Inflammatory bowel diseases (IBDs) comprise Crohn s

GASTROENTEROLOGY 2011;141:217 226 Activation of the Receptor NKG2D Leads to Production of Th17 Cytokines in CD4 T Cells of Patients With Crohn s Disease BENJAMIN PARIENTE,* IULIA MOCAN,* MATTHIEU CAMUS,* CHARLES ANTOINE DUTERTRE, JULIEN ETTERSPERGER,* PIERRE CATTAN, JEAN MARC GORNET, NICOLAS DULPHY,* DOMINIQUE CHARRON,* MARC LÉMANN, ANTOINE TOUBERT,* and MATTHIEU ALLEZ*, *INSERM, Equipe AVENIR U940 Hôpital Saint-Louis, Paris; INSERM, UMRS 872, Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, Paris; and Departments of Surgery and Gastroenterology, Hôpital Saint-Louis, Université Denis-Diderot Paris 7, Paris, France BACKGROUND & AIMS: The natural killer group 2 member D (NKG2D) is a stimulatory receptor expressed on a subset of mucosal and peripheral CD4 T cells in patients with Crohn s disease (CD) and other inflammatory diseases. Ligand activation of NKG2D in patients induces CD4 T cells to release T-helper (Th) 1 cytokines and become cytotoxic. We investigated the Th17 cytokines produced by T cells that express NKG2D in blood and intestinal mucosa samples from patients with CD. METHODS: We isolated CD4 T cells from peripheral blood and lamina propria samples of patients with CD or ulcerative colitis (UC) and healthy individuals (controls). We analyzed the phenotype and functions of the CD4 NKG2D T cells and the cytokines they produce in response to NKG2D stimulation. RESULTS: In patients with CD, CD4 T cells that express NKG2D produced high levels of interleukin (IL)-17 and IL-22 and expressed high levels of CCR6, the IL-23 receptor, CD161, and RORC (a transcription factor that regulates expression of Th17 cytokines). CD4 T cells that produced IL-17 expressed high levels of NKG2D and CD161. Costimulation of NKG2D and the T-cell receptor (TCR) significantly increased production of IL-17 and tumor necrosis factor by CD4 T cells, compared with activation of only the TCR. CD4 NKG2D T cells also responded to Th17 polarization. CONCLUSIONS: NKG2D is a functional marker of CD4 T cells that produce IL-17 in patients with CD, via costimulation of the TCR and NKG2D. Reagents developed to block NKG2D might reduce gastrointestinal inflammation in patients with CD. Keywords: Intestine; T-cell Signaling; Immune Response; Inflammatory Bowel Diseases. Inflammatory bowel diseases (IBDs) comprise Crohn s disease (CD) and ulcerative colitis (UC), which are both characterized by uncontrolled immune responses toward the intestinal flora. 1 The pathogenesis of IBD remains elusive but is clearly influenced by genetic and environmental factors. 1 3 Abnormal innate immune responses have been described in CD, including specific defects in the epithelium and in macrophages. 2,4 However, the inflammatory process is T-cell driven, and chronic intestinal inflammation may be due either to impaired regulatory T-cell activity or excessive effector T-cell function. 5 7 Until recently, CD was described as a T-helper (Th) 1 disease, characterized by high interferon (IFN)-, tumor necrosis factor (TNF)-, and interleukin (IL)-12 levels. 8 The latest data highlighted the role of the Th17 subset in CD, as well as their cytokine signature IL-17 (IL-17A). 9,10 A number of murine models of experimental colitis showed the predominant role of Th17 response. Indeed, inhibition of the Th17 but not of the Th1 response was effective in prevention of colitis. 10 In addition, transfer of Th17 cells induced more severe disease than Th1 cells did in SCID mice. 11 Th17 cells have characteristic features, like elevated expression of IL-23 receptor (IL-23R), CCR6 integrin, and ROR t transcription factor in mice or RORC in humans. 12 The IL-23 cytokine is predominantly involved in Th17 development of previously activated cells. Moreover, the IL-23R gene has been identified as an IBD susceptibility gene, with an uncommon coding variant that confers strong protection against CD. 13 Also in CD, IL-22 has been shown to be elevated in the inflamed mucosa and exhibits proinflammatory properties. 14 Notably, Th17 cells have been reported to be the main producers of IL-22. 15 Recent data have shown an abnormal expression of natural killer (NK) receptors on T cells in patients with CD. 16,17 Kleinschek et al have described a subset of CD4 T cells involved in chronic intestinal inflammation in CD that carry the C-type lectin-like NK receptor CD161. 18 These CD4 CD161 T cells display an activated Th17 phenotype, with increased expression of IL-17, CCR6, and IL-23R. Circulating CD161 Th17 cells are imprinted for gut homing, as indicated by high levels of integrin 7 expression. We have recently identified a subset of effector CD4 T cells mediating inflammatory response in CD and expressing the NK activating receptor natural killer group 2 member D (NKG2D). 16 CD4 NKG2D T cells are functionally active through interactions with NKG2D ligands. The nonclassic major histocompatibility complex class 1 like molecules Abbreviations used in this paper: HC, healthy controls; IFN, interferon; IL, interleukin; LP, lamina propria; LPL, lamina propria lymphocytes; NK, natural killer; NKG2D, natural killer group 2 member D; PB, peripheral blood; PBL, peripheral blood lymphocytes; PMA, phorbol myristate acetate; Q-RT-PCR, quantitative real-time reverse-transcription polymerase chain reaction; TCR, T-cell receptor; Th, T-helper; TNF, tumor necrosis factor. 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2011.03.061

