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Phenotypic and functional features of human Th17 cells

Phenotypic and functional features of human Th17 cells ARTICLE Phenotypic and functional features of human Th17 cells 1 1 1 Francesco Annunziato, Lorenzo Cosmi, Veronica Santarlasci, 1 1 1 1 Laura Maggi, Francesco Liotta, Benedetta Mazzinghi, Eliana Parente, 1 1 1 2 Lucia Fil ì , Simona Ferri, Francesca Frosali, Francesco Giudici, 1 1 2 1 Paola Romagnani, Paola Parronchi, Francesco Tonelli, Enrico Maggi, and Sergio Romagnani Center for Research, Transfer and High Education on Chronic, Infl ammatory, Degenerative and Neoplastic Disorders (DENOTHE) and Department of Pathophysiology, University of Florence, Florence 50134, Italy T helper (Th) 17 cells represent a novel subset of CD4 T cells that are protective against extracellular microbes, but are responsible for autoimmune disorders in mice. However, their properties in humans are only partially known. We demonstrate the presence of Th17 cells, some of which produce both interleukin (IL)-17 and interferon (IFN)-  (Th17/Th1), in the gut of patients with Crohn ’ s disease. Both Th17 and Th17/Th1 clones showed selective expression of IL-23R, CCR6, and the transcription factor ROR  t, and they exhibited similar functional features, such as the ability to help B cells, low cytotoxicity, and poor suscepti- bility to regulation by autologous regulatory T cells. Interestingly, these subsets also ex- pressed the Th1-transcription factor T-bet, and stimulation of these cells in the presence of IL-12 down-regulated the expression of ROR  t and the production of IL-17, but in- duced IFN-  . These effects were partially inhibited in presence of IL-23. Similar receptor expression and functional capabilities were observed in freshly derived IL-17 – producing peripheral blood and tonsillar CD4 T cells. The demonstration of selective markers for human Th17 cells may help us to understand their pathogenic role. Moreover, the identifi - cation of a subset of cells sharing features of both Th1 and Th17, which can arise from the modulation of Th17 cells by IL-12, may raise new issues concerning developmental and/or functional relationships between Th17 and Th1. The adaptive eff ector CD4 Th-mediated im- conditions than Th1 or Th2, which have been CORRESPONDENCE Sergio Romagnani: m une response is highly heterogenous, based found to be the early presence at the time of [email protected] .it on the development of distinct subsets that antigen presentation to the naive Th of IL-12 are characterized by diff erent profi les of cy- or -4, respectively ( 7 ). Initial fi ndings reported Abbreviations used: Ab, anti- body; CD, Crohn ’ s disease; tokine production. Initially, two polarized forms that, in the presence of Borrelia burgdorferi , IL-17 CIA, collagen-induced arthritis; of Th eff ectors, type 1 (Th1) or type 2 (Th2), could be produced by T cells independent EAE, experimental autoimmune were identifi ed in both mice and humans ( 1, 2 ). of the production of Th1 or Th2 cytokines ( 8 ). encephalomyelitis; PB, periph- Th1 cells produce IFN-  and are mainly de- However, the major breakthrough leading to eral blood; RA, rheumatoid arthritis. voted to protection against intracellular mi- discovery of Th17 lineage came from mouse crobes, whereas Th2 cells produce IL-4, -5, -9, models of autoimmunity. Experimental auto- and -13 and are involved in the protection im mune encephalomyelitis (EAE) and colla- against gastrointestinal nematodes, but are also gen-in duced arthritis (CIA), two prototypical responsible for allergic disorders ( 3, 4 ). A third autoimmune mouse models, have historically type of Th that is able to produce both Th1 been associated with unchecked Th1 responses, and Th2 cytokines, type 0 (Th0), has also been based on studies in which disease development detected ( 5 ). More recently, a novel subset of was ablated by treatment with neutralizing Th, which is distinct from Th1, Th2, and Th0 anti bodies (Ab) specifi c for IL-12p40 or gene- cells and is called Th17, has been described ( 6 ). targeted mice defi cient in the p40 subunit of Th17 cells originate under diff erent polarizing IL-12 ( 9, 10 ). However, with the discovery that a new IL-12 family member, IL-23, shares F. Annunziato and L. Cosmi contributed equally to this paper. the p40 subunit with IL-12 but comprises of JEM © The Rockefeller University Press $15.00 Vol. 204, No. 8, August 6, 2007 1849-1861 www.jem.org/cgi/doi/ 10.1084/jem.20070663 The Journal of Experimental Medicine another subunit, p19, which is diff erent from the p35 subunit synovial fl uids, and synovial biopsies of most rheumatoid of IL-12 ( 11 ), it was found that both EAE and CIA were arthritis (RA) patients, whereas both are absent in osteoar- ablated in mice defi cient in IL-23, but not IL-12 ( 12, 13 ). thritis ( 24, 25 ). IL-17 has also been detected in the sera and Moreover, mice lacking the IL-12R complex also succumbed diseased tissues of patients with systemic lupus erythematosus to EAE ( 14 ), suggesting that IL-23, and not IL-12, is critically ( 26 ), in systemic sclerosis ( 27 ), and in sera and colonic biop- linked to autoimmunity, at least in these models. Although sies of infl ammatory bowel disorders ( 28, 29 ), as well as in the IL-23 appeared to be required for Th17-mediated immuno- aff ected skin of subjects with nickel-induced contact derma- pathology, more recent reports indicate that it is not required titis or psoriasis ( 30 ). However, all of these studies were per- for Th17 commitment, but instead appears to be important formed by assessing the presence of mRNA for IL-17 in for amplifying and/or