Published November 18, 2015, doi:10.4049/jimmunol.1501102 The Journal of Immunology

Group 2 Innate Lymphoid Cells Express Functional NKp30 Inducing Type 2 Production

Maryam Salimi,* Luzheng Xue,† Helen Jolin,‡ Clare Hardman,‡ David J. Cousins,x,{ Andrew N. J. McKenzie,‡ and Graham S. Ogg*

Group 2 innate lymphoid cells (ILC2) are important in effector functions for eliciting allergic inflammation, parasite defense, ep- ithelial repair, and lipid homeostasis. ILC2 lack rearranged Ag-specific receptors, and although many soluble factors such as cy- tokines and lipid mediators can influence ILC2, direct interaction of these cells with the microenvironment and other cells has been less explored. Natural cytotoxicity receptors are expressed by subsets of group 1 ILC and group 3 ILC and thought to be important for their effector function, but they have not been shown to be expressed by ILC2. Therefore, we sought to investigate the expression and functional properties of the natural cytotoxicity receptor NKp30 on human ILC2. A subset of ex vivo and cultured ILC2 express NKp30 that upon interaction with its cognate activatory -H6 induces rapid production of type 2 . This interaction can be blocked by NKp30 blocking Ab and an inhibitory ligand, galectin-3. Higher expression of B7-H6 was observed in lesional skin biopsies of patients with atopic dermatitis, and incubation of keratinocytes with proinflammatory and type 2 cytokines upregulated B7-H6, leading to increased ILC2 cytokine production. NKp30–B7-H6 interaction is a novel cell contact mechanism that mediates activation of ILC2 and identifies a potential target for the development of novel therapeutics for atopic dermatitis and other atopic diseases. The Journal of Immunology, 2016, 196: 000–000.

nnate lymphoid cells (ILC) are novel effectors of the immune GATA-3 (4) and retinoic acid–related orphan receptor (ROR) a system that contribute to diverse forms of inflammation and transcription factors (5), and we and others have shown that hu- I defense (1). They depend on the transcriptional repressor ID2 man skin ILC2 produce type 2 cytokines IL-4, IL-5, and IL-13 in and cytokines that signal through the common g-chain of IL-2. response to IL-33, IL-25, thymic stromal lymphopoietin (TSLP), The lack of expression of common lineage markers and expression and PGD2 (6, 7). Group 3 ILC are RORgt-dependent and comprise of IL-7Ra are shared features in this family. Based on their lymphoid tissue inducers, as well as natural cytotoxicity receptor phenotype and function they have been divided into three main (NCR)+ and NCR2 group 3 ILC that express IL-22 alone or in groups (2). Group 1 ILC include NK cells and group 1 ILC that combination with IL-17, respectively (8–10). produce IFN-g and TNF-a (3). Group 2 ILC (ILC2) express family members do not possess a rearranged Ag-specific receptor and are thought to rely on a combination of activating and in- hibitory signals for their effector functions, but the mechanisms *Medical Research Council Human Immunology Unit, Weatherall Institute of Mo- lecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, are unclear (11). Several studies have evaluated the expression of United Kingdom; †Oxford National Institute for Health Research Biomedical Re- the NCRs, NKp44 (NCR2 and CD336), NKp46 (NCR1 and search Centre, Translational Immunology Laboratory, Nuffield Department of Med- CD335), and NKp30 (NCR3 and CD337) on group 1 and 3 ILC in icine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom; ‡Medical Research Council Laboratory of Molecular Biology, Cambridge humans, but the expression of these markers has not yet been x CB2 0QH, United Kingdom; Department of Infection, Immunity and Inflammation, demonstrated on ILC2 in humans (12). In this study, we sought to National Institute for Health Research Leicester Respiratory Biomedical Research Unit, University of Leicester, Leicester LE3 9QP, United Kingdom; and {Medical investigate the expression and function of NKp30 on human ILC2. Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma, King’s NKp30 is an activating type I Ig-like transmembrane receptor College London, London WC2R 2LS, United Kingdom that is not present in mice, but is found mainly on human NK cells ORCIDs: 0000-0003-3821-9596 (D.J.C.); 0000-0002-3097-045X (G.S.O.). (13). It has one Ig-like extracellular domain that connects to the Received for publication May 13, 2015. Accepted for publication October 20, 2015. transmembrane region by a short 6-aa stem. It is encoded in the This work was supported by the Medical Research Council, the Barrie Trust, the extremely polymorphic telomeric end of the class III region of National Institute for Health Research Biomedical Research Centre, and Janssen the human MHC (3, 14). of exon 4 gives Pharmaceuticals. G.S.O. also acknowledges the support of the National Institute for Health Research Clinical Research Network. A.N.J.M. was supported by the rise to three main distinct isoforms, NKp30a, NKp30b, and Medical Research Council and Janssen Pharmaceuticals. D.J.C. was supported by NKp30c. Upon interaction with its ligands, isoforms a and b are grants from the Medical Research Council, Asthma UK, and the National Institute for thought to convey cytotoxicity responses, release of IFN-g and Health Research Leicester Respiratory Biomedical Research Unit and by the Na- tional Institute for Health Research Biomedical Research Centre based at Guy’s and TNF-a by NK cells, and trigger maturation (3, 15, St. Thomas’ National Health Service Foundation Trust and King’s College London. 16), whereas splice variant NKp30c reduces IFN-g production and Address correspondence and reprint requests to Prof. Graham S. Ogg, University of cytotoxicity and increases the production of IL-10 by NK cells (3). Oxford, Cutaneous Immunology Group, Weatherall Institute of Molecular Medicine, A prevalent expression of isoform c on NK cells has been seen in John Radcliffe Hospital, Headley Way, Oxford OX3 9DS, U.K. E-mail address: [email protected] patients with gastrointestinal stromal tumor and is associated with The online version of this article contains supplemental material. reduced survival due to defective IFN-g, TNF-a, and IL-12 pro- Abbreviations used in this article: AD, atopic dermatitis; BAT3, HLA-B–associated duction, defective NK–dendritic cell dialog, and increased im- transcript 3; ILC, innate lymphoid cell; ILC2, group 2 innate lymphoid cell; NCR, munosuppression and IL-10 production (3). Several physiological, natural cytotoxicity receptor; rh, recombinant human; ROR, retinoic acid–related tumor, and viral markers have been identified as NKp30 ligands, orphan receptor; TSLP, thymic stromal lymphopoietin. including CMV tegument pp65 (17), Duffy binding–like-1a Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00 domain of Plasmodium falciparum erythrocyte membrane protein-1

