are representative of results from five patients (in two experiments, cells were stained for CD3, but not for CD4 or gd-TCR). The results obtained for three to five patients are summarized in (D). Bars indicate the mean 6 SEM. characterized by high CD3 and gd-TCR expression (referred to as Similar to mouse E7 transgenic skin, IL-17–producing cells in CD3highgd-TCRhigh), CD8+ T cells, and invariant NKT cells were human CIN2/3 lesions were mostly CD3+ T cells (89.0% 6 3.9%) also found to produce IL-17 in K14E7 skin (Fig. 4A, 4B). IL-17 (Fig. 4C, 4D). CD4+ T cells were the major producers of IL-17 in production in K14E7 transgenic gd T cell–deficient skin was re- CIN2/3 lesions (58.2% 6 12.2%), whereas only a few gd T cells duced but not abolished compared with gd T cell–replete skin produced IL-17 (Fig. 4C, 4D). (Supplemental Fig. 1A). Furthermore, K14E7 transgenic gd T cell– deficient skin grafts were not rejected by nontransgenic recipients IL-17 production in K14E7 skin is induced by IL-23 and IL-1b, (Supplemental Fig. 1B), demonstrating that gd T cells were not but not IL-18 essential for K14E7 graft survival, and that IL-17 levels in the IL-23, IL-1b, and IL-18 are known inducers of IL-17 (8, 26). absence of gd T cells were sufficient to allow for graft survival. K14E7 skin expressed elevated levels of IL-23 (Fig. 2) and IL-18 2254 IMMUNOREGULATION BY IL-17 IN SKIN

(27) compared with wild-type skin, whereas IL-1b is produced at study is corroborated by reports on elevated IL-17 concentrations similar, low levels in both (Supplemental Fig. 2). To test a possible in cervical secretion specimens of women with persistent high- contribution of IL-23, IL-1b, and IL-18 to induction of the enhanced risk HPV infection compared with specimens from HPV-negative IL-17 production in K14E7 skin, we blocked cytokine function by controls (15), as well as increased numbers of IL-17+ cells in addition of neutralizing mAbs to IL-23p19, IL-12/23p40, IL-1b, uterine cervical cancer and CIN tissue compared with cervical tis- and IL-18 to skin explant culture medium (Fig. 5). Neutralization sue from healthy controls (16). Baseline cytokine expression in of IL-23p19, IL-12/23p40 (Fig. 5A), and IL-1b (Fig. 5B) led to human cervical tissue differs between individuals most likely due a signiﬁcant reduction of IL-17 concentrations compared with to genetic, environmental, and hormonal differences. To control isotype Ab-treated explants of the same K14E7 mice. In contrast, for such variables in our study, we compared premalignant and neutralization of IL-18 did not affect IL-17 production (Fig. 5C). histologically normal cervical specimens from the same patients, Together, these results indicate that IL-23 and IL-1b, but not IL-18, excluding an impact of subject-speciﬁc differences in baseline contribute to production of IL-17 in K14E7 skin. IL-17 expression between premalignant and control specimens. We next sought to investigate whether IL-23 or IL-1b expressed This observation contrasts with previous studies in which control in K14E7 skin is required for K14E7 graft survival. To this specimens were sourced from unrelated healthy subjects (15, 16). end, skin from K14E7 transgenic IL-12/23p402/2 mice, K14E7 Persistent infection of basal epithelium with high-risk HPV is transgenic IL-1R12/2 mice, and K14E7 transgenic littermate associated with the expression of the HPV protein E7, which drives controls heterozygous for IL-12/23p40 or IL-1R1 was grafted onto epithelial hyperproliferation (28). To model HPV-associated dis- wild-type recipients. None of the grafts were rejected (Fig. 6A, ease in mice, which are not infected by HPV, transgenic mice that

