IL-1β and IL-23 Promote Extrathymic Commitment of CD27 +CD122− δγ T Cells to δγ T17 Cells

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 30, 2017, doi:10.4049/jimmunol.1700287 The Journal of Immunology

IL-1b and IL-23 Promote Extrathymic Commitment of CD27+CD1222 gd T Cells to gdT17 Cells

Andreas Muschaweckh,*,1 Franziska Petermann,*,1,2 and Thomas Korn*,†

gdT17 cells are a subset of gd T cells committed to IL-17 production and are characterized by the expression of IL-23R and CCR6 and lack of CD27 expression. gdT17 cells are believed to arise within a narrow time window during prenatal thymic development. In agreement with this concept, we show in this study that adult Rag12/2 recipient mice of Il23rgfp/+ (IL-23R reporter) bone marrow selectively lack IL-23R+ gdT17 cells. Despite their absence in secondary lymphoid tissues during homeostasis, gdT17 cells emerge in bone marrow chimeric mice upon induction of skin inflammation by topical treatment with imiquimod cream (Aldara). We demonstrate that IL-1b and IL-23 together are able to promote the development of bona fide gdT17 cells from peripheral CD1222IL-23R2 gd T cells, whereas CD122+ gd T cells fail to convert into gdT17 cells and remain stable IFN-g producers (gdT1 +

cells). IL-23 is instrumental in expanding extrathymically generated gdT17 cells. In particular, TCR-Vg4 chain–expressing Downloaded from CD1222IL-23R2 gd T cells are induced to express IL-23R and IL-17 outside the thymus during skin inflammation. In contrast, TCR-Vg1+ gd T cells largely resist this process because prior TCR engagement in the thymus has initiated their commitment to the gdT1 lineage. In summary, our data reveal that the peripheral pool of gd T cells retains a considerable degree of plasticity because it harbors “naive” precursors, which can be induced to produce IL-17 and replenish peripheral niches that are usually occupied by thymus-derived gdT17 cells. The Journal of Immunology, 2017, 199: 000–000. http://www.jimmunol.org/ unctional phenotypes of gd T cells, i.e., IFN-g–producing Thus, other cues, including Notch-dependent pathways but also gdT1 cells and IL-17–producing gdT17 cells, are gener- signals, might be determinants of gdT17 commitment. F ated during thymic development. gdT17 cells can be For example, the Notch–Hes1 pathway and the noncanonical identified by a series of surface markers. IL-23R expression (RelB) NF-kB pathway as well as, in a gd thymocyte–extrinsic strictly segregates with IL-17 expression in gd T cells (1). In manner, NIK and RelA expression in the thymus are required for addition, all IL-17–producing gd T cells are contained within the the generation of gdT17 cells (6–8). In contrast, IRF4, which is 2 CD27 gd T cell compartment and express CCR6 (2, 3). Whether critically required for Th17 cell differentiation, is dispensable for a thymocyte is committed to the gdT1 versus the gdT17 lineage is the development of gdT17 cells (9). Although probably irrelevant

most likely dependent on various cues. gd TCR engagement has for the de novo generation of gdT17 cells in the thymus, STAT3 is by guest on September 27, 2021 been suggested to dictate the commitment of thymocytes to the required for the expansion of gdT17 cells, and the selective ex- gdT1 lineage (4). In contrast, gdT17 cell development appears to pansion of gdT17 cells by IL-7 was reported to depend on STAT3 be independent of TCR engagement as IL-17 production is de- (10). In contrast, IL-17 production most likely from ab thymo- tected in a subset of thymocytes that are poised to become gd cytes might negatively regulate gdT17 generation and was pro- T cells but have not yet rearranged their d and g TCR (5). posed to be responsible for shutting down gdT17 generation in the adult thymus (5). Once exported from the thymus, gdT1 cells and gdT17 cells *Klinikum Rechts der Isar, Neurologische Klinik, Technische Universita¨tMunchen,€ populate secondary lymphoid organs and certain peripheral tissue † 81675 Munich, Germany; and Munich Cluster for Systems Neurology (SyNergy), niches. For example, the dermis harbors gdT17 cells that expand 81377 Munich, Germany 1 upon exposure of the skin to imiquimod cream (Aldara) and cause A.M. and F.P. contributed equally to this work. a -like pathology (11). Recently, it was demonstrated that 2 Current address: Lymphocyte Cell Biology Section, National Institute of early after birth, the dermis mostly contains TCR-Vg6+ gdT17 and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD. cells, whereas in adult individuals more TCR-Vg4+ than TCR- ORCIDs: 0000-0002-6870-1378 (A.M.); 0000-0002-3145-2304 (F.P.); 0000-0002- + + 3633-0955 (T.K.). Vg6 gdT17 cells populate the dermis (12). TCR-Vg6 gdT17 Received for publication February 23, 2017. Accepted for publication August 4, cells are thymically imprinted to home to the dermis, whereas 2017. TCR-Vg4+ gdT17 cells acquire their skin-homing properties T.K. was supported by a Heisenberg award from the Deutsche Forschungsgemein- extrathymically. Yet, both subsets of dermal gd T cells are pre- schaft (KO2964/3-2 Heisenberg Sachbeihilfe). This work was supported by the imprinted to produce IL-17 and not IFN-g. gdT17 cells exhibit a Deutsche Forschungsgemeinschaft (SFB1054/B06, TR128/A06, A07), the European Research Council (CoG 647215), and within the framework of the Munich Cluster permissive epigenetic landscape and can convert into IFN-g pro- for Systems Neurology (SyNergy) (EXC 1010). ducers in vitro and in vivo in the context of distinct inflamma- Address correspondence and reprint requests to Dr. Thomas Korn, Klinikum Rechts tory