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501102 2 ILC2 EXPRESS FUNCTIONAL NKp30 RECEPTOR

(18), NF HLA-B–associated transcript 3 (BAT3) (19), and tumor- purchased from eBioscience, R&D Systems, and Mabtech, respectively, associated cell surface protein B7-H6 (20). and carried out according to manufacturers’ instructions. In this study, we investigate the expression of NKp30 on ILC2 ex Immunohistochemistry vivo and on cultured ILC2. Using quantitative PCR we identify the Anti–B7-H6 Ab (Abcam, ab121794), isotype control (Abcam, ab37416), splice variants of NKp30 and show that incubation of ILC2 with mouse anti-rabbit HRP (344002, MaxDiscovery), and anti–Galectin-3 plate-bound B7-H6 or cell lines expressing this protein induced Ab (AF1154, Bio-Techne) were used to stain formalin-fixed, paraffin- production of type 2 cytokines. This interaction can be inhibited by embedded skin tissue sections from healthy donors and adult atopic der- NKp30-blocking Abs and the soluble blocking ligand, galectin-3. matitis (AD) patients with moderate–severe disease. The diaminobenzidine We further established that activation of NKp30 induces the ca- signal was quantified using the “Fiji” version of ImageJ. nonical pathway of NF-kB signaling. We identify a functionally Isolation of epidermal cells important activatory cell contact receptor for ILC2, showing the Skin biopsies from healthy donors were cut into wide strips and incubated involvement of NKp30 in ILC2-induced type 2 immune responses. overnight in 2 U/ml dispase at 4˚C. Epidermal sheet was peeled from the dermis by forceps and incubated for 15 min in 0.5% trypsin plus 0.02% EDTA at 37˚C. The mixture was then pipetted repeatedly and passed Materials and Methods through a 40-mm strainer. Cell culture Suction blister technique PBMCs were isolated from healthy adult donors under local ethics approval (National Research Ethics Service Committee South Central, Oxford C, 09/ Suction blister cups were applied to the lesional and nonlesional skin of H0606/71). ILC2 were isolated and cultured as previously described (6). adult AD patients with moderate–severe disease, and not on systemic Briefly, the lineage (CD3, CD4, CD8, CD14, CD19, CD56, CD11c, therapy, at a vacuum pressure of 250–450 mmHg. Blisters were formed CD11b, CD123, and FcεRI)–negative, CD45+, CD127+, CRTH2+ ILC2 within 30–90 min. Twenty-four hours later fluid was aspirated using a 30- population was sorted into 96-well plates at the density of 100 cells per gauge needle. Fluids were then centrifuged at 1500 rpm for 5 min at 4˚C well and resuspended in MLR of gamma-irradiated PBMCs from three and the concentration of cytokines was measured by multiplex array healthy volunteers (2 3 106 cells/ml) coupled with 100 IU/ml IL-2. After (Luminex). 4–6 wk, the growing wells were tested by flow cytometry staining and resorted until a pure population of lineage2CRTH2+IL-7Ra+ ILC2 was Statistical analysis achieved (Supplemental Fig. 1A). The keratinocyte line (HaCaT) was The t tests were performed using GraphPad Prism version 6.00 (GraphPad cultured in tissue culture flasks (Corning, Corning, NY) in DMEM media Software, San Diego, CA). supplemented with 10% FCS at 37˚C with 5% CO2 and split on reaching confluence (approximately every 3–4 d). K562, Jurkat, and THP-1 cell lines were cultured in RPMI 1640 supplemented with 10% FCS, amino Results acids (MEM nonessential amino acids solution 11140-050; Life Tech- A large subset of ILC2 express the natural cytotoxicity receptor nologies) and HEPES (83264; Sigma-Aldrich). Cells were maintained at NKp30 0.2 3 106/ml density. For HaCaT incubation with cytokines, IFN-g was used at the concentration of 300 U/ml (21–24). All other cytokines were To investigate recognition strategies that ILC2 employ to sense the used at a concentration of 100 ng/ml (25). microenvironment, the expression of