218 PARIENTE ET AL GASTROENTEROLOGY Vol. 141, No. 1 MICA and MICB, together with ULBPs (UL16 binding proteins), constitute major ligands of NKG2D in humans. Their increased expression has been correlated with cellular stress 19,20 and with a subsequent NKG2D-driven CD4 T- cell cytotoxicity in autoimmune diseases. 16,21 Nevertheless, their functional implication in CD deserves further studies. CD4 NKG2D T cells expand in the lamina propria (LP) and the peripheral blood (PB) of patients with CD, but not in patients with UC or in healthy controls (HCs). CD4 NKG2D T cells exhibit specific cytotoxic activity and produce IFN- in the presence of MICA-bearing cells. 16 The implication of CD4 NKG2D T cells in gut inflammation has been further shown in a murine model of transfer-induced colitis. 22,23 In these studies, administration of a specific NKG2D blocking antibody decreased NKG2D expression on CD4 T cells and attenuated the development of colitis, highlighting NKG2D as a possible therapeutic target in IBD. The purpose of our study was to further characterize the functional properties of CD4 NKG2D T cells and their relationship with Th17 cells as well as CD4 T cells expressing CD161. We show that CD4 NKG2D T cells represent a major source of IL-17 in CD and have typical features of Th17 cells, including high CD161 expression. In patients with CD, IL-17 producing CD4 T cells preferentially expressed NKG2D and CD161 at their surface, with NKG2D being more specific for this population. NKG2D stimulation by its ligands significantly enhances the IL-17 secretion triggered by the T-cell receptor (TCR). Altogether, these results highlight the functional plasticity of CD4 T-cell subsets in CD and identify CD4 NKG2D CD161 cells as a major proinflammatory T-cell population. Targeting NKG2D might have a significant impact on Th17 pathways in CD and gut inflammation. Patients and Methods Patients With IBD and Controls Forty-nine patients with moderate to severe active CD, 12 patients with active UC, and 15 HCs were included in this retrospective study. All of the patients were hospitalized in the Department of Gastroenterology at Hôpital Saint-Louis in Paris, France. Patient characteristics are given in Table 1. Among the 49 patients with CD, 20 had ileal (n 17) or colonic resections (n 3). This study was approved by the Ethical Committee of Hôpital Saint- Louis (IRB 00003835), and all subjects gave written informed consent. Isolation of Intestinal and Blood Lymphocytes LP lymphocytes (LPLs) and PB lymphocytes (PBLs) were isolated with the same method as we previously described. 16 Techniques are detailed in Supplementary Materials and Methods. Multiparametric Flow Cytometry For surface staining, lymphocytes (PBLs, LPLs) were resuspended in fluorescence-activated cell sorter buffer (phosphate-buffered saline, 5% fetal calf serum) and incubated for 30 Table 1. Clinical Characteristics of Patients With CD and UC Patients With Patients CD With UC (n 49) (n 12) Age (y) a 34.9 12.5 37 20.5 Sex (M/F) 15/34 7/10 Location of lesions Ileum only 18 Ileum and colon 18 Colon 12 12 Anus only 16 Indication of surgical resection (n 20) Small bowel obstruction 15 Abscess 5 Active disease Harvey Bradshow a 8.7 5.5 Truelove Witts b 3 (1 4) C-reactive protein (mg/l) b 49.2 (3 170) 11 (3 39) Ongoing treatments Corticosteroids 8 1 Azathioprine, 6-mercaptopurine, 15 6 methotrexate Infliximab, Adalimumab 5 1 a Mean SD. b Median (extremes). minutes at 4 C with different mixtures of antibodies. The surface antibody mixtures included antibodies conjugated with fluorescein isothiocyanate, phycoerythrin, phycoerythrin-cy5, phycoerythrin-cy7, allophycocyanin, allophycocyanin-h7, Pacific Blue, or AmCyan and directed against CD3, CD8, CD4, CD161 (BD Biosciences, Le-Pont-De-Claix, France), NKG2D (Beckman Coulter, Miami, FL), IL-23R and CCR6 (R&D Systems, Abingdon, England), and relevant isotype controls (R&D Systems, Abingdon, England). For functional studies, lymphocytes (PBLs, LPLs) were stimulated for 4 hours with 25 ng/ml phorbol myristate acetate (PMA) and 1 g/ml ionomycin and incubated with 5 g/ml brefeldin A before intracellular staining. Cells were stained with CD3, CD4, CD8, NKG2D, and CD161 as shown previously. Cells were subsequently permeabilized in a saline buffer containing 0.1% saponin for 5 minutes. Cells were then incubated with fluorescein isothiocyanate conjugated antibodies directed against IL-17A (Clinisciences, Montrouge, France), Alexa 647 anti IFN- (BD Biosciences) and allophycocyanin anti IL-22 (R&D Systems), and relevant isotype controls. Eight-color analyses were performed using BD FACS Canto-II and FACS Diva software (BD Biosciences). Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction Analysis LPLs from 4 patients with active CD were stained for surface CD3, CD4, and NKG2D, and different T-cell populations (CD4, CD4 NKG2D, and CD4 NKG2D ) were sorted using a BD FACSAria II cell sorter (Becton Dickinson, San Jose, CA). Total RNA from purified populations was extracted using a QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany). Quantitative real-time reverse-transcription polymerase chain

July 2011 NKG2D DRIVES IL-17 RELEASE IN CD 219 reaction (Q-RT-PCR) was performed with 40 ng of each RNA using the TaqMan RNA-to-Ct 1-Step Kit (Applied Biosystems, Foster City, CA) and TaqMan Gene Expression Assays (Applied Biosystems). The primers were RORC (Hs01076112_m1) and IL-17A (Hs00174383_m1). Relative quantification was achieved by normalizing each result to the quantification of CD3e RNA obtained with the following primers and probe: CD3_S: CCC CAG AGG AAG CAA ACC A, CD3_AS: CTT GCT CCA GTA GTA AAC CAG CAG, and CD3_P: VIC-TGT GTG AGA ACT GCA TGG A-MGBNFQ. Costimulation of TCR and NKG2D on LPLs and PBLs From Patients With CD Fresh PBLs or LPLs from 7 patients with CD were cultured with murine mastocytoma cell line P815 alone or bearing one of the NKG2D ligands (MICA, MICB, ULBP1, ULBP2, or ULBP3), coated with agonist CD3 and/or agonist CD28 antibodies or their correspondent isotypes. Thus, 2 10 5 of each of the 5 different P815 cell lines were first