environments (13). In contrast, IFN-g–producing gdT1 cells der Isar, Neurologische Klinik, Technische Universita¨tMunchen,€ Ismaninger Strasse have been proposed to exhibit an epigenetic signature indica- 22, 81675 Munich, Germany. E-mail address: [email protected] tive of a “terminally committed” phenotype which precludes The online version of this article contains supplemental material. plasticity to convert into IL-17 producers (13). It is commonly Abbreviation used in this article: DN, double negative. believed that gdT17 cells develop in the thymus during an early This article is distributed under The American Association of Immunologists, Inc., ontogenetic window shortly before birth and, therefore, the Reuse Terms and Conditions for Author Choice articles. possibility of a secondary de novo generation of gdT17 cells Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 outside the thymus has thus far been dismissed. Rather, it is

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700287 2 EXTRATHYMIC DIFFERENTIATION OF gdT17 CELLS thought that postnatally, the initial pool of gdT17 cells is main- Flow cytometry tained through self-renewal, perhaps in response to homeostatic cy- Single-cell suspensions were incubated with Fc-blocking Abs (aCD16/ tokine cues including IL-7 (10). aCD32; BD Biosciences) and subsequently stained with 1:100 dilutions of gdT17 cells have most recently been shown to participate in the the respective surface marker Abs for 30 min at 4˚C. Staining was carried immunopathology of a variety of inflammatory diseases in epi- out with Abs to CD3 (145-2C11), CD4 (RM4-5), and CD27 (LG.3A10) thelial barrier tissues, including inflammatory bowel disease, from BD Biosciences; CD45.1 (A20), CD45.2 (104), and CD122 (TM-b1) from eBioscience; CCR6 (140706, R&D), TCR gd (GL-3), TCR-Vg1 psoriasis, and asthma (11, 14, 15), but also in spondylarthropathies (2.11), and TCR-Vg4 (UC3-10A6) from BioLegend. The Tonegawa no- and CNS inflammation in mouse models (1, 16) and in humans menclature was used to designate TCR-Vg chains throughout the text. The (17–19). Because the presence of gdT17 cells in certain tissues staining panels always included dead cell staining dyes (7-AAD [BD might set the threshold for these tissues to respond directly to Biosciences] or Aqua [Invitrogen]). After staining, cells were washed and analyzed using a CyAn FACS machine (Beckman Coulter) or a BD pathogen-associated molecular patterns and indirectly to the FACSAria III (BD Biosciences). proinflammatory cytokine IL-23, the regulation of gdT17 cell populations during homeostasis and in inflammation is highly Intracellular cytokine and staining relevant in the context of immune interventions in autoimmune Cells were stimulated in culture medium containing PMA (50 ng/ml; diseases, chronic inflammation, and inflammation associated with Sigma), ionomycin (1 mg/ml; Sigma-Aldrich), and monensin (GolgiStop, malignant disease. In this study, we show that under homeostatic 1 ml/ml; BD Biosciences) at 37˚C and 10% CO2 for 4 h. After staining of conditions, the adult thymus has largely lost its capacity to pro- surface markers, cells were fixed and permeabilized (Cytofix/Cytoperm and Perm/Wash Buffer; BD Biosciences), followed by staining with duce gdT17 cells. We found that thymic development of gdT17 mAbs to mouse IL-17A (TC11-18H10) (BD Biosciences) and to mouse cells may be partially restricted by sensing IL-2. However, under IFN-g (XMG1.2) and fluorocytometric analysis (CYAN; Beckmann Downloaded from inflammatory conditions, gdT17 cells can also be de novo gen- Coulter). For ROR-gt staining, cells were fixed on ice in 2% formaldehyde erated in the peripheral immune compartment from IL-23R2 (diluted from Histofix 4%; Carl Roth, Karlsruhe, Germany) for 1 h fol- lowedbystainingwithanti-mouseROR-gt Abs (B2D; eBioscience) precursor gd T cells. Notably, IL-23 is indispensable for this diluted in 13 eBioscience Permeabilization Buffer (eBioscience), as process. De novo–generated gdT17 cells are bona fide gdT17 cells previously described (25). and participate in inducing immunopathology in an in vivo model Quantitative PCR analysis of psoriasis. http://www.jimmunol.org/ For real-time PCR analysis, cells were homogenized using QiaShredder, Materials and Methods and RNA was isolated using the RNeasy Mini or RNeasy Micro Kit (all Mice Qiagen) and transcribed into cDNA using the TaqMan Reverse Tran- scription Reagents Kit (Life Technologies) according to the manufacturer’s Nur77GFP reporter mice (20) and Il23rgfp/+ mice (21) were previously instructions. Primers and probes were purchased from Life Technologies; described. Because the GFP-reporter cassette is knocked into the endog- the assays were performed on 96-well reaction plates (Life Technologies) enous Il23r locus, Il23rgfp/gfp mice are functional Il23r2/2 mice. The Il17f- and analyzed using a StepOne System from Life Technologies. In all ex- Creeyfp fate mapping system to track current and historic IL-17F– periments, Actb was used as the reference to normalize gene ex- producing cells in mice has been published (22, 23). C57BL/6 mice, pression. CD45.1 congenic C57BL/6 mice (B6.SJL-PtprcaPepcb/BoyJ, stock num- by guest on September 27, 2021 ber 002014), Il2ra2/2 mice, and Rag12/2 mice were obtained from the In vitro gd T cell differentiation Jackson Laboratory. CD45.1 congenic mice from the Jackson Laboratory Pooled cell suspensions derived from peripheral lymph nodes and spleen of do not carry the Sox13 mutation that is associated with decreased en- Il23rgfp/+ mice were sorted (based on GFP expression) into