NKp30 on ILC2 was exam- Abs ined. Cultured ILC2 lines were stained for NKp30 (NCR3) and high levels of NKp30 expression were detected (94.6%, Fig. 1A). For FACS surface staining the cells were labeled with the following anti- However, IL-2 is known to induce upregulation of NKp30 on NK human Abs purchased from BioLegend unless stated otherwise: CD3 (SK7, BD Biosciences), CD19 (SJ25C1, BD Biosciences), CD123 (FAB301C, cells, and its presence in the culture media could potentially induce R&D Systems), CD11b (DCIS1/18), CD11c (BU15, Abcam), CD8 (RPA- upregulation of NKp30 by ILC2 (26, 27). Therefore to evaluate the 2 T8), FcεRI (AER-37 [CRA-1]), CD14 (MwP9, BD Biosciences), CD4 expression of this receptor ex vivo, lineage (CD3, CD4, CD8, (MEM-241), CD45 (H130), ICOS (C398.4A), CD56 (B159), CRTH2 CD14, CD19, CD56, IL3R, FcεRI, CD11b, CD11c) cells that ex- (BM16, Miltenyi Biotec), IL-7Ra (A019D5), Live/Dead violet (L34955, press CD45, CD127 (IL-7Ra), and CRTH2 from peripheral blood Invitrogen), NKp30 (clone AF29-4D12), NKp30 blocking Ab (clone 210845, 32/36 6 R&D Systems; AF29-4D12, Miltenyi Biotec), phospho-IkBa (Ser ; Cell of healthy donors were analyzed and 52.4 11.5% of human ILC2 Signaling Technology, 9246), anti–B7-H6 Ab (ab121794), B7-H6 blocking express NKp30 on their surface ex vivo (n = 7, Fig. 1B, with gating Ab (17BL.3), CD68 (Y1/82A), Siglec-8 (7C9), and CD16 (3G8). in Supplemental Fig. 1A; the expression of NKp30 on NK cells is Quantitative RT-PCR shown as a reference). Supplemental Fig. 1B shows the expression of NCRs on group 1, 2, and 3 ILC ex vivo. RNA extraction was performed using an RNeasy Plus mini (Qiagen, 74134) and TurboCapture 96 mRNA kit (Qiagen, 72251). cDNAwas prepared ILC2 predominantly express isoform c of NKp30 using an Omniscript RT kit. The following expression assays were purchased from Applied Biosystems: GATA3 (Hs00231122_m1), IL-5 Alternative splicing of exon 4 of the NCR3 gene gives rise to (Hs01548712_g1), IL-13 (Hs00174379_m1), GAPDH (Hs99999905_m1), different isoforms. In NK cells, cross-linking of each isoform IL-4 (Hs00174122_m1), RORa (HS00536545_m1), NKp30a (Hs01553310- induces a distinct signaling pathway and a different pattern of g1), NKp30b (Hs01561746-g1), and NKp30c (Hs01553311-g). cytokine production. NKp30a and NKp30b isoforms are classified B7-H6 plate bound assay as immunostimulatory whereas NKp30c is thought to be immu- noregulatory (3). To determine the expression of NKp30 isoforms Coat Corning Costar 9018 (Nunc MaxiSorp) were coated with indicated on ILC2, the mRNA levels of NKp30a, b, and c isoforms in concentration of recombinant human (rh)B7-H6 Fc chimera protein (R&D Systems, 7144-B7-050) or control protein overnight at 4˚C. ILC2 (5 3 104) cultured ILC2 were measured with quantitative PCR. The data were cultured on B7-H6 or isotype control–coated plates. After 24 h the were normalized to the ubiquitously expressed GAPDH house- supernatants were collected for cytokine analysis using ELISA or cytokine keeping gene, and ILC2 were found to predominantly express bead array. Where indicated the cells were preincubated with 10 mg/ml NKp30c (Fig. 1C). Taken together, these data raised the possibility Galectin-1 (CF 1152-GA-050/CF, Bio-Techne), Galectin-2 (1153-GA-050, Bio-Techne), and Galectin-3 (10289-HNAE-E-SIB, Stratech) for 1 h be- that NKp30 may play a role in ILC2 function. fore culture with plate-bound rhB7-H6 or cytokine-treated HaCaT cells. B7-H6 induces production of type 2 cytokines by ILC2 ELISA and ELISPOT B7-H6 is a self-ligand that is expressed on the surface of some Human IL-13 ELISA Ready-SET-Go! (88-7439-86), human IL-13 ELISA tumor cell lines (14). It belongs to the B7 family of receptors that DuoSet (DY213-05), and human IL-13 ELISpotBASIC (3470-2A) kits were encode ligands for CD28 and CTLA-4 (B7-1 and B7-2), but unlike The Journal of Immunology 3