incubated in 200 L of culture medium with 1 g/ml anti-cd3 or immunoglobulin G1 or 5 g/ml anti-cd28 for 15 minutes at 37 C. Lymphocytes were added to P815 cells at a 1:1 ratio for 5 hours and in the presence of 5 g/ml brefeldin A for the last 4 hours. Allophycocyanin CD107a specific antibody was added at the beginning of the culture. Cells were subsequently collected, and detection of IL-17A, IFN-, and TNF- producing cells was performed as described previously. Th1 and Th17 Cytokine Polarization Tests on LPLs From Patients With CD LP mononuclear cells freshly isolated from the intestinal mucosa of 5 patients with active CD and 5 HCs were cultured with anti-cd2/anti-cd3/anti-cd28 activation beads (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Cocktails of IL-15 (15 ng/ml), IL-12 (2 ng/ml) for Th1, and IL-1 (50 ng/ml) and IL-23 (10 ng/ml) for Th17 polarization were added. After 3 days of culture, cells were stimulated for 4 hours with 25 ng/ml PMA and 1 g/ml ionomycin and incubated with 5 g/ml brefeldin A. Cells were subsequently stained for surface markers and cytokine secretion as described previously. Statistical Analysis Flow cytometry data are expressed as mean percentages of expression SD. Differences between independent groups were traced with the use of the Mann Whitney exact test for nonnormally distributed values. The correlation between the expressions of different markers was calculated using Spearman s test ( ). A P value of.05 was considered significant. Results CD4 NKG2D T Cells Have a Th17 Pattern in CD The presence of peripheral IL-17 producing T cells was evaluated by flow cytometry in 42 patients with CD, 9 patients with UC, and 10 HCs. The percentage of PB CD4 T cells producing IL-17 was significantly higher in patients with CD (3.4% 2.9%) in comparison with patients with UC (1% 1.1%, P.007) and HCs (0.8% 0.4%, P.004) (Supplementary Figure 1A and B). Also, the proportion of CD4 T cells producing IL-17 was higher in the LP (11.3% 7.2%) than in the PB in patients with CD (3.8% 2.3%, P.002, n 16) (Figure 1A). Notably, IL-17 intracellular staining was low in CD8 LPLs (1.9% 0.9%, n 16). Analysis of IL-17 production by intracellular staining was correlated with phenotypic characteristics of mucosal and peripheral T cells in CD. NKG2D expression on PB CD4 T cells was significantly increased in patients with CD (4.2% 2.7%) as compared with patients with UC (2.7% 1.1%, P.015) and HCs (2.1% 1.3%, P.001) (Supplementary Figure 2). In patients with CD, NKG2D expression on LP CD4 T cells reached 5.5% 2.5%. In CD, LP CD4 NKG2D T cells were highly positive for IL-17 intracellular staining (34.5% 25.3%) and expressed significantly higher levels of IL-17 than their CD4 NKG2D counterparts (8.8% 6%, P.001, n 16) (Figure 1B and D). Also, PB CD4 NKG2D T cells were highly positive for IL-17 (22.1% 22.2%) as compared with CD4 NKG2D T cells (2.2% 2.1%, P.0001, n 42) (Figure 1C and E). Moreover, the expression of NKG2D on PB CD4 T cells was correlated with IL-17 production (Spearman correlation test r 0.39, P.01, n 42). We investigated, by Q-RT-PCR, whether the enhanced secretion of the IL-17 cytokine in the gated CD4 NKG2D population was reflected by the IL-17 gene expression in transcripts from the purified populations. As shown in Figure 1F, messenger RNAs of IL-17 were expressed preferentially in LP CD4 NKG2D T cells compared with the CD4 NKG2D subset (P.02). We were also interested in whether the enhanced secretion of IL-17 in the gated LP CD4 NKG2D population was related to the expression of RORC, a required transcription factor for Th17 differentiation. Q-RT-PCR analysis in sorted cells revealed that LP CD4 NKG2D T cells expressed significantly higher messenger RNA of RORC than their CD4 NKG2D counterparts (P.02, Figure 1F). To complete the analysis of the Th17 profile of the CD4 NKG2D population, we analyzed the expression of different receptors known to be usually expressed by Th17 cells. 12 CCR6 integrin expression was preferentially expressed by LP CD4 NKG2D T cells (14.8% 7.8%) as compared with CD4 NKG2D T cells (3.9% 2.2%, P.0001, n 12) and CD8 NKG2D T cells (2.3% 1.3%, P.0001) (Figure 2A). PB CD4 NKG2D lymphocytes also expressed higher CCR6 staining (19.3% 12.8%) as compared with CD4 NKG2D (7.4% 7%, P.0001, n 27) and CD8 NKG2D T cells (3.8% 6.2%, P.0001). Furthermore, LP CD4 NKG2D T cells showed a higher frequency for IL-23R staining (21% 11.7%) than their negative counterparts (3.4% 3.3%, P.001) and CD8 NKG2D cells (7.5% 5%, P.001) (Figure 2B). IL-23R expression was also particularly increased on

220 PARIENTE ET AL GASTROENTEROLOGY Vol. 141, No. 1 Figure 1. CD4 NKG2D T cells are a major source of IL-17 in CD. (A D) PBLs (n 42) and LPLs (n 16) were isolated from patients with active CD and stimulated with PMA/ionomycin. Intracellular staining for IL-17 was analyzed by flow cytometry on gated CD3 CD4, CD4 NKG2D, and CD4 NKG2D T-cell populations. Scattergrams show percentages of overall IL-17 positive cells among (A) LP and PB CD4 T cells (n 13), (B) LP CD4 NKG2D and CD4 NKG2D T cells (n 16), and (C) PB CD4 NKG2D and CD4 NKG2D T cells (n 42). Median values are indicated by a bar. *P.05, **P.005. (D and E) These figures represent flow cytometric analysis of IL-17 intracellular staining in (D) LP and (E) PB CD4 NKG2D (right dot plot) and CD4 NKG2D (left dot plot) T cells in 2 patients. Plots are representative of (D) 16 and (E) 42 independent patients with CD. Isotype control antibody staining was used for gate settings. (F) LP mononuclear cells from patients with active CD (n 4) were fluorescence-activated cell sorted for CD4, CD4 NKG2D, and CD4 NKG2D T cells, and gene expression was profiled. RORC (white bars) and IL-17A (black bars) gene expression was assessed by Q-RT-PCR and normalized to CD3e RNA. *P.05.