IL-23R+ and dogenous frequencies of gdT17 cells (24). Analysis of fetal thymic Il2 IL-23R2 gd T cells; or, as indicated, into IL-23R2CD122+ or IL-23R2 transcript levels was performed in embryos of timed-pregnant mice. All CD1222 gd T cells; or according to TCR-Vg expression by FACS (BD animals were kept in a conventional, specific pathogen-free facility at the FACSAria III). Purified gd T cells were stimulated in 96-well U-bottom Technical University of Munich, and all experiments were carried out in plates (20,000 cells per well) for 5 d with 10,000 mouse anti-CD3/-CD28– accordance with the guidelines prescribed by the Bavarian state authorities coated T-Activator Dynabeads (Life Technologies) in 10% FCS containing (Az 55.2-1-54-2532-29-13 and 55.2-1-54-2532-1-15). DMEM supplemented with 5 3 1025 M 2-ME, 1 mM sodium pyruvate, Generation of chimeric mice nonessential amino acids, L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Recombinant were added to the cultures as indi- For the generation of bone marrow chimeras or fetal liver chimeras, Rag12/2 cated: 10 ng/ml mouse IL-1b, 50 ng/ml mouse IL-6, 10 ng/ml mouse recipient mice were lethally irradiated. The 7 Gy total dose was delivered IL-23, and 100 ng/ml mouse IL-21 (R&D Systems). as two 3.5-Gy doses 3 h apart. The donor bone marrow was always de- pleted of CD90+ cells prior to transfer to get rid of residual mature ab and Statistical analysis 3 6 3 7 gd T cells. A total of 5 10 (or 1 10 when indicated) bone marrow Statistical evaluations were performed using GraphPad Prism 6 software. cells were injected i.v. into recipients 1 d after irradiation. For fetal liver The unpaired Student t test was used for comparison of two populations. cell transplantation, fetal mice were prepared at day 13.5 of gestation. The paired Student t test was used for mean fluorescence intensity com- 3 6 Liver cells (9 10 ) were transferred 1 d after irradiation. Reconstituted parison between different TCR-Vg chain subsets derived from the same mice were maintained on antibiotic water (Enrofloxacin, Bayer, 0.1 mg/ml) mouse. Multiple comparisons were performed using one-way or two-way for 2 wk after cell transfer. Reconstitution of the hematopoietic system was ANOVA, followed by post hoc tests (Tukey). A p value ,0.05 was con- tested in the peripheral blood. sidered significant. Induction of psoriasiform plaques The mouse back was shaved and depilated (Veet; Reckitt Benckiser). A dose Results of 50 mg of Aldara (5% IMQ cream; 3M Pharmaceuticals) or vehicle The adult thymus fails to support gdT17 cell generation control (Vaseline [white], European Pharmacopoeia; Bombastus Werke, Freital, Germany) was applied on the back for 4–5 consecutive days. During thymic development, gdT17 cells are precommitted to Back skin thickness was measured daily in triplicates using a micrometer express the IL-23R and acquire the potential to produce IL-17, (Mitutoyo). whereas gdT1 cells are believed to be terminally committed to Preparation of lymphocytes produce IFN-g but not IL-17. gdT17 cells leave the thymus of mice in a “wave” around embryonic day 18. After birth the thymic To obtain single-cell suspensions from thymi, spleens, and peripheral lymph nodes, tissues were dissected and grated through a nylon mesh with 70-mm export of gdT17 cells has been reported to wane (2). To challenge pores (Falcon). RBC lysis was performed using BD Pharmlyse Buffer (BD this observation in a bone marrow transplantation setting, we Biosciences). grafted Il23rgfp/+ bone marrow, which reports IL-23R expression The Journal of Immunology 3 by expression of GFP into irradiated adult Rag12/2 recipient lymphoid organs (27). Because the transcriptional profiles of Th17 mice. Although these bone marrow chimeras were not deficient in cells and gdT17 cells are similar in many aspects (28, 29), we Th17 cells and exhibited regular fractions of gd T cells within the investigated whether IL-2 was a relevant determinant of thymic CD3+ T cell compartment (Fig. 1A, 1B), gdT17 cells were es- gdT17 development. Thymic IL-2 expression decreases before sentially absent in the thymus, lymph nodes, or spleen (Fig. 1C). birth and resumes postnatally (Supplemental Fig. 2A). As the To test whether the thymic milieu was repulsive for the develop- prenatal trough in thymic IL-2 availability coincides with the ment of gdT17 cells because of the ontogenetic “age” of thymo- proposed time window of output of TCR-Vg4+ gdT17 cells (30), it cytes, we compared the development of gdT17 cells in the was possible that increased sensing of IL-2 within the adult thy- lymphoid compartments of Rag12/2 mice transplanted with adult mus would prevent the commitment of gd thymocytes toward the bone marrow or fetal liver cells (26). We found that Rag12/2 gdT17 lineage. Therefore, we generated mixed bone marrow recipient mice failed to generate gdT17 cells irrespective of chimeric mice by transferring wild-type plus Il2ra2/2 bone mar- whether they were reconstituted with adult bone marrow or fetal row into irradiated Rag12/2 recipients. Indeed, after full recon- liver cells (Fig. 1D). In contrast to bone marrow or fetal liver stitution, the fraction of thymic gdT17 cells (as measured by precursor cells, cotransferred neonatal thymocytes, which had CCR6 expression in CD272 gd T cells [2, 3]) was slightly in- already received their thymic imprinting, contained gdT17 cells creased in the IL-2Ra–deficient cell compartment as compared and were readily able to populate the secondary lymphoid tissue with its wild-type counterpart (Supplemental Fig. 2B). However, of Rag12/2 recipient mice (Supplemental Fig. 1, see also Ref. 26). despite this facilitated commitment of Il2ra2/2 thymocytes to the IL-2 plays an inhibitory role during early commitment of gdT17 phenotype, the peripheral lymph node gdT17 population conventional naive CD4+ T cells to the Th17 lineage in secondary was not rescued in mixed bone marrow chimeras in the steady Downloaded from http://www.jimmunol.org/