6B), suggesting that IL-12/23p40 or IL-1R1 signaling alone, al- express HPV16 E7 under control of the K14 promoter in basal Downloaded from though contributing to IL-17 induction in K14E7 skin, is not epithelial cells (designated K14E7) were used (17). These mice sufﬁcient to suppress an effective E7-directed immune response, exhibit epithelial hyperplasia that closely models premalignant most likely due to the residual IL-17 production observed in the lesions in chronic HPV infection (17). Similar to our ﬁndings in E7 transgenic grafts (Fig. 6C). human HPV-infected, hyperplastic cervical tissue, E7 transgenic, hyperplastic skin featured higher IL-17 production and elevated

Discussion numbers of IL-17–producing cells compared with wild-type skin. http://www.jimmunol.org/ Local immunosuppression is considered a major contributing Furthermore, production of IL-23, which is a major inducer of factor for persistent infection of epithelial tissues with high-risk IL-17 in humans and mice (8), was also increased in the majority of HPV and associated epithelial hyperplasia (5), which is an es- CIN2/3 compared with control specimens, and signiﬁcantly ele- sential step in the development of HPV-associated cancers (28). vated in HPV16 E7 transgenic skin. These phenotypical similarities IL-17 is a pleiotropic cytokine whose role in HPV-associated encourage the use of the K14E7 mouse as a model of HPV- premalignant disease is unknown. This study identiﬁes IL-17 as associated hyperplasia. a critical immunosuppressive determinant in HPV-associated, epi- We have previously shown that immunocompetent hosts fail thelial hyperplasia. to reject K14E7 transgenic skin grafts (24), and that this is not The observed increased IL-17 production in hyperplastic, pre- dependent on Foxp3+ regulatory T cells (29). In this study, we by guest on September 24, 2021 malignant CIN2/3 lesions compared with control specimens in our demonstrate that IL-17 produced in K14E7 skin grafts suppresses

FIGURE 5. IL-23 and IL-1b, but not IL-18, induce IL-17 production in K14E7 skin. IL-17 concentrations in the supernatants of K14E7 skin explants cultured in the presence of (A) anti–IL-23, IL-12/23p40 (2 mg/ ml, n = 6 mice); (B) anti–IL-1b (10 mg/ml, n = 12 mice); and (C) anti– IL-18 (10 mg/ml, n = 6 mice) neutralizing or matched isotype control Ab for 40 h, measured by ELISA. Pooled results from two to three independent experiments. Groups were compared by paired t test; *p , 0.05, **p , 0.01, ***p , 0.001. n.s., not signiﬁcant. The Journal of Immunology 2255

FIGURE 6. IL-1 and IL-23 are not required for survival of K14E7 skin grafts. Kaplan–Meier survival curves of (A)E7+IL-12p402/2 and littermate control E7+IL-12p40+/2 grafts and (B) E7+IL-1R12/2 and littermate control E7+IL-1R1+/2 grafts on C57BL/6 recipients. Numbers shown indicate the numbers of donor and recipient mice. Pooled results from three and two independent experiments, respectively. (C) IL-17 concentrations in the supernatants of K14E7 transgenic IL-12p40+/2 (n =6 mice), IL-12p402/2 (n = 6 mice), IL- +/2 2/2

1R1 (n = 6 mice), IL-1R1 (n =5 Downloaded from mice), IL-17+/2 (n = 6 mice), and IL- 172/2 (n = 5 mice) skin explants, measured by ELISA. Pooled results from two independent experiments per group. Bars indicate the mean 6 SEM. Groups were compared by unpaired t test; *p ,

0.05, **p , 0.01. n.s., not signiﬁcant. http://www.jimmunol.org/