stabilizing the Th17 phenotype ( 15, 16 ). tissues and/or measuring IL-17 in biological fl uids. Only two The concomitant activity of TGF-  and IL-6 appears to be reports have shown the production of IL-17 by small num- necessary for initiation of Th17 diff erentiation ( 17, 18 ). bers of T cell clones generated from the skin of subjects with Moreover, IL-1  and TNF-  were found to amplify the contact dermatitis ( 30 ) or the synovial membranes and syno- Th17 response induced by TGF-  and IL-6, but could not vial fl uid of subjects with RA ( 31 ); however, no additional substitute for either of these cytokines ( 19 ). Finally, an analy- information on the phenotypic and functional features of sis of transcription-factor expression by IL-17 – producing human IL-17 – producing T cells were provided. CD4 T cells showed that Th17 eff ectors lacked expression In this study, we demonstrate the existence of remark- of T-bet and Hlx in comparison to Th1-polarized cells, and able proportions of Th17 and of IFN-  – producing Th17 of GATA-3 compared with Th2-polarized populations, sup- (Th17/Th1) in the gut of subjects with Crohn ’ s disease (CD). porting and extending previous fi ndings that identifi ed Th17 When expanded in vitro and cloned, Th17 and Th17/Th1 as a product of an eff ector lineage distinct from Th1 and Th2 clones showed selective expression of IL-23R, CCR6, and ( 20, 21 ). The latter fi nding was supported by the recent dem- the transcription factor ROR  t. Both Th17 and Th17/Th1 onstration that the orphan nuclear receptor ROR  t directs clones showed an excellent ability to help B cells, low cyto- the diff erentiation program of mouse Th17 ( 22 ). toxic potential, and reduced susceptibility to suppression by Most of the knowledge of Th17 available so far originates autologous CD4Foxp3 regulatory T (T reg) cells. Similar from studies performed in experimental animal models, receptor expression and functional capabilities were observed whereas very little information on Th17 exists in humans. in IL-17 – producing T cell clones obtained from normal gut, A higher number of IL-17 mRNA – expressing cells were found as well as in freshly derived PB and tonsillar IL-17 – producing by using in situ hybridization in the cerebrospinal fl uid than in CD4 T cells. Interestingly, both Th17 and Th17/Th1 clones peripheral blood (PB) from patients with multiple sclerosis ( 23 ). also expressed the Th1-related transcription factor T-bet, and Moreover both IL-17 and -23p19 are present in the sera, their culturing in the presence of IL-12 down-regulated the Figure 1. Ex vivo identifi cation of human Th17 and Th17/Th1. (A) CD4 T cells from PB of healthy subjects, healthy gut areas of subjects with colon carcinoma, and PB and disease-affected gut areas of subjects with CD were assessed by intracellular staining with fl ow cytometry for their ability to produce IFN-  (black), IL-4 (gray), or IL-17 (white) after stimulation with PMA plus ionomycin. (B) Proportions of IL-17 – producing cells able to also produce IFN-  (black) or IL-4 (gray) among CD4 T cells from PB or gut of subjects with CD. Columns in A and B represent mean values (  the SEM) obtained from 10 different subjects for each group. a versus c, P  0.05; b versus c, P  0.05; d versus e, P  0.05. (C) Representative fl ow cytometric analyses in PB and gut of one subject with CD. 1850 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE expression of ROR  t and IL-17 and up-regulated the pro- with colon carcinoma or from PB of subjects with CD, whereas duction of IFN-  , suggesting that, at least in humans, a devel- they were signifi cantly higher in disease-aff ected gut areas of opmental and/or functional relationship between Th17 and the same subjects with CD ( Fig. 1 A ). Comparable propor- Th1 may exist. tions of IL-17 – producing cells were found among CD4 T lymphocytes derived from the disease-aff ected gut areas RESULTS of fi ve subjects suff ering from ulcerative colitis (unpublished Ex vivo demonstration of IL-17 – producing CD4 T cells data). A minor, but consistent, proportion of IL-17 – producing in subjects with CD, and their in vitro expansion and cloning CD4 T cells derived from PB of healthy subjects or The presence of IL-17 – producing cells was evaluated by fl ow healthy gut areas (not depicted), as well as from PB or disease- cytometry on CD4 T cells from PB of 10 healthy individu- aff ected gut areas of subjects with CD ( Fig. 1, B and C ), als, as well as from PB and disease-aff ected areas of 10 subjects also exhibited the ability to produce IFN-  , whereas cells with CD, after their stimulation with PMA and ionomycin. producing both IL-17 and -4 were virtually undetectable. As an additional control, CD4 T cells derived from ap- Notably, proportions of Th17 also producing IFN-  were parently healthy gut areas of 10 subjects who underwent higher among gut T cells than PB CD4 T cells from sub- colectomy because of colon carcinoma were also assessed. jects with CD ( Fig. 1, B and C ). IL-17 – producing CD4 Proportions of Th17 were consistently  1% in PB from T cells were found to be contained within the CD45RO healthy individuals, and only slightly higher among CD4 population, and they all appeared to be TCR    cells (un- T cells derived from apparently healthy gut areas of subjects published data). Figure 2. Cytokine production by in vitro – expanded CD4 T cells from PB and gut and by T cell clones from gut of subjects with CD. (A) CD4 T cells from PB or disease-affected gut areas of 10 subjects with CD were stimulated for 7 d with anti-CD3 plus anti-CD28 mAb and IL-2, and their ability to produce IFN-  (black), IL-4 (gray), or IL-17 (white) was assessed (see Fig. 1 legend). Columns represent the mean values (  the SEM). a versus b, P  0.05. (B) Representative fl ow cytometric analyses on expanded PB or gut CD4 T cells from the same CD subject. (C) A total number of 217 T cell clones (circles) were obtained from CD4 T cells of disease-affected gut areas of two subjects with CD and assessed by fl ow cytometry for intracellular synthesis of IFN-  , IL-4, and IL-17 after stimulation with PMA plus ionomycin. Production of cytokines by each clone was arbitrarily considered as noteworthy when the proportion of producing T cell blasts was  20%. (D) Representative fl ow cytometric analyses of cytokine production for each type of clone. JEM VOL. 204, August 6, 2007 1851 To better investigate the phenotypic and functional fea- tures of IL-17 – producing CD4 T cells, those present in PB or disease-aff ected gut areas of the 10 subjects with CD were expanded in vitro by stimulation for 7 d with a mixture of anti-CD3 and -CD28 mAb in the presence of IL-2. Under these conditions, remarkable proportions of CD4 T-blasts derived from both sources showed the ability to produce IL-17 after their stimulation with PMA plus ionomycin, with those derived from gut being signifi cantly higher ( Fig. 2 A ). Notably, in agreement with the aforementioned ex vivo fi ndings, some of these cells produced both IL-17 and IFN-  . In contrast, virtually no CD4 T-blasts producing both IL-17 and -4 were observed ( Fig. 2 B ). T-blasts from two randomly selected cultures derived from the disease-aff ected gut of subjects with CD were cloned under conditions of limiting dilution and expanded in the presence of alloge- neic irradiated feeder cells and IL-2. 217 CD4 T cell clones were obtained, and the cytokine production (IL-4, IFN-  , and IL-17) by each clone was evaluated by fl ow cytometry after stimulation with PMA plus ionomycin. T cell clones producing IL-17 alone ( n 11), IFN-  alone ( n 72), or Figure 3. IL-23R and -12R  2 expression by Th17 and Th17/Th1 IL-4 alone ( n 7) were classifi ed as Th17, Th1, and Th2, clones and their role in cell proliferation. IL-12R  2 (A) and -23R (B) respectively, whereas those producing both IL-4 and IFN-  , mRNA levels were measured in Th0, Th1, Th2, Th17, and Th17/Th1 clones. but not IL-17 ( n 66), were classifi ed as Th0, and those Columns represent mean values (  the SEM) obtained in 10 clones from each producing both IL-17 and IFN-  ( n 50) or both IL-17 type, except Th2, where they represent 7 clones. (A) d versus a, P  0.05; and -4 ( n 0) were classifi ed as Th17/Th1 or Th17/Th2, d versus b, P ns; d versus c, P  0.05; d versus e, P ns; e versus a, P  0.05; e versus b, P ns; e versus c, P  0.05. (B) i versus f, P  0.01; respectively. 11 clones apparently did not produce any of the i versus g, P  0.05; i versus h, P  0.05; i versus l, P ns; l versus f, three cytokines ( Fig. 2, C and D ). P  0.001; l versus g, P  0.05; l versus h, P  0.05. (C) Proliferative response of Th17 and Th17/Th1 clones after stimulation for 4 d with anti- Th17 and Th17/Th1 clones selectively express IL-23R CD3 plus anti-CD28 Ab without or with the addition of IL-2 (black), IL-12 and CCR6 (gray), IL-15 (white), or IL-23 (striped). Columns represent mean values The expression of IL-12R  2 and IL23-R in Th17 and (  the SEM) of mitogenic indexes (ratio between cpm obtained in the Th17/Th1 clones was fi rst examined and compared with that presence of anti-CD3/28 Ab with the indicated cytokine and cpm obtained found in the other types of T cell clones. IL-12R  2 was ex- with anti-CD3/CD28 Ab alone), obtained by testing seven clones from each type. pressed by all types of clones ( Fig. 3 A ), whereas IL-23R ap- peared to be selectively expressed by Th17 and Th17/Th1 clones ( Fig. 3 B ). However, this receptor was apparently not involved in Th17 or Th17/Th1 cell proliferation because Th17 and Th17/Th1 clones exhibit similar IL-23 did not exert any proliferative activity on both types of functional properties clones (not depicted), even after their stimulation with anti- The ability of Th17 and Th17/Th1 clones to help Ab pro- CD3 plus anti-CD28 Ab ( Fig. 3 C ). In contrast, the addition duction by B cells and to display cytotoxic activity, as well as of IL-2, IL-12, and mainly of IL-15, potentiated the anti- their susceptibility to the suppressive activity of an autologous CD3/28 Ab induced proliferation of both Th17 and Th17/ CD4CD25Foxp3 T cell clone were then evaluated and Th1 clones ( Fig. 3 C ). Among chemokine R, a high expres- compared with the same activities assessed on Th1 and Th2 sion of CXCR4 and CXCR6 by Th17 and Th17/Th1 clones clones. Both Th17 and Th17/Th1 clones showed the ability was shared with Th1 or Th2 clones (unpublished data). Sig- to induce B cell production of IgM, IgG, and IgA, but not of nifi cantly higher levels of CCR4 ( Fig. 4 A ) and CCR5 ( Fig. IgE, with the latter appearing to be a selective property of Th2 4 B ) were found in Th17 clones in comparison with all other clones ( Fig. 5 A ). In regard to the cytotoxic potential, Th17, types of clones, whereas Th17 and Th17/Th1 apparently Th17/Th1, and, as expected, Th2 clones exhibited granzyme lacked CXCR3-A, CXCR3-B, CCR3, CCR8 and CCR9 A expression at levels signifi cantly lower than those of Th1 (not depicted). In contrast, CCR6 was selectively expressed clones, with the granzyme levels of Th17/Th1 clones higher by both Th17 and Th17/Th1, but not by Th1, Th2 or Th0, than those present in Th17 and Th2 clones ( Fig. 5 B ). More- clones ( Fig. 4 C ). CCR6 expressed by Th17 clones was func- over, the cytotoxic ability of Th17 and Th17/Th1 clones tionally active inasmuch as its