FIGURE 1. ILC2 express natural cytotoxicity receptor NKp30. Cul- tured ILC2 (A)orexvivoNKcells and ILC2 (B) were examined for NKp30 expression by flow cytom- etry. Black and dotted lines are NKp30 staining of ILC2 and NK cells, respectively. Filled histogram is isotype control staining. ILC2 were gated as lineage2 (CD3, CD4, CD8, CD14, CD19, CD56, CD11b, CD11c, IL-3R, FcεRI), CD45+,IL-7Ra+, CRTH2+. NK cells were gated as CD32CD45+CD56+. Representative example stains of seven donors are shown. (C) Expression of different isoforms of NKp30 on ILC2 was measured by quantitative RT-PCR. mRNA levels were normalized to the housekeeping gene GAPDH (n =9). *p , 0.05.

other members of the B7 family it is thought to be selective for pression of BAT3, another NKp30 ligand, on the surface of the NKp30 and does not bind CD28 or other NCRs. It is composed of tumor cell lines (Fig. 2D). 2 Ig extracellular domains (a distal V-like and a proximal C-like Similar to its effect on ILC2 interaction with plate-bound rhB7- domain) with an adjacent phase 1 intron. Both the front and back H6, Galectin-3 had an inhibitory effect on NKp30-mediated in- b sheets of NKp30 bind to the V-like domain of B7-H6 via hy- teraction of ILC2 and K562 (Supplemental Fig 1C). Comparing drophobic interactions (14, 28). To investigate whether NKp30 K562-mediated activation of ILC2 with PGD2 and IL-33 activa- expressed on ILC2 mediates effector functions, ILC2 were treated tion showed that K562 cells are as potent inducers of IL-13 ex- with a rhB7-H6 Fc chimeric protein, a selective NKp30 ligand. pression by ILC2 as are PGD2 and IL-33 (Fig. 2E). To investigate Cross-linking NKp30 receptor on ILC2 by plate-bound B7-H6 pro- whether IL-33 or PGD2 can modify NKp30, we stimulated ILC2 tein induced production of increased amounts of IL-13 (Fig. 2A). with increasing concentrations of IL-33 and PGD2. No significant Galectin-3 is a b-galactosidase–binding protein expressed on the change was observed in the expression of NKp30 isoforms on surface of tumor cells and promotes proliferation and metastasis. ILC2 following stimulation with IL-33 and PGD2 (Supplemental Galectin-3 interacts with N-glycosylated sites (Asn42,Asn121)of Fig. 2A). To examine the effect of NKp30-mediated activation of NKp30 ectodomain and interferes with ligand binding (29). Incu- ILC2 on the expression of cytokine receptors and CRTH2, we bation of ILC2 for 1 h with soluble NKp30-blocking ligand, incubated ILC2 with plate-bound B7-H6 and isolated mRNA at Galectin-3, before performing the B7-H6 plate-bound assay re- various time points. Three hours after incubation with B7-H6 the duced the expression of IL-13 (Fig. 2B) whereas it did not affect mRNA levels encoding the IL-33 receptor (ST-2) and PGD2 re- PGD2 stimulation, which is mediated through CRTH2 (data not ceptor (CRTH2) were significantly reduced (Fig. 2F). IL-25 re- shown). The inhibitory effect of Galectin-3 was not observed with ceptor (IL-17RB) expression was moderately reduced. Following Galectin-1 and Galectin-2 (Fig. 2B). Next, we screened a panel of initial downregulation, the ST2 mRNA level increased above the tumor cell lines for their ability to activate ILC2, including cells basal level. To test whether K562 also induced expression of other with differential expression of HLA class I molecules to investi- cytokines, we used multiplex cytokine analysis. Activation of gate whether HLA may influence ILC2 function through interac- ILC2 by K562 cells induced production of IL-2, IL-3, IL-4, IL-5, tion with nonpolymorphic HLA-binding receptors. The human B IL-13, IL-8, and GM-CSF (Fig. 3A) but did not increase pro- lymphoblastoid cell line .221 lacks expression of surface class I. duction of IFN-g and IL-10 (data not shown). Analysis of the level Native .221 cells or .221 cells transfected with specific HLA of these cytokines in the blister fluid of skin of patients with AD variants did not induce production of IL-13 by ILC2 (Fig. 2C). In confirmed the biological significance of the levels of produced contrast, K562 cells (HLAlow myelogenous leukemia line), THP-1 cytokines by activated ILC2 (Supplemental Fig. 2B). cells (monocytic leukemia line), and Jurkat cells (T cell leukemia To confirm that the ILC2 activation is mediated by NKp30, we line) induced IL-13 expression by ILC2 (Fig. 2C). Further analysis incubated the cells with increasing concentrations of NKp30- showed that these cell lines all express B7-H6 on their cell sur- specific Ab for 1 h before culture with the K562 cell line. Block- face, whereas the .221 cell line was negative. There was no ex- ing NKp30 reduced the K562-induced cytokine production in a 4 ILC2 EXPRESS FUNCTIONAL NKp30 RECEPTOR