FIGURE 1. Generation of gdT17 cells is impaired in the adult thymus. Bone marrow chimeric (BMC) mice were constructed by transplanting bone marrow cells from Il23rgfp/+ mutant mice into lethally irradiated adult Rag12/2 mice. After full recon- stitution of the peripheral blood com- by guest on September 27, 2021 partment, thymus (THY), peripheral lymph nodes (LN), and spleen (SPL) of BMC mice were analyzed by flow cytometry and compared with the lymphoid organs of cohoused Il23rgfp/+ mutant mice. (A) Frequencies of CD4+ IL-17+ T cells after ex vivo PMA/ ionomycin stimulation. (B) Frequen- cies of gd T cells within the CD3+ T cell compartment. (C) Frequencies of IL-23R (GFP)+ gd T cells within the gd T cell compartment. (D) Fraction of IL-23R (GFP)+ gd T cells within the gd T cell compartment of Il23rgfp/+ mutant mice in comparison with irra- diated chimeric Rag12/2 mice recon- stituted with either bone marrow cells from adult Il23rgfp/+ donors (Il23rgfp/+ BMC) or fetal liver cells from Il23rgfp/+ donors (Il23rgfp/+ fetal liver chimera [FLC]). Each symbol represents an individual mouse and bars indicate mean 6 SD. Unpaired Student t test (A–C); one-way ANOVA with the Tukey multiple comparison test (D). The data are presentative of at least three independent experiments. **p , 0.01, ***p , 0.001, ****p , 0.0001. 4 EXTRATHYMIC DIFFERENTIATION OF gdT17 CELLS state (Supplemental Fig. 2B). Together, these data suggest that To directly test this idea in vivo, we established a competitive IL-2 may contribute to restraining the commitment of gd thymocytes setting comparing the efficacy of induction/expansion of gdT17 to the gdT17 lineage in the adult thymus, but may also be required cells in Il23rgfp/+ (IL-23–responsive) versus Il23rgfp/gfp (IL-23– for their maintenance in secondary lymphoid tissues. nonresponsive) precursors in the context of skin inflammation induced by Aldara, which leads to the production of IL-23 in the Inflammation induces de novo generation of gdT17 cells in skin (32). Mixed bone marrow chimeras (Il23rgfp/+ plus secondary lymphoid tissues Il23rgfp/gfp → Rag12/2) were treated with Aldara. As expected, We and others have previously reported that gdT17 cells massively the mixed bone marrow chimeric animals developed significant expand under inflammatory conditions and contribute to IL-17 skin thickening as compared with Vaseline-treated controls production in various organ systems (1, 31). Because adult (Fig. 4A). Notably, the large majority of gdT17 cells in the skin- Rag12/2 recipients of Il23rgfp/+ bone marrow lacked gdT17 cells draining lymph nodes were recruited from GFP+ Il23rgfp/+ gd under homeostatic conditions, we expected these mice to be T cells, whereas GFP+ Il23rgfp/gfp gd T cells were induced but largely resistant to gdT17-dependent, psoriasis-like skin inflam- failed to expand; resulting in a large bias of the chimerism in the mation induced by application of the imiquimod-containing cream GFP+ gd T cell compartment (as well as the IL-17+ gd T cell Aldara. Surprisingly, we found that Il23rgfp/+ → Rag12/2 bone compartment) in the skin-draining lymph nodes in favor of marrow chimeric mice were susceptible to Aldara-induced skin Il23rgfp/+ and at the expense of Il23rgfp/gfp gd T cells (Fig. 4B, inflammation almost to the same extent as non–bone marrow 4C). Accordingly, the absolute number of GFP+ gd T cells in the chimeric Il23rgfp/+ mutant mice, as indicated by a substantial in- draining lymph nodes was significantly smaller in the CD45.1+ crease in skin thickness and formation of psoriasis-like lesions (Il23rgfp/gfp) compartment as compared with the CD45.2+ Downloaded from (Fig. 2A). Importantly, in contrast to vehicle-treated controls, the (Il23rgfp/+) compartment (Fig. 4D). Together, these data indicate skin-draining lymph nodes of Aldara-treated bone marrow chi- that, after establishment of IL-23 responsiveness in gd T cells, meras harbored marked fractions and absolute numbers of IL-23R IL-23 is crucial for the expansion of gdT17 cells in vivo. (GFP)+ gd T cells (Fig. 2B), which were bona fide gdT17 cells because they expressed ROR-gt and produced IL-17 (Fig. 2C). The potential to differentiate into gdT17 cells in the peripheral immune compartment is largely restricted to IL-23R2CD1222 These findings prompted the hypothesis that the inflammatory http://www.jimmunol.org/ milieu might promote the de novo generation of gdT17 cells in gd T cells the peripheral immune compartment, either through imprinting of gdT1 cells were previously characterized as CD27+ gd T cells (2), the gdT17 transcriptional profile in uncommitted precursors or whereas another report referred to them as CD122+ gd T cells through reprogramming of gdT1 cells (contained within the (33). When we sorted IL-23R2CD122+ and IL-23R2CD1222 IL-23R2 gd T cell compartment). gd T cells from the peripheral immune compartment of Il23rgfp/+ mice to high purity (Fig. 5A) and compared their capacity to TCR stimulation together with IL-1b and IL-23 de novo induce + 2 “convert” into IL-23R (GFP) gdT17 cells, we found that only gdT17 cells from IL-23R (GFP) gd T cells CD1222 gd T cells but not their CD122+ counterparts could be To test the possibility of de novo generation of