July 2011 NKG2D DRIVES IL-17 RELEASE IN CD 221 the main characteristics of mucosal effector T lymphocytes in CD is the presence of some receptors also expressed on NK cells, such as NKG2D or CD161. 16,18 We show that CD161 expression was significantly increased on PB CD4 T cells isolated from patients with CD (21.3% 9.8%) as compared with patients with UC (12.5% 6.2%, P.006) and HCs (6.9% 3%, P.011) (Figure 2D). CD161 expression was also significantly increased on CD4 NKG2D as compared with CD4 NKG2D (P.0001) or CD8 NKG2D T cells (P.0001) (Figure 2E). Thus, we show that, in a human immune-mediated inflammatory disorder, CD4 NKG2D T cells are a major source of IL-17 and highly express CCR6, IL-23R, and CD161 as compared with CD4 NKG2D T cells. We next analyzed the coexpression of NKG2D and CD161 on IL-17 producing T cells. There was a higher proportion of CD4 T cells expressing both NKG2D and CD161 among IL-17 positive as compared with IL-17 negative T cells (52.5% 23.7% vs 4.5% 3.2%, P.0001) (Figure 3A). The vast majority of IL-17 positive CD4 T cells expressed NKG2D (72.2% 10.1%) as well as CD161 (66.6% 22.1%) (Figure 3C). Of note, CD4 IL-17 negative T cells also significantly expressed CD161 (21.4% 7.5%), while the expression of NKG2D was less than 5% (Figure 3B). Thus, NKG2D appears more specific of IL- 17 producing CD4 T cells in CD and better defines this population than CD161 expression. Figure 2. CD4 NKG2D T cells exhibit Th17 and NK markers. Flow cytometry analysis of CCR6, IL-23R, CD161, and NKG2D expression was performed on (A and B) LPLs (n 12) and (C E) PBLs (n 26) isolated from patients with active CD. Scattergrams indicate percentages of (A) CCR6 and (B) IL-23R expression on CD3 CD4 NKG2D, CD4 NKG2D, and CD8 NKG2D LP T-cell populations. (C) This figure represents flow cytometric analysis of IL-23R staining in PB CD4 NKG2D and CD4 NKG2D T cells in one patient. The plot is representative of n 26 independent CD patients. Isotype control antibody staining was used for gate settings. (D) Graphics indicate proportions of PB CD4 T cells expressing CD161 in patients with CD (n 26) as compared with patients with UC (n 12) and HCs (n 10). (E) Scattergrams indicate percentages of CD161-positive cells among CD3 CD4 NKG2D, CD4 NKG2D, and CD8 NKG2D PB T-cell populations. Median values are indicated by a bar.*p.05, **P.005. the PB CD4 NKG2D T cells as compared with CD4 NKG2D or CD8 NKG2D T cells (both with P.0001) (Figure 2C). NKG2D Better Identifies the Th17 Subset Than CD161 in CD It has been previously shown that Th17 cells express CD161 in CD. 18 Separate studies showed that one of NKG2D Is Functionally Active Upon Ligand Interactions in CD We performed functional analysis targeting NKG2D by its different ligands transfected in a murine mastocytoma cell line P815, with or without a concomitant stimulation of the TCR. Functional assays of cytokine production (IL-17, IFN-, and TNF- ) and cytotoxic activity (CD107a) were performed using T cells from patients with CD stimulated by P815 cells alone or bearing NKG2D ligands (NKG2DL) (MICA, MICB, ULBP1, ULBP2, or ULBP3) and coated with an agonist anti-cd3 antibody at suboptimal concentrations or its corresponding isotype control. In CD4 T cells, stimulation through TCR alone or through both TCR and NKG2D (via interaction with MICA) significantly increased IL-17 (P.05), IFN- (P.05), and TNF- (P.005) production and induced little cytotoxicity as compared with the isotype control (Figure 4A C). Importantly, stimulation of both NKG2D and TCR led to a significant increase in IL-17 (P.05) and TNF- (P.05) production on CD4 T cells as compared with TCR activation alone (Figure 4A C). To assess the relative importance of NKG2D activation in IL-17 secretion in CD4 NKG2D T cells, we performed functional analysis targeting TCR and/or CD28. In CD4 NKG2D T cells, stimulation of both TCR and NKG2D (via interaction with MICA) significantly increased IL-17 production as compared with TCR alone or both TCR and CD28 (42.7% 13.3%, 27.1% 14.2%, and 19.8% 16.4%, respectively, both P.05) (Figure 4D). Notably, stimulation through both TCR and CD28 did