graft rejection. Reduced levels of IL-17 in E7 transgenic grafts myeloid cells and their subsequent development into MDSC as deficient in IL-12/23p40, IL-1R1, or gd T cells were still sufficient a possible mechanism responsible for impaired host effector T cell by guest on September 24, 2021 to protect against graft rejection, indicating a strong potency of responses to K14E7 skin grafts, particularly as IDO expression is this cytokine in inhibiting host effector functions against E7 as elevated in CIN2/3 lesions, and in K14E7 skin, and contributes to a foreign Ag. These findings in the mouse model of HPV-induced local immune regulation (43). However, numbers of CD11b+Gr-1int hyperplasia suggest similar immunosuppressive functions for IL- MDSCweresimilarinK14E7andE7+IL-172/2 grafts (Supplemental 17 in HPV-associated premalignant disease in humans. This hy- Fig. 3), indicating that recruitment of MDSC into E7+ skin is pothesis is further supported by studies reporting associations of IL-17 independent. polymorphisms in the genes encoding IL-17 and IL-12/23p40 with Similar to our findings for IL-17, we have previously reported CIN and cervical cancer (30–32). that IFN-g production in K14E7 skin had immune-suppressive Rejection of K14E7 skin grafts is mediated by Ag-specific host effects in the skin-grafting model (25). This suggested that IL- cytotoxic CD8+ T cells (33), suggesting that excessive IL-17 17 and IFN-g production in K14E7 skin might be functionally production in the hyperplastic graft locally inhibits host cyto- linked, as previously shown in other studies. The successful che- toxic effector T cell functions. In line with this, some studies have motherapeutic treatment of some transplantable and induced tu- reported immune regulatory functions of IL-17 in epithelial dis- mor models in mice required both IL-17 and IFN-g, with IL-17 eases. In eczematous mice infected with vaccinia virus, IL-17 production preceding IFN-g production (44, 45). In this study, reduced the cytotoxic activity of NK cells in the infected tissue, however, we demonstrate that IFN-g production in K14E7 skin is thus hampering viral clearance (34). In the CD45RBhigh T cell independent of IL-17 and vice versa. This conclusion is further transfer model of colitis, IL-17 was shown to negatively regu- corroborated by the finding that, in K14E7 skin, IL-18 is required late Th1 responses in the gut (35). Furthermore, in the lung, IL-17 for production of IFN-g, but not IL-17, whereas IL-23 and IL-1b exerted immune regulatory functions upon Ag challenge during induce IL-17, but not IFN-g production (Fig. 5) (27). The IL-17– the effector phase of allergic asthma by recruiting eosinophils and mediated immune suppression thus represents a novel, IFN-g– inhibiting chemokine synthesis in vivo and by inhibiting dendritic independent mechanism of immune suppression in HPV- cells in vitro (36). IL-17 contributed to the formation and pro- associated hyperplasia. gression of pancreatic neoplasia (37), and, in mouse models of The major producers of IL-17 in K14E7 skin were CD3int ovarian cancer, carcinogen-induced skin cancer and lung cancer gd-TCRint gd T cells and CD4 T cells. Notably, the majority of IL- were shown to enhance tumor development and growth in asso- 17–producing cells were detected in the K14E7 transgenic epi- ciation with the recruitment of CD11b+ myeloid cells (38–41). dermis, despite IL-17–producing gd T cells displaying a dermal These can develop into myeloid-derived suppressor cells (MDSC) phenotype, characterized by intermediate expression of