gdT17 cells, we induced to express IL-23R (GFP) and IL-17 in response to IL-1b by guest on September 27, 2021 isolated IL-23R2 gd T cells from lymph nodes and spleen of naive plus IL-23 (Fig. 5A). To test whether CD1222 gd T cells also Il23rgfp/+ mice by flow cytometric sorting to high purity (Fig. 3A) represented the gd T cell population that was induced to express and stimulated these gd T cells in the presence of anti-CD3/anti- IL-23R and IL-17 in our bone marrow chimeric mice (Fig. 1), we CD28–coated beads under neutral conditions, or in the presence of sorted IL-23R (GFP)2CD122+ and IL-23R (GFP)2CD1222 gd various cytokines. Similar to any individual cytokine tested, IL-23 T cells from the secondary lymphoid tissue of unmanipulated alone failed to induce a stable population of gdT17 cells, whereas Il23rgfp/+ → Rag12/2 bone marrow chimeras and exposed them to only the combination of IL-1b plus IL-23 resulted in the sustained IL-1b and IL-23 in vitro. Similar as in gd T cells directly isolated induction of IL-23R+ gd T cells in the precursor IL-23R2 gd from Il23rgfp/+ mice, only CD1222 but not CD122+ gd T cells T cell population (Fig. 3B). “Induced” gd T cells were bona fide from bone marrow chimeras were induced to express IL-17 gdT17 cells because they expressed ROR-gt and IL-17 (Fig. 3C). (Fig. 5B). Thus, IL-23R2CD1222 gd T cells compose a pop- IL-1b alone was sufficient to induce minute amounts of IL-23R ulation of gd T cells that can be induced to express IL-17 and (GFP) in IL-23R2 gd T cells in the presence and absence of TCR commit to the gdT17 lineage outside the thymus. triggering (Fig. 3D), suggesting that after initial induction of 2 IL-23 responsiveness in IL-23R2 gd T cells by IL-1b, IL-23 then The TCR-Vg chain of CD122 gd T cells determines their expanded this population and perhaps also stabilized their func- potential to express IL-17 tional phenotype. Within the in vitro–induced gdT17 cells, TCR-Vg4+ and TCR- Vg6+ gd T cells (identified in this article as Vg12Vg42) were IL-23 is indispensable for the expansion of induced gdT17 significantly enriched over Vg1+ gd T cells, which dominated the cells in vivo pool of IL-23R2CD1222 gd T cells ex vivo (Fig. 6A, 6B). To test We consider IL-23 as a for IL-23R+ gd T cells because whether the possibility to differentiate into gdT17 cells indeed Il23rgfp/gfp mice that entirely lack a functional IL-23R still harbor segregated with the expression of distinct TCR-Vg chains, we GFP+ (IL-23R “wannabe”–expressing) gd T cells, which are sorted Vg1+ and Vg4+ as well as “Vg double-negative (DN)” cells equally capable of producing IL-17 as their GFP+ Il23rgfp/+ coun- from the peripheral IL-23R (GFP)2CD1222 gd T cell compart- terparts (Supplemental Fig. 3). Although the fraction of GFP+IL- ment of Il23rgfp/+ mice and cultured them in the presence of IL-1b 17+ gd T cells in the thymus is similar in Il23rgfp/+ and Il23rgfp/gfp plus IL-23. Here, Vg1+ gd T cells were only poorly induced to (= Il23r2/2) mice, the fraction of gdT17 cells in secondary lym- express IL-23R (GFP), whereas the majority of Vg4+ gd T cells as phoid tissue is much higher in Il23rgfp/+ than in Il23rgfp/gfp mice well as Vg DN gained IL-23R and IL-17 expression upon (Supplemental Fig. 3). Together with our in vitro data, these find- stimulation with IL-1b and IL-23 (Fig. 6C). Consistent with this ings suggest that the function of IL-23 is essentially to expand gd finding, IL-23R (GFP)+ gd T cells converted by in vitro culture T cells which have been previously induced to express IL-23R. with IL-1b and IL-23 from Il23rgfp/+ → Rag12/2 bone marrow The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Aldara-induced skin inflammation leads to the accumulation of bona fide gdT17 cells in skin-draining lymph nodes of Il23rgfp/+ BMC mice. Il23rgfp/+ BMC mice were either treated with Vaseline or exposed to imiquimod cream (Aldara). Il23rgfp/+ mutant mice served as positive control. (A) Clinical appearance and measurement of skin thickness. Symbols represent the mean of five mice per group, error bars indicate SD. (B) Fraction and absolute number of CD272IL-23R (GFP)+ gd T cells in skin-draining lymph nodes of Vaseline (Vas) versus Aldara (Ald) treated Il23rgfp/+ BMC mice and Aldara-treated Il23rgfp/+ mutant controls. Each symbol shows an individual mouse and bars indicate mean + SD. (C) IL-17 (top row) and ROR-gt (bottom row) expression in skin-draining lymph node gd T cells on day 4 after treatment, as determined by intracellular staining of skin-draining LN cells with or without prior PMA/ionomycin stimulation, respectively. Representative of three independent experiments. chimera–derived IL-23R2CD1222 gd T cells, as well as IL-23R or historic production of IL-17 in CCR62CD1222 gd T cells (GFP)+ gd T cells induced outside the thymus in vivo in Il23rgfp/+ (corresponding to the IL-23R2CD1222 gd T cell fraction in → Rag12/2 bone marrow chimeras, were highly biased toward the Il23rgfp/+ mice) of unmanipulated Il17f-Creeyfp reporter mice ex expression of Vg4attheexpenseofVg1, which was the dominating vivo (Supplemental Fig. 4A), it is unlikely that IL-23R2CD1222 Vg chain in IL-23R2CD1222 precursor gd Tcells(Fig.6D).Taken Vg4+ or Vg6+ gd T cells descend from previous gdT17 cells. together, these data suggest that IL-23R2CD1222Vg4+ gd T cells, None of the auxilliary transcription factors of the Th17 profile, but not their Vg1+ counterparts, can be committed to the gdT17 which have recently been described to be dispensable for the lineage in the peripheral immune compartment. development of gdT17 cells (29), were regulated (Fig. 