ligand, CCL20, induced calcium was signifi cantly lower than that of Th1 clones ( Fig. 5 C ). infl ux in Th17, but not in Th1, clones, whereas the CCR9 li- Finally, the ability of Th17 and Th17/Th1 clones to prolifer- gand, CCL25, did not exert any eff ect ( Fig. 4 D ). ate in response to allogeneic stimulation in the presence of 1852 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE an autologous CD4CD25 T reg cell clone was assessed. This clone expressed Foxp3, did not produce cytokines in response to stimulation with PMA plus ionomycin ( Fig. 6 A ), and showed the ability to suppress the proliferative response of autologous CD25 T cells, as well as that of Th1 or Th2 clones ( Fig. 6 B ). In contrast, Th17 and Th17/Th1 exhibited a signifi cantly lower susceptibility to the suppressive activity of the same autologous T reg clone ( Fig. 6 B ). IL-12 down-regulates ROR  t and IL-17 expression, but up-regulates T-bet and IFN-  expression, in Th17 clones We asked what was the relationship between Th17 and Th17/Th1, as both types of cells were already found ex vivo, and thus the latter could not result from a possible artifact caused by T cell culturing in vitro. To this end, we fi rst as- sessed the expression of T-bet, which is a transcription factor that has been found to be selective for Th1 in both mice and humans ( 32 ), and of ROR  t, which is a transcription factor that has been found to be selective for mouse Th17 ( 21 ). As control, the expression of GATA-3, which is a transcription factor selective for both mouse and human Th2 ( 32 ), was also assessed. As expected, Th2 clones selectively expressed GATA-3 and Th1 clones selectively expressed T-bet. In con- trast, both Th17 and Th17/Th1 clones were found to ex- press either ROR  t or T-bet ( Fig. 7, A – C ). The presence of ROR  t and T-bet proteins in Th17 clones was then assessed by confocal microscopy. Whereas ROR  t was expressed in Th17, but not in Th1, clones ( Fig. 7 D ), T-bet protein could be detected in both Th1 and Th17 clones ( Fig. 7 E ). Therefore, we asked whether Th17/Th1 could derive from each of the single Th1 or Th17 subset under particular environmental conditions. To answer this question, Th1 and Th17 clones were stimulated with anti-CD3 plus anti-CD28 Ab and IL-2 in the absence or presence of IL-12, and the ex- pression of ROR  t and T-bet, as well as the cytokine pro- duction profi le, of these clones were evaluated after 7 d of culture. IL-12 down-regulated ROR  t and up-regulated T-bet mRNA levels ( Fig. 8 A ), and induced a signifi cant increase of IFN-  and a reduction of IL-17, as shown by measuring cytokine mRNA levels ( Fig. 8 B ), cytokine con- centrations into cell-free supernatants by ELISA ( Fig. 8 C ), or the proportions of IFN-  – and IL-17 – producing cells by fl ow cytometry after stimulation with PMA plus ionomycin ( Fig. 8, D and E ). We fi nally asked whether the IL-12 – induced eff ects on cytokine production on Th17 could be infl uenced by the presence of IL-23. To this end, three Th17 and three Th1 clones were stimulated for 7 d with anti- CD3/28 Ab and IL-2 in presence of IL-12, -23, or -12 plus -23. Figure 4. Selective expression of functionally active CCR6 by Th17 The addition of IL-12 strongly increased the concentrations and Th17/Th1 clones. CCR4 (A), CCR5 (B), and CCR6 (C) mRNA levels in Th0, Th1, Th2, Th17, and Th17/Th1 clones. Columns represent mean values (  the SEM) obtained in 10 clones from each type, except Th2, where they represent 7 clones. (A) d versus a, P  0.005; d versus b, P  0.05; d versus c, P  0.001; d versus e, P ns; e versus a, P  0.05; d versus c, P  0.05; d versus e, P  0.005; e versus a, P ns; e versus b, e versus b, P  0.05; e versus c, P  0.05. (D) Cells from one Th17 and one P ns; e versus c, P ns. (B) d versus a, P  0.001; d versus b, P  0.05; Th1 clone were incubated with Fluo-4 and stimulated with medium alone, d versus c, P  0.05; d versus e, P  0.01; e versus a, P  0.05; e versus b, the CCR6 ligand, CCL20, the CCR9 ligand, CCL25, or ionomycin. Similar P ns; e versus c, P ns. (C) d versus a, P  0.0001; d versus b, P  0.001; results were obtained with other two Th17 and three Th17/Th1 clones. JEM VOL. 204, August 6, 2007 1853 Figure 5. B cell helper ability and cytotoxic potential by Th17 and Th17/Th1 clones. (A) Measurement by ELISA of IgM, IgG, IgA, and IgE levels in supernatants of B cells cocultured for 7 d with autologous anti-CD3 Ab-stimulated Th1, Th2, Th17, or Th17/Th1 clones. Columns represent mean values (  the SEM) of fi ve different clones from each group. b versus a, P  0.05; c versus a, P  0.05; b versus c, P ns. (B) Granzyme A expression by different types of clones, as assessed by fl ow cytometry. Columns represent mean values (  the SEM) obtained with seven different clones of each type. c versus a, P  0.005; c versus b, P ns; c versus d, P  0.05; d versus a, P  0.01; d versus b, P ns. (C) Cytotoxic activity by different types of clones, as assessed by detection of AnnexinV binding on target cells P815 with fl ow cytometry. Columns represent mean values (  the SEM) obtained with six different clones from each group. c versus a, P  0.01; c versus b, P ns; c versus d, P  0.05; d versus a, P  0.05; d versus b, P ns. of IFN-  into cell-free supernatants of Th17 clones, and even proportions of Th17 clonal cells that acquired the ability to more into those of Th1 clones ( Figs. 8 C and 9 A ), whereas produce IFN-  in response to IL-12, and their degree of the addition of IL-23 alone did not exhibit any eff ect on proliferation ( Fig. 9 B ). IFN-  production by either Th17 or Th1 clones ( Fig. 9 A ). Receptor expression and functional properties of Th17 