AB

C D

E

F

FIGURE 2. NKp30 ligand B7-H6 activates ILC2. (A) IL-13 production by ILC2 was measured by ELISA after 24 h of culture with increasing con- centrations of rhB7-H6 or protein control (n = 5). Statistical comparisons were made compared with ILC2 alone. (B) IL-13 production by ILC2 was measured by ELISA 4 h after culture with 5 mg/ml plate-bound B7-H6 with and without 1 h preincubation with soluble Galectin-1 (10 mg/ml), Galectin-2 (10 mg/ml), and Galectin-3 (10 mg/ml) (n = 4). (C) ILC2 were cultured with tumor cell lines for 24 h and production of IL-13 cytokine was measured by ELISA (n = 5). Statistical comparisons were made compared with ILC2 alone control. (D) Flow cytometry was used to examine expression of B7-H6 and BAT3 protein on the surface of K562, THP-1, .221, and Jurkat tumor cell lines. (E) IL-13 production by ILC2 was measured by (Figure legend continues) The Journal of Immunology 5 dose-dependent manner (Fig. 3A, Supplemental Fig. 2C). Incu- However B7-H6 is known to be upregulated under inflammatory bating K562 cells with B7-H6–blocking Ab (30) for 1 h before conditions (32). Therefore, we incubated the HaCaT cell line with culturing with ILC2 significantly inhibited production of IL-13 IFN-g, TNF-a, epithelial cytokines, mast cell products, and type 2 (Fig. 3B). cytokines. NKp30-mediated activation of ILC2 is contact-dependent, as Consistent with earlier reports, the proinflammatory cytokines supernatant from cultured tumor cell lines could not activate ILC2 IFN-g and TNF-a induced significant upregulation of B7-H6. (Fig. 3C) and indeed 24-plex and 7-plex analysis of K562 su- Interestingly, type 2 cytokines IL-13 and IL-4 also significantly pernatant did not reveal any known ILC2-stimulating cytokines upregulated B7-H6, whereas epithelial cytokines (IL-25, IL-33, or chemokines (ILC2-stimulating cytokines are shown in and TSLP) and the mast cell derivative PGD2 did not signifi- Supplemental Fig. 2D). In fact, ILC2 produced a similar pattern of cantly upregulate cell surface expression of B7-H6 on HaCaT cells cytokines either by K562-mediated cross-linking of NKp30 re- (Fig. 4C). We next treated HaCaT cells with increasing concen- ceptor or plate-bound rhB7-H6 activation, but rhB7-H6 provided a trations of IFN-g or IL-13 and then washed, trypsinized, counted, stronger signal (Supplemental Fig. 2E). and subsequently cultured the cells with ILC2 for 24 h. The ability To confirm the NKp30-mediated rapid effector functions of ILC2 of ILC2 to produce IL-13 was correlated with the dose of IFN-g ex vivo, we incubated freshly isolated ILC2 from the blood of or IL-13 used to treat HaCaT cells (Fig. 4D). Interestingly, healthy donors with the K562 tumor cell line and showed that K562 preincubation with soluble Galectin-3 for 1 h before culturing alone does not produce IL-13, but it can indeed activate ILC2 and with IL-13– and IFN-g–treated HaCaT cells reduced the ex- induce their production of IL-13 (Fig. 3D). pression of IL-13 (Fig. 4E). Galectin-3 is expressed by myeloid- As discussed above, NK cells express different isoforms of derived cells (Ref. 33 and Supplemental Fig. 3A) at similar NKp30, whereas ILC2 mainly express the NKp30c isoform. To levels in healthy skin and lesional biopsies of AD patients as compare the cytokine production profile of activated NK cells and shown by objective analysis of Galectin-3 staining of five donors ILC2, we incubated both cell populations with rhB7-H6. Upon (Supplemental Fig 3C). interaction with B7-H6, NK cells produced significant amounts of Rapid NKp30 mediated activation of ILC2 triggers the IFN-g, TNF-a, and IL-10 whereas ILC2 did not. ILC2 mainly canonical pathway of NF-kB activation produced type 2 cytokines, IL-13 and IL-5. Although NK cells also expressed these cytokines, the ability of ILC2 in producing All