7A). Finally, we directly tested the correlation of differential ROR-gt Engagement of the gd TCR contributes to the commitment of expression in CD1222Vg4+ versus CD1222Vg1+ gd T cells with gd T cells to distinct functional lineages previous TCR triggering by investigating adult Nur77GFP reporter To further investigate the idea that the Vg repertoire was a de- mice. We did not detect any differences in the extent of TCR terminant of the commitment of gd T cells to either gdT1 or engagement between these two subsets in the peripheral immune gdT17 cells, we assessed the expression of key transcription compartment. However, in the thymus, CD1222Vg1+ gd T cells factors in highly purified CD1222Vg1+ versus CD1222Vg4+ gd showed a high fraction of Nur77GFP+ cells, which was essentially T cells. We found that Vg1+ gd T cells resembled CD122+ gd absent in CD1222Vg4+ gd T cells (Fig. 7B, Supplemental Fig. T cells (bona fide gdT1 cells), whereas Vg4+ gd T cells expressed 4B). Thus, TCR engagement in CD1222Vg1+ gd thymocytes low levels of ROR-gt and the gdT17-associated scavenger re- might prevent the expression of ROR-gt and poise these cells ceptor Scart2 (34) (Fig. 7A). Because we did not observe current for the gdT1 lineage. Conversely, lack of TCR engagement in 6 EXTRATHYMIC DIFFERENTIATION OF gdT17 CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. IL-1b plus IL-23 induce gdT17 cells de novo from IL-23R2 gd T cells. (A) IL-23R (GFP)2 and IL-23R (GFP)+ gd T cells were isolated from lymph node (LN) and spleen (SPL) cell suspensions of unmanipulated Il23rgfp/+ mutant mice by flow cytometric sorting and cultured for 5 d in the presence of anti-CD3/anti-CD28–coated beads and the indicated cytokines. (B) Analysis of CD27 and IL-23R (GFP) expression after 5 d of culture. Data are representative of three independent experiments. (C) IL-17 and ROR-gt expression in sorted IL-23R (GFP)+ (left column) and IL-23R (GFP)2 gd T cells (right column) after 5 d culture with anti-CD3/anti-CD28–coated beads and IL-1b plus IL-23. Representative plots out of three independent ex- periments are shown. (D) IL-23R (GFP)2 gd T cells were sorted and cultured in the presence of either IL-1b alone, anti-CD3/anti-CD28–coated beads alone, or IL-1b plus anti-CD3/anti-CD28–coated beads. At the indicated time points, IL-23R (GFP) induction was measured by flow cytometry. Symbols represent the mean of duplicate wells and error bars indicate SD. Representative plots showing IL-23R (GFP) expression in gd T cells 18 h after start of culture. Data are representative of one out of two independent experiments. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. IL-23R signaling is required for the accumulation of gdT17 cells upon Aldara-induced skin inflammation. Mixed bone marrow chimeras (MBMC) were generated by transferring equal fractions of congenically marked bone marrow cells from Il23rgfp/+ (CD45.2) and Il23rgfp/gfp (CD45.1) mutant donor mice into lethally irradiated adult Rag12/2 recipients. After full reconstitution of the peripheral blood immune cell compartment, blood chimerism CD45.2 (Il23rgfp/+)/CD45.1 (Il23rgfp/gfp) ratio was ∼40:60 (data not shown). MBMC were then treated with Aldara or sham treated with Vaseline. (A) Change in skin thickness in Vaseline- versus Aldara-treated MBMC. Symbols show mean + SD of five animals per group. (B and C) Skin- draining lymph node gd T cells were analyzed for CD27 and IL-23R (GFP) expression (B) and intracellular expression of IFN-g and IL-17 (C) followed by assessment of the CD45.2/CD45.1 chimerism in the indicated compartments. (D) Fractions and absolute numbers of IL-23R expressers in the draining lymph node CD45.2+ Il23rgfp/+ compartment and fractions and absolute numbers of IL-23R wannabe expressers in the draining lymph node CD45.1+ Il23rgfp/gfp compartment of MBMC on day 4 posttreatment with either Vaseline or Aldara. Symbols in the graph depict individual mice and bars indicate mean + SD. Two-way ANOVA with the Tukey multiple comparison test. Representative of two independent experiments. **p , 0.01, ****p , 0.0001. 8 EXTRATHYMIC DIFFERENTIATION OF gdT17 CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. IL-23R (GFP)2CD1222 but not CD122+ gd T cells can be induced to express IL-17 outside the thymus. (A) gd T cells from spleen (SPL) and lymph nodes (LN) were sorted from unmanipulated Il23rgfp/+ mutant mice on the basis of IL-23R (GFP) and CD122 expression into IL-23R (GFP)2 CD122+ (CD122+) and IL-23R (GFP)2CD1222 (DN) subsets, tested for intracellular expression of IFN-g and IL-17 (postsort), and cultured in the presence of anti-CD3/anti-CD28–coated beads and IL-1b plus IL-23. IL-23R (GFP) induction and expression of IL-17 and IFN-g were measured 5 d postculture by flow cytometry. Representative data out of three independent experiments are shown. Symbols in graph depict individual replicate wells and bars show mean + SD. One-way ANOVA with the Tukey multiple comparison test. ****p , 0.0001. (B) Analogously, IL-23R (GFP)+, IL-23R (GFP)2CD122+, and IL-23R (GFP)2CD1222 (DN) gd T cells were purified from SPL and LN of unmanipulated Il23rgfp/+ → Rag12/2 bone marrow chimeras and subjected to in vitro culture with anti-CD3/-CD28 beads and IL-1b plus IL-23. IL-23R (GFP) induction and expression of IL-17 and IFN-g were measured in the indicated gates 5 d postculture by flow cytometry. Representative data out of two independent experiments are shown. Symbols in graph depict individual replicate wells and bars show mean + SD. One-way ANOVA with the Tukey multiple comparison test, **p , 0.01, ****p , 0.0001. n.d., not detected.