However, the addition of IL-23 partially reduced the IL-12 – induced increase in the production of IFN-  ( Fig. 9 A ), the clones derived from CD patients are shared by Th17 clones from normal gut and by freshly derived Th17 cells Because the majority of experiments were performed on T cell clones derived from disease-aff ected areas of gut from patients with CD, we asked whether the observed pheno- typic and functional features of Th17 cells were particular to the disease or whether they were shared by T cell clones derived from normal gut. As shown in Fig. 10 A , Th17 and Th17/Th1 clones derived under the same experimental conditions from normal gut showed nearly selective CCR6, IL-23R, and ROR  t expression. Despite exhibiting IL-23R, even these clones did not proliferate in response to IL-23, both in the absence or presence of anti-CD3/CD28 Ab (un- published data). Moreover, they displayed excellent B cell helper ability for IgM, IgG, and IgA, but not IgE, production and reduced cytolytic capacity, as Th17 clones derived from diseased areas of subjects with CD (unpublished data). We fi nally asked whether the phenotypic and functional features of Th17 clones resulted from an artifact introduced by the cloning procedures or they were also shared by freshly derived Th17 cells. The ex vivo existence of T cells able to produce both IL-17 and IFN-  , in addition to those able to produce IL-17 alone, has already been shown in Fig. 1 . How- ever, in subsequent experiments, we assessed the presence of Figure 6. Low susceptibility of Th17 and Th17/Th1 clones to the Th17 cells in human PB and tonsil CD4 T cell suspensions suppressive activity of an autologous T reg cell clone. (A) Foxp3 and by taking advantage of the observation performed on Th17 CD25 expression by the T reg cell clone (left) and its inability to produce clones of their apparently selective expression of CCR6. A IL-4 and IFN-  in response to PMA plus ionomycin (right), as detected by small, but consistently detectable, number of CCR6 CD4 fl ow cytometry. (B) Suppression by the T reg cell clone of the proliferative T cells was observed in both human PB and tonsils by fl ow response of autologous CD4CD25 T cells stimulated with allogeneic cytometry. CCR6 and CCR6 CD4 T cells were then irradiated PBMC, as well as of an autologous Th1 or Th2, but not Th17 or separated by immunomagnetic cell sorting and assessed for Th17/Th1, clone. Columns represent mean values ( ± the SEM) of percent- age of inhibition of the proliferative response in triplicate determinations. their ability to produce IL-17 and IFN-  in response to 1854 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE Figure 7. Expression of T-bet and ROR  t by Th17 and Th17/Th1 clones. ROR  t (A), T-bet (B), and GATA-3 (C) mRNA levels were measured in Th0, Th1, Th2, Th17, and Th17/Th1 clones. Columns represent mean values (  the SEM) in 10 clones from each type, except Th2, where they represent 7 clones. (A) d versus a, P  0.0001; d versus b, P  0.005; d versus c, P  0.05; d versus e, P ns; e versus a, P  0.001; e versus b, P  0.01; e versus c, P  0.05. (B) d versus a, P ns; d versus b, P ns; d versus c, P  0.05; d versus e, P ns; e versus a, P ns; e versus b, P ns; e versus c, P ns; b versus c, P  0.05. (C) d versus a, P ns; d versus b, P ns; d versus c, P  0.05; e versus a, P  0.001; e versus b, P ns; e versus c, P  0.001; b versus c, P  0.001. ROR  t (D) and T-bet (E) protein expression, as assessed by confocal microscopy. Data from one Th17 and one Th1 clone are reported. Similar pictures were obtained in other two Th17 and two Th1 clones. Bars, 10 m. TO-PRO-3 (red) was used to counterstain the nuclei. stimulation with PMA plus ionomycin. Cell sorting enabled least in mice, Th17 cells arise as a part of mucosal host defense a strong enrichment for CCR6 T cells (consistently  90%), and their major role seems to be protection against infections whereas virtually no CCR6 cells were detected in the sustained by extracellular bacteria ( 33 – 35 ), but under certain CCR6-depleted CD4 T cell population (unpublished data). circumstances they can also be involved in the pathogenesis Notably, all T cells producing IL-17 alone or IL-17 plus IFN-  of chronic infl ammatory disorders, including some models of in response to PMA plus ionomycin were contained within autoimmune diseases ( 9 – 14 ). Notably, in these models, IFN- the CCR6-enriched population, whereas CCR6-depleted T produced by Th1 cells, which are crucial for the protection cells could be induced to produce IFN-  , but not IL-17 ( Fig. against intracellular bacteria, does not appear to be patho- 10 B , top). Moreover, IL-23R and ROR  t mRNA levels in genic, but instead appears protective, as inhibition of IFN- CCR6-enriched and -depleted T cells were measured. Levels signaling enhances the development of pathogenic Th17 and of both IL-23R and ROR  t mRNA were found to be sig- exacerbates autoimmunity ( 20 ). Even the neutralization of IL-4 nifi cantly higher in the CCR6-enriched than in the CCR6- produced by Th2 is critical in neutralizing the development of depleted population from either PB or tonsils ( Fig. 10 B , IL-17; however, neither IFN-  nor IL-4 seem to be eff ective bottom). Again, despite the presence of IL-23R, even freshly on already established Th17 ( 20 ). derived IL-17 – producing CD4 T cells did not proliferate Currently, very little is known about human Th17. Some in response to IL-23, both in the absence or presence of anti- studies have shown that the presence of IL-17 mRNA or IL-17 CD3/CD28 mAbs (unpublished data). protein in tissues or biological fl uids of subjects with diff erent autoimmune disorders ( 23 – 27 ) or other chronic infl amma- DISCUSSION tory diseases ( 28 – 30 ). The results of this study provide evi- Several studies demonstrate the existence in experimental an- dence of the existence of increased numbers of CD4 T cells imal models of a novel subset of Th eff ectors that are distinct producing IL-17 in the disease-aff ected gut areas of subjects from the classic Th1 and Th2, and that have been named with CD, compared with either PB or apparently healthy gut Th17 because of their ability to produce IL-17. These cells areas. Based on this fi nding, IL-17 – producing CD4 T cells represent a distinct lineage that originates mainly in the pres- derived from the gut of CD subjects were expanded in vitro ence of TGF-  and IL-6 and need the presence of IL-23 for and cloned to get an amount of these cells suitable for pheno- their expansion and/or maintenance. IL-23 is a member of typic and functional investigations. the IL-12 cytokine family, which shares the p40 subunit with The fi rst information produced by our study was that a IL-12 and diff ers in the expression of p19 instead of p35. At remarkable proportion of IL-17 – producing CD4 T cells JEM VOL. 204, August 6, 2007 1855 Figure 8. Effects of IL-12 on ROR  t and T-bet expression and on IFN-  and IL-17 production by Th17 clones. (A) Decreased ROR  t and increased T-bet mRNA expression in fi ve Th17 clones after stimulation for 7 d with anti-CD3 plus anti-CD28 Ab and IL-2, in the absence (black) or presence (gray) of IL-12. a versus b, P  0.05; c versus d, P  0.05. (B) Decreased IL-17 and increased IFN-  mRNA levels in Th17 clones cultured under the condi- tions described in A. Columns represent mean values (  the SEM). a versus b, P  0.01; c versus d, P  0.01. (C) Reduced levels of IL-17 and increased levels of IFN-  in supernatants of Th17 clones, as measured by ELISA. a versus b, P  0.05; c versus d, P  0.001. (D) Decreased proportions of IL-17 – producing and increased proportions of IFN-  – producing cells in response to PMA plus ionomycin by Th17 clones. a versus b, P  0.05; c versus d, P  0.05. (E) Flow cytometric analysis of IL-17 and IFN-  production by one representative Th17 clone. share the ability to produce IFN-  , whereas CD4 T cells gut mucosa and to get Th17 clones, is apparently at variance producing both IL-17 and -4 were never observed. The cells with recent results showing that IL-2 constrains generation producing both IL-17 and IFN-  , which we named Th17/ of mouse Th17 cells ( 36 ). The reason for such a discrepancy Th1, were not the result of an in vitro artifact, as about one is presently not clear. Among chemokine R, both Th17 and third of ex vivo – derived IL-17 – producing CD4 T cells Th17/Th1 clones apparently lacked CXCR3-A, CXCR3-B, already showed a double IL-17IFN-   phenotype. When CCR3, CCR8, and CCR9, but exhibited high levels of examined at clonal level, Th17 and Th17/Th1 shared dif- CXCR4 and CXCR6. Th17 clones also expressed signif- ferent phenotypic properties from the other types of clones, icantly higher levels of CCR4 and CCR5 than the other some of which appearing to be selective. For example, only types of clones, and both Th17 and Th17/Th1 selectively Th17 and Th17/Th1 clones exhibited the expression of expressed CCR6. CCR4 expression has been associated with IL-23R. Interestingly, the IL-23R did not apparently play any the ability of cells to traffi c into peripheral tissues ( 37 ). CCR6 role in the expansion of these cell subsets, because they did has been found to be expressed by B cells, DCs, and memory, not proliferate in response to IL-23 alone, and IL-23 did not but not naive, T cells. However, its ligand, MIP-3  /CCL20 potentiate their anti-CD3/CD28 Ab – induced prolifera- does not exert chemotactic activity on B cells, but only on tion. Thus, it is probable that IL-23 is not important for hu- memory T cells ( 38 ). Interestingly, however, CCR6 expres- man Th17 expansion, but rather for their survival and/or sion by memory T cells is lost after their prolonged TCR maintenance. In contrast, under the same conditions, anti- triggering ( 39 ). Thus, the selective expression of CCR6 by CD3/28 – stimulated Th17 and Th17/Th1 clones proliferated Th17 and Th17/Th1, but not by Th1, Th2, or Th0, clones in response to IL-2, -12, or -15. The ability of IL-2 to pro- may mean that Th17 and Th17/Th1 are the only memory mote the proliferation of human Th17, a fi nding that was T cells that continue to express CCR6 even after prolonged also supported by the observation that IL-2 was the growth antigen activation, thereby maintaining the possibility of factor used in this study to expand Th17 from both PB or recruitment in response to MIP-3  /CCL20. This fi nding may 1856 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE portant role of IL-17 – producing cells in the maintenance of the infl ammatory processes in autoimmune disorders. The receptor expression and functional capabilities ob- served in T cell clones derived from the aff ected areas of gut from subjects with CD were neither related to their patho- logical source nor to some in vitro artifact referable to the cloning procedure. Indeed, similar receptor expression and function were found in T cell clones derived from healthy gut areas of subjects who underwent colectomy because of colon carcinoma. More importantly, the existence of Th17 and Th17/Th1 cells, as well as their expression of CCR6, IL-23R, and ROR  t was also observed in freshly derived CD4 T cells from gut, PB, and tonsil. In this respect, it is of note that sorting of CCR6 cells allowed to obtain popu- lations strongly enriched in IL-17 – producing cells, suggesting that