NKp30 isoform monomers have -charged residues in type 2 cytokines was significantly higher (Fig. 3E). their transmembrane regions that couple with aspartate amino acids Therefore, NKp30 expressed on ILC2 is functionally active and in CD3z homodimers or CD3z/FcRg heterodimers by a salt bridge mediates interaction between ILC2 and immortal laboratory tumor (34). CD3z is an ITAM-bearing adaptor molecule essential for cell lines, suggesting ILC2 may have a role in the interaction with transmitting downstream signaling (14). Isoform b is constantly malignant or stressed host cells. associated with CD3z whereas isoform c shows a weaker inter- action. NKp30 cross-linking triggers the canonical pathway of B7-H6 is expressed on basal epidermis in normal tissue and NF-kB activation. NF-kB dimers are bound to the inhibitory suprabasal epidermis in lesional skin biopsies of AD patients protein IkB. The IkB kinase complex (IKK) consists of IKKa, Several studies have reported the expression of B7-H6 on tumor cell IKKb, and regulatory protein NEMO and phosphorylates IkB. lines. To investigate whether B7-H6 is also expressed in nonma- Phosphorylated IkB undergoes proteasomal degradation that re- lignant tissue, sections of healthy skin and lesional skin biopsies of leases NF-kB to translocate to the nucleus (35). To study the patients with AD were evaluated. Interestingly, we found expression signaling pathway involved in NKp30-mediated ILC2 activation, of B7-H6 protein in healthy skin tissue, but this was confined to intracellular FACS analysis of these cells confirmed the increase the basal layer of epidermis. In contrast, lesional skin biopsies in phosphorylation of IkB (Fig. 5A). To evaluate whether the from adult patients with AD showed B7-H6 expression increase in production of cytokines was due to the release of throughout the suprabasal layers of the epidermis and myeloid- preformed cytokines or to an increase in , we derived populations in the dermis (Fig. 4A, Supplemental Fig. incubated ILC2 with plate-bound B7-H6 protein for up to 24 h, 3A). Objective quantification of immunohistochemistry staining which increased production of IL-13 protein (Fig. 5B) and in- of B7-H6 expression in skin biopsies of healthy controls and AD creased mRNA expression of IL-13, IL-4, IL-5, and AREG and patients is shown in Fig. 4B. enhanced expression of GATA-3 (Fig. 5C) but did not affect RORa It is therefore plausible to speculate that widespread expression expression (data not shown). of B7-H6 in the skin of patients with AD can lead to NKp30- mediated activation of ILC2. This finding is compatible with our Discussion earlier observation that ILC2 resident in the skin of patients with In 1999, Moretta and colleagues (36) identified natural cyto- AD show an activated phenotype (6). The presence of ILC2 in the toxicity receptors with activating properties that can trigger an epidermis (Ref. 31) and Supplemental Fig. 3B) favors their mi- immune response on recognition of cognate cellular and viral gration into or retention in the epidermis and supports their pos- ligands and therefore play an important role in NK cell antitumor sible interaction with keratinocytes. and antiviral cytotoxicity. Although NCRs have been detected on To further understand whether the widespread expression of B7- type 1 and type 3 innate lymphoid cells, their specific functions H6 in patients’ skin contributes to the NKp30-mediated activation have not yet been fully elucidated (37, 38). In the present study, of ILC2, the effect of the immortalized human keratinocyte cell we identified and characterized NKp30 expression on group 2 line (HaCaT) on ILC2 activation was tested. A relatively low innate lymphoid cells, showing an important role in induction of expression of B7-H6 (Fig. 4C) was observed in HaCaT cells. type 2 cytokines.