CD1222Vg4+ gd thymocytes saves these cells from premature extent of immunopathology resulting from inflammation. In this lineage commitment in the thymus and allows for the induction of study, we confirmed that gdT17 cells are not produced by the adult a gdT17 transcriptional program outside the thymus. thymus. In contrast to Th17 cell commitment, IL-2 has only a In summary, although the adult thymus ceases producing gdT17 minor inhibitory function during thymic development of gdT17 cells, bona fide gdT17 cells can be generated in the peripheral cells. However, gdT17 cells can be de novo generated extra- immune compartment from those IL-23R2 precursors that are not thymically by conversion of IL-23R2 gd T cells into bona fide already poised for IFN-g production by TCR engagement in the gdT17 cells in response to IL-1b plus IL-23, indicating an un- thymus. anticipated plasticity of a subset of IL-23R2 gd T cells that are CD1222 and express TCR-Vg4 or TCR-Vg6. Discussion Thymic development of gd T cells occurs according to waves of gdT17 cells are gatekeepers of inflammatory responses at epi- gd TCR rearrangement during embryogenesis. Thymic generation thelial surfaces and perhaps also in the CNS (35, 36). It is an of TCR-Vg5–positive gd T cells peaks at embryonic day 14 and emerging concept that the steady-state population of certain tissue TCR-Vg5–positive gd T cells populate the epidermis of mice as niches with gdT17 cells might dictate their responsiveness to dendritic epidermal T cells. TCR-Vg6–positive gd T cells are pathogen-associated molecular patterns, IL-23, eventually the generated around embryonic day 17 and home to urothelium and The Journal of Immunology 9 dermis, and TCR-Vg4–positive gd T cells peak at embryonic day wave and TCR-Vg4 is overrepresented in gdT17 cells in sec- 18.5 and home to bronchial epithelium as well as secondary ondary lymphoid tissues. More recently, populations of patho- lymphoid tissues (37). An alternative concept uses functional genic TCR-Vg6+ gdT17 cells were described in the dermis and definitions for different developmental waves of gd T cells. Here, joints of mice (12, 40). Although the TCR-Vg chain repertoire of thymic export of dendritic epidermal T cells is followed by the gdT17 cells appears to be restricted, it is unlikely that commit- gdT17 wave and the gdT1 wave (30, 38, 39). Notably, the time ment of gd T cells to the production of IL-17 is based on the window of gdT17 export largely coincides with the TCR-Vg4 thymic engagement of distinct gd TCRs because the ability to Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Vg4+ and Vg6+ gd T cells but not Vg1+ gd T cells are extrathymically induced to express IL-23R and IL-17. (A) Pie charts illustrate the relative fractions of Vg1+ and Vg4+ as well as Vg12Vg42 (Vg DN) cells in constitutively IL-23R (GFP)–expressing gd T cells as well as in IL-23R (GFP)+ gd T cells converted in vitro from IL-23R (GFP)2CD1222 (DN) gd T cell precursors after 5 d culture in the presence of IL-1b plus IL-23. (B) Pie charts of relative fractions of Vg1- and Vg4-expressing as well as Vg12Vg42 (Vg DN) cells in the indicated gd T cell subsets directly ex vivo. (C) IL-23R (GFP)2 CD1222 (DN) gd T cells were sorted into Vg1+,Vg4+, and Vg12Vg42 (Vg DN) subsets and cultured in the presence of anti-CD3/anti-CD28–coated beads and IL-1b plus IL-23. IL-23R (GFP) induction and intracellular cytokines (upon PMA/ionomycin stimulation) were measured 5 d postculture by flow cytometry. Representative data of one out of three independent experiments are shown. Symbols in graphs show the fraction of CD272IL-23R (GFP)+ gd T cells in individual replicate wells and bars indicate mean + SD. One-way ANOVA with the Tukey multiple comparison test. ****p , 0.0001. (D) Pie chart of Vg TCR chains used by IL-23R (GFP)2CD1222 gd T cells isolated from the lymph nodes of Il23rgfp/+ → Rag12/2 bone marrow chimeras after complete reconstitution directly ex vivo and after in vitro culture of the same cells with anti-CD3/anti-CD28 and IL-1b plus IL-23 (gate on converted IL-23R [GFP]+ cells). In the far right pie chart, the Vg TCR chain usage of “in vivo converted” IL-23R (GFP)+ gd T cells isolated from the draining lymph nodes of Aldara-treated Il23rgfp/+ → Rag12/2 bone marrow chimeras is shown. Mean of five individual mice and representative of two independent experiments. 10 EXTRATHYMIC DIFFERENTIATION OF gdT17 CELLS Downloaded from