222 PARIENTE ET AL GASTROENTEROLOGY Vol. 141, No. 1 Figure 3. NKG2D expression identifies IL-17 producing cells. PBLs were stimulated with PMA/ionomycin before IL-17 intracellular staining. Analysis of IL-17 production was gated on CD4 T cells. (A) This figure is representative of analysis performed in 10 patients with active CD. NKG2D and CD161 expression in IL-17 negative (left dot plot) and IL-17 positive (right dot plot) CD4 T cells in one patient is shown. (B and C) Expressions of CD161 (gray bars) and NKG2D (black bars) on(b) CD4 IL-17 and (C) CD4 IL-17 lymphocytes from 10 patients with CD are plotted. not significantly increase IL-17 production as compared with TCR alone (P.8) (Figure 4D). In contrast, stimulation of both TCR and CD28 increased IFN- secretion in CD4 NK2GD T cells (data not shown). NKG2D stimulation through its different ligands expressed on the P815 cell line is different from patient to patient (maximal secretion with MICA in 2 patients, MICB in 3 patients, or ULBP2 in 2 patients) (Supplementary Figure 3). Nevertheless, stimulation through each NKG2D ligand and TCR induced higher cytokine production, respectively, to TCR alone. The analysis of the IL-17/TNF- costaining during the NKG2D-TCR costimulation of lymphocytes showed that all IL-17 secreting CD4 T cells were also TNF- positive (Figure 4E). Cell cytotoxicity (CD107a surface expression) was also increased by the NKG2D and TCR costimulation (Figure 4E). We could thus show that NKG2D activation with TCR costimulation specifically enhances cytokine secretion and cell cytotoxicity in CD4 lymphocytes in patients with CD. Th17 Polarization of CD4 NKG2D T Cells We have previously described that CD4 NKG2D T cells secrete Th1 cytokines, 16 and we show here that they also present a Th17 profile. It has already been shown that under polarizing conditions, Th17 cells can present Th1 functions. 24 We decided to test the behavior of LP CD4 NKG2D T cells under the Th1 and Th17 polarizing action of IL-12, IL-23, and IL-1 cytokines in the presence or absence of IL-15. As we have previously shown, IL-15 has a proliferative effect and sustained survival on CD4 NKG2D T cells. 16 Therefore, IL-15 stimulation was considered as our control condition. Under Th17 polarization, IL-17 secretion greatly differed between the 2 CD4 subsets. In LP CD4 NKG2D T cells, IL-17 production was significantly higher in the presence of IL-23 as compared with the IL-15 condition (12.2 4.1 and 8.7 2.9, respectively, P.05) (Figure 5). In contrast, under stimulation with IL-23, LP CD4 NKG2D T cells failed to induce significant levels of IL-17 as compared with the IL-15 condition (P.3) (Figure 5), underlining the unique responsiveness of CD4 NKG2D T cells to Th17-promoting signals. Likewise, IL-17 production was increased in the presence of IL-1 as compared with the IL-15 condition in the LP CD4 NKG2D subset but not in the CD4 NKG2D subset (Figure 5). Under Th1 polarization (IL-12 alone or in combination with IL-15), IFN- secretion was significantly increased as compared with the IL-15 condition alone in both LP CD4 NKG2D and CD4 NKG2D T-cell subsets (P.05) (data not shown). Our experiments indicate that the CD4 NKG2D population responds to Th1 conditions but is significantly more sensitive to Th17 polarization than CD4 NKG2D lymphocytes. As opposed to that in patients with CD, there was no significant difference in IL-17 expression on CD4 NKG2D T cells under IL-23 stimulation as compared with IL-15 stimulation in HCs (P.5). To confirm that NKG2D specifically stained Th17 cells in patients with CD as compared with HCs, we analyzed the expression of NKG2D on IL-17 producing CD4 T cells under IL-23

July 2011 NKG2D DRIVES IL-17 RELEASE IN CD 223 Figure 4. NKG2D costimulation significantly enhances IL-17 secretion triggered by the TCR in CD4 T cells. (A D) PBLs isolated from 7 patients with CD were cultured in the presence of P815 cell lines transfected or not with MICA coated with an immunoglobulin G1 antibody (isotype) or with an anti-cd3 agonist antibody at 1 g/ml concentration and/or with an anti-cd28 antibody at 5 g/ml concentration. Analysis of (A) IL-17, (B) IFN-, and (C) TNF- intracellular staining was gated on CD3 CD4 T cells. (D) Analysis of IL-17 intracellular staining was gated on CD3 CD4 NKG2D T cells. Histograms represent percentage of CD4 T cells expressing IL-17 under different stimulations. *P.05, **P.005. (E) In this experiment, PBLs of one patient were cultured in the presence of P815 cell lines transfected or not with ULBP2, coated with an IgG1 antibody (isotype) or with an anti-cd3 agonist antibody. IL-17 production versus IFN- and TNF- production and CD107a expression were analyzed in gated CD3 CD4 T cells. The dot plots are representative of 7 independent experiments.