ELISA after 4 h stimulation with 10, 30, and 100 ng/ml IL-33, 10, 30, and 100 nM PGD2,and 1:1, 2:1, and 3:1 ILC2/K562. (F) mRNA expression of ST2 (IL- 33R), IL-17RB (IL-25R), and CRTH2 following culture with rhB7-H6 for indicated times. Statistical comparisons were made compared with ILC2 alone control. *p , 0.05, **p , 0.01, ****p , 0.0001. 6 ILC2 EXPRESS FUNCTIONAL NKp30 RECEPTOR

FIGURE 3. Cross-linking of NKp30 receptor on ILC2 increases production of type 2 cytokines. (A) Cytokine production was measured by cytokine multiplex analysis of ILC2 supernatant following 4 h incubation with K562 tumor cell in the absence or presence of anti-NKp30 Ab. (B) Production of IL-13 by ILC2 after incubation with K562 for 4 h with and without preincubation with B7-H6-blocking or isotype control Ab ( 5mg/ml). (C) IL-13 production by ILC2 was measured using ELISA following 24 h incubation with K562 cells or supernatant from K562 cells (n =3).(D) Freshly isolated ILC2 were cocultured with K562 tumor cells for 24 h and IL-13 cytokine production was measured by ELISPOT (SFU, spot-forming units; figure is representative of three experiments). (E) Cytokine analysis of supernatant of NK cells and ILC2 following 4 h of incubation with B7-H6. *p , 0.05, **p , 0.01, ****p , 0.0001. The Journal of Immunology 7