2 2 + + A 2 FIGURE 7. The transcriptional profile of IL-23R (GFP) CD122 Vg4 but not Vg1 gd T cells is poised for gdT17 cell fate commitment. ( ) IL-23R http://www.jimmunol.org/ CD1222 gd T cells from spleen (SPL) and lymph node (LN) of Il23rgfp/+ mice were sorted into Vg1+ (DN Vg1+) versus Vg4+ (DN Vg4+) subsets and relative expression of the indicated genes was determined via quantitative PCR. Sorted IL-23R (GFP)+ gdT17 cells as well as IL-23R2CD122+ gdT1 cells + (CD122 ) served as controls. Gene expression was normalized to the expression of b-actin. Graphs show the fold change in expression (log2 ratio) comparing DN Vg4+ with DN Vg1+ gd T cells (left) and IL-23R (GFP)+ gdT17 cells with IL-23R (GFP)2CD122+ gdT1 cells. Bars show mean + SD (n =4). Because Rorc expression was below detection limit in CD122+ gdT1 cells, a Ct value of 40 (= maximum number of cycles) was used for calculation. (B) Nur77GFP expression in Vg1+ (black histogram) versus Vg4+ (gray shaded histogram) subsets of CCR6-CD1222 gd T cells derived from thymus (THY), LN, or SPL of Nur77GFP reporter mice. Representative histograms (left) and mean fluorescence intensity (MFI) of Nur77GFP in Vg1+ and Vg4+ CCR62 CD1222 gd T cells in the thymus (right). Paired Student t test. **p , 0.01, n = 4 individual mice. by guest on September 27, 2021 produce IL-17 from gd thymocytes is imprinted prior to TCR-Vg In contrast to the current understanding that gd T cells are chain rearrangement (5). committed to their cytokine phenotype in the thymus with limited The molecular underpinning of gdT17 commitment in the thy- plasticity to revert to IL-17 production, we found in this study that mus is only partly understood. In this study, we found that IL-2 IL-17–producing gd T cells can be de novo induced in secondary partially constrains gdT17 cell development in the thymus. In a lymphoid tissues. Under inflammatory conditions, i.e., in the previous report, IL-2 has been proposed to support (rather than presence of IL-1b and IL-23, IL-23R2 gd T cells can be induced constrain) the maintenance of gdT17 cells (33). However, these to express IL-23R and respond to IL-23 by expansion. In adult observations were made in IL-2–deficient or IL-2Ra–deficient mice individuals, TCR-Vg4+ gdT17 cells are strongly expanded in the that suffer from a lymphoproliferative syndrome, which represents a presence of IL-23. The de novo induction of gdT17 cells in the systemic inflammatory scenario where IL-2 might indeed promote peripheral immune compartment would be in line with the idea the expansion of gdT17 cells in the peripheral immune compart- that the gd T cell population in secondary lymphoid tissue con- ment. In addition, as in conventional ab T cells, thymic egress of tains uncommitted gd T cells. It has recently been suggested that gd T cells is controlled by S1P1 in a KLF2-dependent manner (41). naive (uncommitted) gd T cells are continuously produced from As IL-2 stimulation in vitro abrogates S1P1 expression in gd Tcells embryonic day 16 and, upon recognition of the “model Ag” PE, (data not shown), the inability to sense IL-2 could result in in- might be able to initiate IL-17 production without prior commit- creased thymic egress due to sustained surface expression of S1P1. ment to the gdT17 lineage in the thymus (30, 43). Uncommitted However, in mixed bone marrow chimeras, the fractions the gdT17 gd T cells in secondary lymphoid organs are CD44lowCD62Lhigh. cells in the thymus were higher within the IL-2Ra–deficient than All of the CD44low gd T cells in naive mice lack IL-23R ex- the wild-type compartment, suggesting that IL-2 restricts the thymic pression and CD44low gd T cells can indeed be induced to express development of gdT17 cells, for example by binding of STAT5 to IL-23R (data not shown). gdT17 hallmark genes as has been demonstrated in Th17 cells (27). IL-23R2TCR-Vg1+ gd T cells largely resist conversion into Recently, IL-15 has been reported to suppress the generation of gdT17 cells, whereas CD1222IL-23R2 TCR-Vg4+ or Vg6+ gd gdT17 cells. IL-15 shares CD122 and CD132 as molecules T cells readily adopt a gdT17 phenotype in response to IL-1b plus with IL-2 and thus, like IL-2, also belongs to the family of common IL-23. The mechanistic underpinning of the differential potential g-chain cytokines. While suppressing gdT17 cells, IL-15 has been of IL-23R2 gd T cells with various TCR-Vg chains to acquire a shown to promote gdT1 development in the thymus by induction of gdT17 transcriptional profile is currently unclear. Interestingly, T-bet in CD8aa precursors (42). Altogether, it is intriguing to TCR-Vg1+ but not TCR-Vg4+ gd thymocytes appear to have speculate that common g-chain cytokines might be an important sensed a TCR signal in the thymus, suggesting that TCR ligation determinant in dictating the balance of gdT1 versus gdT17 cells in the thymus skews gd thymocytes toward an IFN-g–producing during thymic development. phenotype. Similar observations were made previously in an The Journal of Immunology 11

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