224 PARIENTE ET AL GASTROENTEROLOGY Vol. 141, No. 1 The CD4 NKG2D Population Shows Functional Heterogeneity Ex Vivo Th17 (IL-17 positive, IFN- negative), Th22 (IL-17 negative, IL-22 positive, IFN- negative), and Th1 (IL-17 negative, IFN- positive) cells are detected in CD lesions and implicated in CD pathogenesis. 8,9,14 Considering single Th-positive cells, Th17, Th22, and Th1 represent 30% 17%, 11.5% 10%, and 12% 5%, respectively, of the LP CD4 NKG2D population. Annunziato et al have identified the presence of a Th1/ Th17 population in the mucosa of patients with CD. 12 We decided to explore the functional heterogeneity of the CD4 NKG2D population ex vivo. IL-17 and IFN- secretion were concomitantly analyzed in mucosal lymphocytes (n 7) and PBLs (n 12) of patients with CD. A higher proportion of Th1/Th17 cells were observed in the CD4 NKG2D T cells than in the CD4 NKG2D pool of PB mononuclear cells (14.9% 11.4% vs 0.8% 0.8%, P.0001) and of LP mononuclear cells (9.5% 9.5% vs 2.2% 2.1%, P.19) (Figure 6). Nevertheless, inside the Figure 5. IL-23 drives IL-17 secretion by CD4 NKG2D T cells. LP mononuclear cells isolated from 5 patients with CD were cultured with anti-cd2/anti-cd3/anti-cd28 activation beads and with the addition of IL15, IL-12, IL-1, and/or IL-23 as indicated. After 3 days of culture, the cells were stimulated with PMA/ionomycin. Intracellular staining for IL-17 was analyzed by flow cytometry on gated CD3 CD4 NKG2D and CD4 NKG2D T-cell populations. Individual symbols represent individual patients with CD to visualize trends per patient. Horizontal bars represent mean values. *P.05. stimulation (in vitro differentiated Th17 cells) in 5 patients with CD and 5 HCs. In patients with CD, IL-17 producing CD4 T cells highly expressed NKG2D (75.8% 4.9%) as compared with IL-17 producing CD4 T cells in HCs (6.5% 1.1%, P.02). CD4 NKG2D T Cells Are a Major Source of IL-22 in CD It has been shown that IL-22 is increased in active CD and promotes proinflammatory gene expression. 14 In our study, a notable fraction of CD4 T cells produced IL-22 in the mucosa (7.4% 4.6%, n 10) and in the PB (6.6% 5.6%, n 19). As observed for IL-17, the LP CD4 NKG2D T cells produced higher levels of IL-22 (17.3% 7.3%) than CD4 NKG2D T cells (6.2% 4.6%, P.001, n 10). Also, PB CD4 NKG2D T cells were highly positive for IL-22 (17.6% 11.9%) as compared with CD4 NKG2D T cells (5.7% 5.5%, P.0004, n 19). We then analyzed the expression of NKG2D on IL- 22 producing T cells. There was a higher proportion of LP CD4 cells expressing NKG2D among lymphocytes secreting IL-22 as compared with IL-22 negative cells (25.2% 16.4% vs 8.4% 3.7%, P.001). Thus, we show that in CD, CD4 NKG2D T cells are a major source of the 2 main Th17 cytokines, IL-17 and IL-22. Figure 6. IL-17/IL-22 and IL-17/IFN- producing cells are mainly found among CD4 NKG2D T cells. LP lymphocytes were stimulated with PMA/ionomycin before IL-17, IL-22, and IFN- intracellular staining. Analysis of IL-17/IL-22 and IL-17/IFN- production was assessed in CD3 CD4 NKG2D, CD4 NKG2D, and CD8 NKG2D LP T cells. This figure is representative of analysis performed in 8 patients with active CD.

July 2011 NKG2D DRIVES IL-17 RELEASE IN CD 225 CD4 NKG2D subset, only a minority (9.5% 9.6%) of IL-17 positive cells express IFN-. Concerning the Th17/Th22 profile, a higher proportion of CD4 NKG2D cells expressed both IL-17 and IL-22 as compared with the expression of IL-17 only (P.018). Importantly, the percentage of CD4 NKG2D cells producing both IL-17 and IL-22 was significantly higher than their CD4 NKG2D counterparts (P.031) (Figure 6). Interestingly, as shown in Figure 6, CD8 NKG2D T cells almost did not produce IL-17 but highly expressed IFN-. Altogether, these results show the heterogeneity of CD4 T cells in CD and highlight the proinflammatory potential of the CD4 NKG2D population, represented by the expression of 2 central Th17 cytokines (IL-17 and IL-22), together with Th1 cytokines. Discussion CD4 T cells expressing NKG2D are specifically up-represented in CD as compared with UC and with HCs and present effector functions by producing high levels of IFN- and TNF- and exhibiting specific cytotoxic properties. 16 Nevertheless, recent studies have shown the major role of the Th17 pathway in CD, which seems to have an advantage over the Th1 functions. 9 12 In the present study, we show that CD4 NKG2D T cells present a pronounced Th17 phenotype and function. Moreover, NKG2D appears to be a more specific marker of Th17 cells than CD161 in CD. Functionally, IL-17 secretion by CD4 T cells is highly dependent of NKG2D activation in CD, as shown by the significant increase of IL-17 production under stimulation through both TCR and NKG2D as compared with TCR alone. One major finding of our study is that CD4 NKG2D lymphocytes are skewed toward a Th17 profile in CD. Indeed, CD4 NKG2D T cells present both functional and phenotypic characteristics of Th17 cells. Half of LP CD4 NKG2D T cells produce IL-17 and/or IL-22, known to be the 2 major Th17 cytokines. 15 Also, RORC, the master transcription factor guiding Th17 differentiation, is expressed preferentially in CD4 NKG2D T cells. We show that NKG2D is present on more than 70% of IL-17 positive CD4 T cells and almost absent in CD4 IL-17 negative cells. Moreover, CD4 NKG2D T cells showed a strong responsiveness to Th17-promoting signals. These results emphasize that NKG2D strongly identifies Th17 cells in CD. In contrast, in HCs, NKG2D and IL-17 expressions on CD4 T cells were significantly lower, and IL-17 producing CD4 T cells slightly expressed NKG2D. We provide evidence that IL-17 production is enhanced by specific interaction between NKG2D and its ligands. NKG2DL are stress-inducible molecules, especially in the context of bacterial infections, and are overexpressed in the mucosa of patients with CD. 25 It has been recently reported that IL-17 production by CD4 T cells in response to intracellular pathogens was NKG2D dependent. 