CD3 and at the tumor site and suppress T cell responses (42). Infiltrates of gd-TCR (46). We hypothesize that HPV16 E7-induced hyper- myeloid cells were also observed in HPV16 E7-driven hyperproliferation of epithelial cells is associated with the production of plastic skin (25), suggesting an IL-17–associated recruitment of cytokines and chemokines that specifically recruit inflammatory 2256 IMMUNOREGULATION BY IL-17 IN SKIN cells to the epidermis. These include T cells that express CCR6 9. Lalor, S. J., L. S. Dungan, C. E. Sutton, S. A. Basdeo, J. M. Fletcher, and K. H. Mills. 2011. Caspase-1-processed cytokines IL-1beta and IL-18 promote (47), a chemokine receptor known for its role in epidermal traf- IL-17 production by gammadelta and CD4 T cells that mediate autoimmunity. J. ficking (48). In line with this, the migration of dermal gd T cells to Immunol. 186: 5738–5748. the epidermis in an IL-23–induced mouse model of psoriasiform 10. Iwakura, Y., H. Ishigame, S. Saijo, and S. Nakae. 2011. Functional specialization of interleukin-17 family members. Immunity 34: 149–162. dermatitis, which also features a hyperplastic epithelium, was also 11. Miossec, P., and J. K. Kolls. 2012. Targeting IL-17 and TH17 cells in chronic mediated by CCR6 (48). It is conceivable that similar mechanisms inflammation. Nat. Rev. Drug Discov. 11: 763–776. mediate the recruitment of inflammatory cells to human hyper- 12. Yeremenko, N., and D. Baeten. 2011. IL-17 in spondyloarthritis: is the T-party over? Arthritis Res. Ther. 13: 115. plastic cervical tissue. Similar to E7 transgenic skin, in human 13. O’Connor, W., Jr., L. A. Zenewicz, and R. A. Flavell. 2010. The dual nature of cervical HPV-associated premalignant lesions, CD4+ T cells and T(H)17 cells: shifting the focus to function. Nat. Immunol. 11: 471–476. gd T cells produced IL-17; however, CD4+ T cells were more 14. Zou, W., and N. P. Restifo. 2010. T(H)17 cells in tumour immunity and immunotherapy. Nat. Rev. Immunol. 10: 248–256. prominent among the IL-17–producing cell population. This 15. Marks, M. A., R. P. Viscidi, K. Chang, M. Silver, A. Burke, R. Howard, and finding is corroborated by a recent study reporting increased P. E. Gravitt. 2011. Differences in the concentration and correlation of cervical + + immune markers among HPV positive and negative perimenopausal women. numbers of CD4 IL-17 cells in CIN and cervical cancer com- Cytokine 56: 798–803. pared with control cervical tissue from healthy donors (16). 16. Hou, F., Z. Li, D. Ma, W. Zhang, Y. Zhang, T. Zhang, B. Kong, and B. Cui. 2012. As the E7 transgene is expressed as an autoantigen in the thymus Distribution of Th17 cells and Foxp3-expressing T cells in tumor- infiltrating lymphocytes in patients with uterine cervical cancer. Clin. Chim. Acta of K14E7 mice (17), IL-17 production by T cells in K14E7 skin is 413: 1848-1854. expected to be E7 Ag independent. Furthermore, the IL-17 pro- 17. Herber, R., A. Liem, H. Pitot, and P. F. Lambert. 1996. Squamous epithelial duction in K14E7 skin occurred downstream of IL-23 and IL-1b. hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene. J. Virol. 70: 1873–1881.