this marker may be useful to get high numbers of Th17 cells and, therefore, of potentially great help in clarifying their pathophysiologic role in humans. Of note, during the revision process of this paper, a study was published ( 40 ) that also reports preferential expression of CCR6 and ROR  t by human PB Th17 cells. Another important fi nding emerging from this study was the apparent discrepancy with some results reported in mice in regard to the relationship between Th17 and Th1. In a fi rst proposed mouse model, it was suggested that the early diff erentiation of Th1 and Th17 from naive CD4+ T cell precursors was shared, and thus Th1 and Th17 diverged contingent upon the selective availability of IL-12 or -23 Figure 9. IL-23 partially inhibits the increase of IFN-  production acting on a common “ Th1 precursor ” or “ pre-Th1 ” inter- and the proliferation induced by IL-12 on Th17 clones. (A) Detection mediate that coexpressed IL-12R and -23R ( 41 ). However, of IFN-  into cell-free supernatants of Th17 and Th1 clones cultured for 7 d with anti-CD3/CD28 and IL-2 (black), plus IL-12 (gray), -23 (white), in a subsequently proposed mouse model, which was based or -12 plus -23 (striped). Columns represent mean values (  the SEM) on the demonstration that the diff erentiation into Th17 de- obtained in three different Th17 and in three different Th1 clones. a versus b, pends upon TGF-  and IL-6 rather than upon IL-23, it was P  0.05; b versus c, P  0.05; d versus e, P  0.05; e versus f, P  0.05. suggested that Th1 and Th17 subsets were not overlapping (B) Flow cytometric analysis of IL-17 and IFN-  production by one and represented distinct lineages ( 19, 20 ). In this study, we representative Th17 clone, and of its proliferation, as assessed by CFDA- do not provide any evidence for the mechanisms responsi- SE content. Similar results were obtained in other two Th17 clones. ble for the diff erentiation of human Th17, as well as for the question of whether in humans, as in mice, Th17 and Th1 have important implications for the long-term maintenance represent distinct lineages. However, we not only demon- of Th17 infl ux, supporting their important pathogenic role strated the ex vivo existence of CD4 T cells able to pro- into the infl amed tissues. duce both IL-17 and IFN-  but we were also able to induce Human Th17 and Th17/Th1 clones also shared some Th17 clones to produce IFN-  in addition to IL-17 after peculiar functional properties. We found that both types of their culturing in the presence of IL-12. The possible rela- clones were able, as Th1 clones, to provide B cell help for the tionship between human Th17 and Th1 was also supported production of IgM, IgG, and IgA, but not IgE, Ab. In contrast, by the demonstration that Th17 clones expressed not only as Th2 clones, they showed poor granzyme A expression and ROR  t, a fi nding that is in agreement with the results re- low cytotoxic capability, thus displaying an intermediate func- ported in mice ( 21 ), but also remarkable amounts of the tional pattern in regard to B cell helper and cytotoxic activity Th1 transcription factor T-bet, and that amounts of both between Th1 and Th2 clones. However, the most impressive T-bet and ROR  t were comparable in human Th17 and functional feature of both Th17 and Th17/Th1 clones was Th17/Th1 clones. their lower susceptibility, in comparison with both Th1 and Interestingly, the ability of IL-12 to induce Th17 cells to Th2 clones, to the suppressive activity of an autologous T reg produce IFN-  , in addition to IL-17 associated with the up- cell clone derived from circulating CD4CD25highFoxp3 regulation of T-bet and the down-regulation of both ROR  t cells. Although the mechanism for the higher resistance of expression and IL-17 production in Th17 clones. This means Th17 and Th17/Th1 to the action of T reg cells is still un- that in humans, even established Th17 clones, independent clear, this fi nding can provide additional support for an im- of whether they have a distinct or common origin with Th1, JEM VOL. 204, August 6, 2007 1857 Figure 10. Th17 clones derived from normal gut, as well as freshly derived IL-17 – producing CD4 T cells, exhibit similar features to gut CD-derived Th17 clones. (A) CCR6, IL-23R, and ROR  t mRNA expression by Th1, Th17, and Th17/Th1 clones derived from normal gut of subjects who underwent colectomy because of colon carcinoma. Columns represent mean values (  the SEM) obtained from fi ve clones of each type. (B) Detection of IL-17 and IFN-  production by CCR6-depleted and CCR6-enriched CD4 T cell populations from PB or tonsil (top). Measurement of IL-23R and ROR  t mRNA levels in CCR6-depleted (black columns) and CCR6-enriched (red columns) CD4 T cells from PB (left) or tonsils (right). Columns represent mean values  the SEM were obtained in three different donors of PB or tonsil. ported in studies performed in mice ( 42, 43 ). Second, T-bet are responsive to IL-12 and are still suffi ciently fl exible to ac- has been found to be required for optimal IL-17 production quire the ability to produce IFN-  . These fi ndings strongly in the presence of IL-23 ( 44 ). Finally, therapeutic admin- suggest that in human Th17 both ROR  t and T-bet can istration of a small interfering RNA specifi c for T-bet sig- play an important modulatory role. The possibility that T-bet – nifi cantly improved the clinical course of established EAE dependent signaling was responsible for the increased pro- by limiting the diff erentiation of autoreactive Th1 and in- duction of IFN-  by our T cell clones was not directly explored hibiting pathogenic Th17 via regulation of IL-23R ( 45 ). in this study, and