FIGURE 4. Higher expression of B7-H6 is found in lesional skin biopsies of AD patients. (A) B7-H6 expression in healthy control (n = 11) and AD lesions (n = 6) was examined by immunohistochemistry using anti–human B7-H6 Ab. Original magnification 320 (inset, original magnification 3100). (B) Quantification of B7-H6 immunohistochemistry signal using the Fiji version of ImageJ in healthy control (n = 11) and atopic patients skin lesions (n = 6). (C) The expression of B7-H6 on the keratinocyte cell line (HaCaT) was evaluated after 72 h of incubation with IFN-g (300 U/ml), TNF-a (100 ng/ml), IL-4

(100 ng/ml), IL-13 (100 ng/ml), IL-25 (100 ng/ml), IL-33 (100 ng/ml), TSLP (100 ng/ml), and PGD2 (100 nM) by flow (Figure legend continues) 8 ILC2 EXPRESS FUNCTIONAL NKp30 RECEPTOR

FIGURE 5. NKp30-mediated activation of ILC2 induces the NF-kB signaling pathway. (A) The expression of phosphorylated IkB in ILC2 was evaluated by flow cytometry after 4 h of culture with K562 cells (data representative of four experiments). (B) IL-13 production by ILC2 in the absence or presence of plate-bound B7-H6 was measured by ELISA, and (C) mRNA expression was quantified using RT-PCR following incubation with plate-bound B7-H6 (n = 2). Statistical comparisons were made with ILC2 alone control. *p , 0.05, **p , 0.01, ***p , 0.001.

NKp30 protein is encoded by the NCR3 gene located in the Another difficulty that limits our understanding of the role of highly polymorphic telomeric end of the class III region (39). NK NKp30 is poor characterization of its ligands and their distribution cell subpopulations can express all three of the most common on normal tissues under homeostatic and inflammatory conditions. isoforms (a, b, and c), and the relative contribution of each re- Two self-ligands have been identified to bind to NKp30: a novel ceptor depends on the expression levels on the cell surface. In- member of B7 family receptors, B7-H6, and a largely intracellular terestingly, ILC2 showed predominant expression of the putative protein, BAT3, which can be expressed on the cell surface under immunomodulatory splice variant, NKp30c, and lower expression certain conditions (14, 20, 28, 51). Consistent with activation of of NKp30a and b isoforms. We show that NKp30 engagement on NKp30-expressing cells through B7-H6, our data demonstrated ILC2 augments the production of type 2 cytokines IL-4, IL-5, IL-13, that plate-bound rhB7-H6 can induce production of IL-13 by ILC2 and GM-CSF as well as other inflammatory cytokines such as IL-2, in a dose-dependent manner. Interestingly, incubation of multiple IL-3, and IL-8. These cytokines have a crucial role in allergic and tumor cell lines with ILC2 showed that cell lines that activated inflammatory conditions, and encoding many of these cyto- ILC2 and induced production of IL-13, such as THP1, K562, and kines are located at the same locus 5q31–33 (40). Jurkat, lacked cell surface staining of BAT3 but maintained B7-H6 Proliferation and differentiation of CD34+ progenitor cells into ba- expression, suggesting that B7-H6 is the main NKp30 ligand be- sophils and mast cells are highly dependent on IL-3. IL-3 also ing recognized on the laboratory tumor cell lines. Indeed, IL-13 regulates dendritic cell differentiation from monocytes (41, 42). IL-8 production was diminished upon prior incubation of ILC2 with is a potent neutrophil chemoattractant (43, 44) and contributes to increasing concentrations of NKp30-blocking Ab or the soluble allergy and severe asthma (45, 46). GM-CSF has been increasingly NKp30-blocking ligand, Galectin-3. We showed that the IL-13 appreciated as a type 2 cytokine (47, 48) and a neutrophil and eo- production occurs rapidly, consistent with an early innate im- sinophil survival factor (49, 50). mune function to respond during the initial phases of an immune

cytometry and compared with isotype control staining. (D) Following 72 h of incubation with increasing concentrations of IFN-g or IL-13, HaCaT cells were washed, trypsinized, and equal numbers were cultured with ILC2 for 24 h. The production of IL-13 was measured in the supernatant by ELISA (n =3). (E) IL-13 production by ILC2 was measured by ELISA after incubation with 10 mg/ml Galectin-3 for 1 h prior to culture with IFN-g–treated (300 U/ml) or IL-13–treated (100 ng/ml) HaCaT cells. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. The Journal of Immunology 9

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