+ Downloaded from Ag-independent IL-17 production by memory CD4 T cells and 18. Itohara, S., P. Mombaerts, J. Lafaille, J. Iacomini, A. Nelson, A. R. Clarke, by gd T cells has been previously described and required IL-23 in M. L. Hooper, A. Farr, and S. Tonegawa. 1993. T cell receptor delta gene mutant combination with IL-1b (49, 50). More recently, gd T cells pu- mice: independent generation of alpha beta T cells and programmed rear- rangements of gamma delta TCR genes. Cell 72: 337–348. rified from lymph nodes of mice with experimental autoimmune 19. Hirota, K., J. H. Duarte, M. Veldhoen, E. Hornsby, Y. Li, D. J. Cua, H. Ahlfors, encephalitis were also found to produce IL-17 following stimu- C. Wilhelm, M. Tolaini, U. Menzel, et al. 2011. Fate mapping of IL-17- producing T cells in inflammatory responses. Nat. Immunol. 12: 255–263. lation with IL-1b or IL-18 in combination with IL-23 (9). In E7 20. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, transgenic, hyperplastic skin, however, IL-18 was not required for J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, and M. K. Gately. 1996. IL- http://www.jimmunol.org/ IL-17 production, reinforcing the idea of IL-17 production being 12-deficient mice are defective in IFN gamma production and type 1 cytokine responses. Immunity 4: 471–481. specifically regulated according to tissue and cellular origin. 21. Glaccum, M. B., K. L. Stocking, K. Charrier, J. L. Smith, C. R. Willis, In summary, our results demonstrate that IL-17 mediates im- C. Maliszewski, D. J. Livingston, J. J. Peschon, and P. J. Morrissey. 1997. munosuppression in HPV-associated epithelial hyperplasia, sug- Phenotypic and functional characterization of mice that lack the type I receptor for IL-1. J. Immunol. 159: 3364–3371. gesting that blocking of IL-17 could be of therapeutic use in 22. Dunn, L. A., M. Evander, R. W. Tindle, A. L. Bulloch, R. L. de Kluyver, persistent infection with high-risk HPV and thus aid prevent G. J. Fernando, P. F. Lambert, and I. H. Frazer. 1997. Presentation of the progression of premalignant lesions to cancer. HPV16E7 protein by skin grafts is insufficient to allow graft rejection in an E7- primed animal. Virology 235: 94–103. 23. Matsumoto, K., G. R. Leggatt, J. Zhong, X. Liu, R. L. de Kluyver, T. Peters, Acknowledgments G. J. Fernando, A. Liem, P. F. Lambert, and I. H. Frazer. 2004. Impaired antigen by guest on September 24, 2021 presentation and effectiveness of combined active/passive immunotherapy for We thank Prof. Lewis Perrin and Dr. Stephen Cattanach (Mater Medical epithelial tumors. J. Natl. Cancer Inst. 96: 1611–1619. Centre, Brisbane, QLD, Australia) for diagnosing and providing human 24. Dunn, G. P., C. M. Koebel, and R. D. Schreiber. 2006. Interferons, immunity and CIN2/3 and control cervical specimens. We further thank Kon Kyparissoudis cancer immunoediting. Nat. Rev. Immunol. 6: 836–848. and Prof. Dale Godfrey (University of Melbourne) for generating the 25. Mattarollo, S. R., A. Rahimpour, A. Choyce, D. I. Godfrey, G. R. Leggatt, and I. H. Frazer. 2010. Invariant NKT cells in hyperplastic skin induce a local im- a-GalCer–loaded mouse CD1d tetramer and the staff of the Biological mune suppressive environment by IFN-gamma production. J. Immunol. 184: Research Facility (Brisbane, QLD, Australia) for excellent technical assis- 1242–1250. tance and animal care. We also acknowledge Lilly Research Laboratories 26. Dungan, L. S., and K. H. Mills. 2011. Caspase-1-processed IL-1 family cyto- for providing IL-23 and IL-17 neutralizing and matched isotype control kines play a vital role in driving innate IL-17. Cytokine 56: 126–132. 27. Gosmann, C., I. H. Frazer, S. R. Mattarollo, and A. Blumenthal. 2014. IL-18, but Abs. not IL-12, induces production of IFN-g in the immunosuppressive environment of HPV16 E7 transgenic hyperplastic skin. J. Invest. Dermatol. DOI: 10.1038/ jid.2014.201. Disclosures 28. Schiffman, M., and N. Wentzensen. 