26 However, CD4 NKG2D T cells have not been identified yet as a direct source of IL-17. Previous studies showed the major role of effector lymphocytes in the uncontrolled immune responses characterizing CD. The balance between effector and regulatory lymphocytes is disturbed in favor of the effector side. 7,27,28 A striking characteristic of effector T lymphocytes in CD is the presence of NK receptors on their surface as NKG2D or CD161. 16,17 Nevertheless, little is known about the associated expression of NK receptors on CD4 T cells in CD. In this study, we point out that NKG2D and CD161 are specifically expressed on the surface of CD4 LPLs and PBLs from patients with CD compared with UC and HCs. Nevertheless, because the expression of CD161 is very important on the CD4 T cells in intestinal mucosa (more than 43%), this could barely be a specific CD marker. On the contrary, only 5% of LP CD4 T cells express NKG2D, and the majority of CD4 NKG2D cells express CD161. Moreover, it has been shown that CD4 CD161 T cells displayed an activated Th17 phenotype in CD. 18 Here, we point out that NKG2D better identifies the Th17 subset than CD161 in CD and identify CD4 CD161 NKG2D T cells as the most selective subset of Th17 cells in CD. We show that during the NKG2D-TCR costimulation of lymphocytes, all IL-17 secreting CD4 T cells also express TNF-. The role of these 2 proinflammatory cytokines in CD has been well shown, but to our knowledge no association has been shown so far between them. These data are of an important relevance in CD because the latest therapies rely mostly on anti TNF- antibodies, and inhibition of the Th-17 pathway represents an attractive therapeutic target. Finally, we point out the cytokine plasticity of the CD4 NKG2D population. CD4 lymphocytes have been initially classified into specific Th lineages, but actually it becomes clear that CD4 T cells show functional flexibility. 29 Indeed, a recent work in human autoimmune arthritis reported that Th17 cells could convert to Th17/Th1 under conditions that mimic the disease site, namely high IL-12 levels. 24 Here we confirm that under IL-12 stimulation, single IL-17 positive CD4 NKG2D T cells could produce the Th1 cytokine IFN-. Annunziato et al have also shown the presence of a Th1/Th17 population in the mucosa of patients with CD. 17 We show here that the CD4 NKG2D T cells are a mixed Th1/Th17/Th22 population in CD. Thereby, beyond production of IL-17, CD4 NKG2D T cells also exhibit a direct inflammatory effect reflected by a high production of IFN- and TNF-. We showed that a differential regulation is achieved by proinflammatory cytokines (IL-23 and IL-12) on IL-17 and IFN- production by CD4 NKG2D T cells. Overall, our study provides a new functional insight in pathogenic CD4 T cells in CD. We show that the subset of CD4 T cells expressing NKG2D in CD represents a major source of IL-17 and has typical features of Th17 cells. Moreover, the IL-17 production is strongly enhanced by the costimulation of the TCR and NKG2D. Our results suggest that the NKG2D pathway could represent a specific therapeutic target in CD, which would influence at

226 PARIENTE ET AL GASTROENTEROLOGY Vol. 141, No. 1 the same time the TNF- and IL-17 proinflammatory actions. Additionally, the neutralizing anti-nkg2d antibodies have already shown their efficacy to reduce inflammatory-induced colitis in murine models as well as other autoimmune models. 22,30 Regarding these results, the neutralization of the CD4 NKG2D T-cell population in patients with CD appears to be a pertinent therapeutic strategy and would justify undertaking a clinical trial using blocking anti-nkg2d antibodies. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2011.03.061. References 1. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 2003;3:521 533. 2. Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med 2009;361:2066 2078. 3. Maeda S, Hsu LC, Liu H, et al. Nod2 mutation in Crohn s disease potentiates NF-kappaB activity and IL-1beta processing. Science 2005;307:734 738. 4. Smith AM, Rahman FZ, Hayee B, et al. Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn s disease. J Exp Med 2009;206: 1883 1897. 5. Kobayashi KS, Chamaillard M, Ogura Y, et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 2005;307:731 734. 6. van Heel DA, Ghosh S, Butler M, et al. Muramyl dipeptide and toll-like receptor sensitivity in NOD2-associated Crohn s disease. Lancet 2005;365:1794 1796. 7. Allez M, Mayer L. Regulatory T cells: peace keepers in the gut. Inflamm Bowel Dis 2004;10:666 676. 8. Fuss IJ, Neurath M, Boirivant M, et al. Disparate CD4 lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J Immunol 1996;157:1261 1270. 9. Fujino S, Andoh A, Bamba S, et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 2003;52:65 70. 10. Noguchi D, Wakita D, Tajima M, et al. Blocking of IL-6 signaling pathway prevents CD4 T cell-mediated colitis in a T(h)17-independent manner. Int Immunol 2007;19:1431 1440. 11. Elson CO, Cong Y, Weaver CT, et al. Monoclonal anti-interleukin 23 reverses active colitis in a T cell-mediated model in mice. Gastroenterology 2007;132:2359 2370. 12. Annunziato F, Cosmi L, Santarlasci V, et al. Phenotypic and functional features of human Th17 cells. J Exp Med 2007;204:1849 1861. 13. Duerr RH, Taylor KD, Brant SR, et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 2006;314:1461 1463. 14. Andoh A, Zhang Z, Inatomi O, et al. Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts. Gastroenterology 2005;129:969 984. 15. Liang SC, Tan XY, Luxenberg DP, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006;203:2271 2279. 16. Allez M, Tieng V, Nakazawa A, et al. CD4 NKG2D T cells in Crohn s disease mediate inflammatory and cytotoxic responses through MICA interactions. Gastroenterology 2007;132:2346 2358. 17. Iiai T, Watanabe H, Suda T, et al. CD161 T (NT) cells exist predominantly in human intestinal epithelium as well as in liver. Clin Exp Immunol 2002;129:92 98. 18. Kleinschek MA, Boniface K, Sadekova S, et al. Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation. J Exp Med 2009;206:525 534. 19. Groh V, Bahram S, Bauer S, et al. Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium. Proc Natl Acad Sci U S A 1996;93: 12445 12450. 20. Cosman D, Mullberg J, Sutherland CL, et al. ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity 2001;14:123 133. 21. Groh V, Bruhl A, El-Gabalawy H, et al. Stimulation of T cell autoreactivity by anomalous expression of NKG2D and its MIC ligands in rheumatoid arthritis. Proc Natl Acad Sci U S A 2003;100:9452 9457. 22. Kjellev S, Haase C, Lundsgaard D, et al. Inhibition of NKG2D receptor function by antibody therapy attenuates transfer-induced colitis in SCID mice. Eur J Immunol 2007;37:1397 1406. 23. Ito Y, Kanai T, Totsuka T, et al. Blockade of NKG2D signaling prevents the development of murine CD4 T cell-mediated colitis. Am J Physiol Gastrointest Liver Physiol 2008;294:G199 G207. 24. Nistala K, Adams S, Cambrook H, et al. Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proc Natl Acad Sci U S A 2010;107:14751 14756. 25. Tieng V, Le Bouguenec C, du Merle L, et al. Binding of Escherichia coli adhesin AfaE to CD55 triggers cell-surface expression of the MHC class I-related molecule MICA. Proc Natl Acad Sci U S A 2002;99:2977 2982. 26. Paidipally P, Periasamy S, Barnes PF, et al. NKG2D-dependent IL-17 production by human T cells in response to an intracellular pathogen. J Immunol 2009;183:1940 1945. 27. Allez M, Brimnes J, Dotan I, et al. Expansion of CD8 T cells with regulatory function after interaction with intestinal epithelial cells. Gastroenterology 2002;123:1516 1526. 28. Brimnes J, Allez M, Dotan I, et al. Defects in CD8 regulatory T cells in the lamina propria of patients with inflammatory bowel disease. J Immunol 2005;174:5814 5822. 29. O Shea JJ, Paul WE. Mechanisms underlying lineage commitment and plasticity of helper CD4 T cells. Science 2010;327:1098 1102. 30. Ogasawara K, Hamerman JA, Ehrlich LR, et al. NKG2D blockade prevents autoimmune diabetes in NOD mice. Immunity 2004;20: 757 767. Received October 16, 2010. Accepted March 25, 2011. Reprint requests Address requests for reprints to: Matthieu Allez, MD, PhD, Equipe AVENIR INSERM U940, Service de Gastroentérologie, Hôpital Saint- Louis, 1, Avenue Claude Vellefaux, 75010 Paris, France. e-mail: matthieu.allez@sls.aphp.fr. Acknowledgments The authors thank Christelle Doliger, Charlene Lasgi, and Niclas Setterblad from the Imagery Department of the Institut Universitaire d Hématologie for their help in cell sorting. The authors also thank Alexander Steinle from the Department of Immunology, Eberhard Karls University of Tuebingen, Germany for provision of P815 NKG2D ligand transfectants. B.P. and I.M. contributed equally to this work. Conflicts of interest The authors disclose no conflicts. Funding Supported by grants from INSERM (contrat AVENIR) and Novo Nordisk.

July 2011 NKG2D DRIVES IL-17 RELEASE IN CD 226.e1 Supplementary Materials and Methods Isolation of LPL and PBL Lymphocytes LPLs were isolated from the 20 surgical specimens as previously described. 16 Surgical specimens were washed extensively with phosphate-buffered saline. The mucosa was stripped off from the submucosa, minced into small pieces, and placed in 1 mmol/l dithiothreitol for 10 minutes at room temperature. The pieces were washed in phosphate-buffered saline and incubated in medium (RPMI 1640) containing 1.5 mmol/l MgCl 2 and 1 mmol/l EDTA for 30 minutes at 37 C and vortexed every 10 minutes. The supernatant containing released intestinal epithelial cells and intraepithelial lymphocytes was removed. LPLs were isolated from the remaining tissue. The tissue was incubated for 1 hour at 37 C in medium containing 1 mg/ml collagenase (Clostridiopeptidase A). The cell suspension was collected, centrifuged, washed, and resuspended in phosphate-buffered saline. PBLs were isolated in all patients with IBD and controls on a Ficoll Hypaque density gradient. Supplementary Figure 1. IL-17 secretion by ex vivo PB CD4 T cells from patients with CD, patients with UC, and HCs. PBLs were isolated from patients with CD (n 42), patients with UC (n 9), and HCs (n 10) and stimulated with PMA/ionomycin. Intracellular staining for IL-17 was analyzed by flow cytometry on gated CD3 CD4 T cells. (A) Scattergrams show the percentages of overall IL-17 positive cells among PB CD4 T cells. Median values are indicated by a bar.*p.05, **P.005. (B) This figure represents flow cytometric analysis of IL-17 intracellular staining in PB CD4 T cells in one patient with CD (left dot plot), one patient with UC (middle dot plot), and one HC (right dot plot). Gates have been set regarding isotype control antibody staining.

226.e2 PARIENTE ET AL GASTROENTEROLOGY Vol. 141, No. 1 Supplementary Figure 2. NKG2D expression by PB CD4 T cells from patients with CD, patients with UC, and HCs. PB lymphocytes were isolated from patients with CD (n 28), patients with UC (n 12), and HCs (n 10). NKG2D staining was analyzed by flow cytometry on gated CD3 CD4 T cells. Scattergrams show the percentages of overall NKG2D-positive cells among PB CD4 T cells. Median values are indicated by a bar. *P.05, **P.005. Supplementary Figure 3. IL-17 production under NKG2D stimulation through its different ligands expressed on the P815 cell line is different from patient to patient. PBLs isolated from patients with CD were cultured in the presence of P815 cell lines transfected or not with MICA, MICB, ULBP1, ULBP2, or ULBP3, coated with an immunoglobulin G1 antibody (isotype) or with an anti-cd3 agonist antibody at 1 g/ml concentration. Analysis of IL-17 intracellular staining was gated on CD3 CD4 T cells. Histograms represent percentage of CD4 T cells expressing IL-17 under stimulation through each ligand in 3 different patients. In each patient, IL-17 production was maximal under stimulation of both NKG2D and TCR when NKG2D was triggered by (A) MICA, (B) MICB, or (C) ULBP2 interaction.