2013. Human papillomavirus infection and The authors have no financial conflicts of interest. the multistage carcinogenesis of cervical cancer. Cancer Epidemiol. Biomarkers Prev. 22: 553-560. 29. Mattarollo, S. R., M. Yong, C. Gosmann, A. Choyce, D. Chan, G. R. Leggatt, References and I. H. Frazer. 2011. NKT cells inhibit antigen-specific effector CD8 T cell induction to skin viral proteins. J. Immunol. 187: 1601–1608. 1. de Martel, C., J. Ferlay, S. Franceschi, J. Vignat, F. Bray, D. Forman, and 30. do Carmo Vasconcelos de Carvalho, V., J. L. de Maceˆdo, C. A. de Lima, M. Plummer. 2012. Global burden of cancers attributable to infections in 2008: M. da Conceiçaõ Gomes de Lima, S. de Andrade Heraćlio, M. Amorim, a review and synthetic analysis. Lancet Oncol. 13: 607–615. M. de Mascena Diniz Maia, A. L. Porto, and P. R. de Souza. 2012. IFN- 2. Forouzanfar, M. H., K. J. Foreman, A. M. Delossantos, R. Lozano, A. D. Lopez, gamma and IL-12B polymorphisms in women with cervical intraepithelial C. J. Murray, and M. Naghavi. 2011. Breast and cervical cancer in 187 countries neoplasia caused by human papillomavirus. Mol. Biol. Rep. 39: 7627–7634. between 1980 and 2010: a systematic analysis. Lancet 378: 1461–1484. 31. Hussain, S. K., M. M. Madeleine, L. G. Johnson, Q. Du, D. A. Galloway, 3. Schiffman, M., P. E. Castle, J. Jeronimo, A. C. Rodriguez, and S. Wacholder. J. R. Daling, M. Malkki, E. W. Petersdorf, and S. M. Schwartz. 2013. Nucleotide 2007. Human papillomavirus and cervical cancer. Lancet 370: 890–907. variation in IL-10 and IL-12 and their receptors and cervical and vulvar cancer 4. Moody, C. A., and L. A. Laimins. 2010. Human papillomavirus oncoproteins: risk: a hybrid case-parent triad and case-control study. Int. J. Cancer 133: 201- pathways to transformation. Nat. Rev. Cancer 10: 550–560. 213. 5. Bhat, P., S. R. Mattarollo, C. Gosmann, I. H. Frazer, and G. R. Leggatt. 2011. 32. Quan, Y., B. Zhou, Y. Wang, R. Duan, K. Wang, Q. Gao, S. Shi, Y. Song, Regulation of immune responses to HPV infection and during HPV-directed L. Zhang, and M. Xi. 2012. Association between IL17 polymorphisms and risk immunotherapy. Immunol. Rev. 239: 85–98. of cervical cancer in Chinese women. Clin. Dev. Immunol. 2012: 258293. 6. Fossiez, F., O. Djossou, P. Chomarat, L. Flores-Romo, S. Ait-Yahia, C. Maat, 33. De Kluyver, R. L., L. Moritz, C. A. Harris, H. Azukizawa, and I. H. Frazer. 2010. J. J. Pin, P. Garrone, E. Garcia, S. Saeland, et al. 1996. T cell interleukin-17 Antigen-specific CD8 T cells can eliminate antigen-bearing keratinocytes with induces stromal cells to produce proinflammatory and hematopoietic cytokines. clonogenic potential via an IFN-gamma-dependent mechanism. J. Invest. Der- J. Exp. Med. 183: 2593–2603. matol. 130: 1841–1848. 7. Mills, K. H. 2008. Induction, function and regulation of IL-17-producing T cells. 34. Kawakami, Y., Y. Tomimori, K. Yumoto, S. Hasegawa, T. Ando, Y. Tagaya, Eur. J. Immunol. 38: 2636–2649. S. Crotty, and T. Kawakami. 2009. Inhibition of NK cell activity by IL-17 allows 8. Cua, D. J., and C. M. Tato. 2010. Innate IL-17-producing cells: the sentinels of vaccinia virus to induce severe skin lesions in a mouse model of eczema vac- the immune system. Nat. Rev. Immunol. 10: 479–489. cinatum. J. Exp. Med. 206: 1219–1225. The Journal of Immunology 2257

35. O’Connor, W., Jr., M. Kamanaka, C. J. Booth, T. Town, S. Nakae, Y. Iwakura, genase activity contributes to local immune suppression in the skin expressing J. K. Kolls, and R. A. Flavell. 2009. A protective function for interleukin 17A in human papillomavirus oncoprotein e7. J. Invest. Dermatol. 133: 2686–2694. T cell-mediated intestinal inflammation. Nat. Immunol. 10: 603–609. 44. Ma, Y., L. Aymeric, C. Locher, S. R. Mattarollo, N. F. Delahaye, P. Pereira, 36. Schnyder-Candrian, S., D. Togbe, I. Couillin, I. Mercier, F. Brombacher, L. Boucontet, L. Apetoh, F. Ghiringhelli, N. Casares, et al. 2011. Contribution of V. Quesniaux, F. Fossiez, B. Ryffel, and B. Schnyder. 2006. Interleukin-17 is IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy. a negative regulator of established allergic asthma. J. Exp. Med. 203: 2715–2725. J. Exp. Med. 208: 491–503. 37. Chang, S. H., S. G. Mirabolfathinejad, H. Katta, A. M. Cumpian, L. Gong, 45. Mattarollo, S. R., S. Loi, H. Duret, Y. Ma, L. Zitvogel, and M. J. Smyth. 2011. M. S. Caetano, S. J. Moghaddam, and C. Dong. 2014. T helper 17 cells play Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of a critical pathogenic role in lung cancer. Proc. Natl. Acad. Sci. USA 111: 5664– established tumors. Cancer Res. 71: 4809–4820. 5669. 46. Cai, Y., X. Shen, C. Ding, C. Qi, K. Li, X. Li, V. R. Jala, H. G. Zhang, T. Wang, 38. Charles, K. A., H. Kulbe, R. Soper, M. Escorcio-Correia, T. Lawrence, J. Zheng, and J. Yan. 2011. Pivotal role of dermal IL-17-producing gd T cells in A. Schultheis, P. Chakravarty, R. G. Thompson, G. Kollias, J. F. Smyth, et al. skin inflammation. Immunity 35: 596–610. 2009. The tumor-promoting actions of TNF-alpha involve TNFR1 and IL-17 in 47. Choyce, A., M. Yong, S. Narayan, S. R. Mattarollo, A. Liem, P. F. Lambert, ovarian cancer in mice and humans. J. Clin. Invest. 119: 3011–3023. I. H. Frazer, and G. R. Leggatt. 2013. Expression of a single, viral oncoprotein in 39. Wang, L., T. Yi, W. Zhang, D. M. Pardoll, and H. Yu. 2010. IL-17 enhances skin epithelium is sufficient to recruit lymphocytes. PLoS One 8: e57798. tumor development in carcinogen-induced skin cancer. Cancer Res. 70: 10112– 48. Mabuchi, T., T. P. Singh, T. Takekoshi, G. F. Jia, X. Wu, M. C. Kao, I. Weiss, 10120. J. M. Farber, and S. T. Hwang. 2013. CCR6 is required for epidermal trafficking 40. He, D., H. Li, N. Yusuf, C. A. Elmets, M. Athar, S. K. Katiyar, and H. Xu. 2012. of gd-T cells in an IL-23-induced model of psoriasiform dermatitis. J. Invest. IL-17 mediated inflammation promotes tumor growth and progression in the Dermatol. 133: 164–171. skin. PLoS One 7: e32126. 49. van Beelen, A. J., Z. Zelinkova, E. W. Taanman-Kueter, F. J. Muller, 41. McAllister, F., J. M. Bailey, J. Alsina, C. J. Nirschl, R. Sharma, H. Fan, D. W. Hommes, S. A. Zaat, M. L. Kapsenberg, and E. C. de Jong. 2007. Y. Rattigan, J. C. Roeser, R. H. Lankapalli, H. Zhang, et al. 2014. Oncogenic Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells Kras activates a hematopoietic-to-epithelial IL-17 signaling axis in preinvasive to promote interleukin-17 production in human memory T cells. Immunity 27: pancreatic neoplasia. Cancer Cell 25: 621–637. 660–669. 42. Gabrilovich, D. I., and S. Nagaraj. 2009. Myeloid-derived suppressor cells as 50. Sutton, C. E., S. J. Lalor, C. M. Sweeney, C. F. Brereton, E. C. Lavelle, and regulators of the immune system. Nat. Rev. Immunol. 9: 162–174. K. H. Mills. 2009. Interleukin-1 and IL-23 induce innate IL-17 production from Downloaded from 43. Mittal, D., A. J. Kassianos, L. S. Tran, A. S. Bergot, C. Gosmann, J. Hofmann, gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity A. Blumenthal, G. R. Leggatt, and I. H. Frazer. 2013. Indoleamine 2,3-dioxy- 31: 331–341. http://www.jimmunol.org/ by guest on September 24, 2021