thus remains unproved. However, it has re- These fi ndings suggest that even in mouse models T-bet and cently been shown that ectopic T-bet expression in mouse ROR  t may play distinct, yet complementary, roles in the Th17 cells can promote IFN-  secretion and decrease IL-17 development of Th17 cells. In this study, the IL-12 – induced production, suggesting that even in mice the Th17 pheno- IFN-  production by Th17 clones was partially inhibited by type is not stable and can give rise to IFN-  production in the contemporaneous presence of IL-23. Because a similar vitro via T-bet – mediated signaling ( 42 ). In this respect, it inhibitory eff ect by IL-23 on the IL-12 – induced IFN-  pro- should be taken into account that other recent data in mice, duction could also be observed in Th1 clones, which do not again, raise the question of the developmental relationship express IL-23R, it is reasonable to suggest that it was mediated between Th1 and Th17. First, the existence of a dual popula- by the ability of IL-23 to bind both IL-23R and IL-12R  1 tion of IL-17- and IFN-  – producing cells has also been re- 1858 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE Immunomagnetic cell sorting. Separation of CCR6 from CCR6 PB via the p40 chain shared between IL-12 and -23, which there- or tonsillar CD4 T cells was performed by immunomagnetic cell sorting, fore display binding competition. Because IFN-  is a well as previously described ( 49 ). known inducer of IL-12R  2 ( 46 ), it can strongly infl uence the susceptibility of Th17 to the activity of IL-12, thus favor- T cell expansion and cloning. Gut-derived and PB T lymphocytes were ing the shifting of these cells toward the Th1 phenotype. This expanded by stimulation for 10 d with 5 g/ml anti-CD3 plus 5 g/ml anti- fi nding may also account for the reported inhibitory activity CD28 mAb (BD Biosciences) and 20 UI/ml IL-2 (Eurocetus). On day 10, T cell blasts were cloned under limiting dilution (0.3 cell/well), as previously of IFN-  on the development of mouse Th17, as well as for reported ( 50 ). T reg clones were generated from CD4CD25Foxp3 T its protective eff ects ( 47 ). lymphocytes obtained from PBMC of one subject with CD. To this end, DCs In conclusion, our data provide the fi rst detailed pheno- were generated from CD14+ cells cultured for 5 d with IL-4 and GM-CSF. typic and functional characterization of human Th17 isolated CD4CD25 or CD25high T cells were obtained from PBMC by cell sort- from the disease-aff ected gut mucosa of subjects with CD, ing. CD4CD25high cells were cultured with autologous DCs (1:1 ratio) and as well as from normal tissues, showing that Th17 display 100 U/ml IL-2 for 10 d and cloned under limiting dilution (0.3 cell/well). distinct functional properties from Th1 or Th2 cells and identi- RNA isolation, cDNA synthesis, and real-time quantitative RT- fying IL-23R, CCR6, and ROR  t as Th17 – specifi c markers. PCR. Total RNA was extracted by using the RNeasy Micro kit (QIAGEN) In addition, we describe a new subset of IFN-  – producing and treated with DNase I to eliminate possible genomic DNA contamina- Th17 sharing features with both Th1 and Th17, which has tion. Taq-Man RT-PCR was performed, as described elsewhere ( 51 ). Prim- not been previously reported in mice. This novel subset ex- ers and probes used were purchased from Applied Biosystems. ists in vivo in humans and can be induced in vitro by stim- ulating Th17 in presence of IL-12, thus raising new issues Proliferation assay. 5 10 total T cell blasts from each clone were stimu- lated with 2 g/ml anti-CD3 plus 2 g/ml anti-CD28 mAb (BD Bio- on the Th17 developmental and/or functional relationship sciences), in the absence or presence of diff erent cytokines (rIL-2, 50 IU/ml; with Th1. rIL-12, 2.5 ng/ml; rIL-15, 7 ng/ml; rIL-23, 20 ng/ml). In T cell suppression experiments, 2 10 T cell blasts from Th1, Th2, Th17, or Th1/Th17 clones MATERIALS AND METHODS were cultured with 4 10 irradiated (9,000 rad) T cell-depleted allogeneic Subjects. Small bowel specimens were obtained from the apparently healthy 4 high PBMC, with or without 2 10 T cell blasts from a CD4+CD25 Foxp3+ areas of 10 subjects who underwent surgery because of colon cancer, the T clone. Cultures were pulsed for the last 8 h with 0.5 Ci (0.0185 MBq) disease-aff ected areas of 10 subjects with CD, and the disease-aff ected areas of H-TdR (GE Healthcare), harvested, and radionuclide uptake was mea- of 5 subjects with ulcerative colitis. PB samples were obtained from the 10 sured by scintillation counting. In some experiments, cells were labeled with subjects with CD and from 13 healthy donors. Tonsillar specimens were ob- CFDA-SE (Invitrogen), as previously described ( 52 ). tained from three children, who were subjected to tonsillectomy because they were aff ected by chronic tonsillitis. The procedures followed in the study Calcium infl ux. 5 10 cells from a Th1 and a Th17 clone were incu- were in accordance with the ethical standards of the Regional Committee on bated in complete medium in the presence of 2.5 M Fluo-4 AM (Invitro- Human Experimentation. gen) for 30 min in the dark at room temperature. After dye loading, cells were washed twice in complete medium and incubated at 37 ° C in the pres- Reagents. The medium used was RPMI 1640 (Seromed) supplemented ence of 5% CO for an additional 30 min. Cells were analyzed by a BD-LS- with 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 2 RII fl ow cytometer using the Diva software (Becton Dickinson). The basal 10 M 2-mercaptoethanol (2-ME; all from Invitrogen), and 10% FCS. fl uorescence intensity was assessed before the stimulus. The stimulation was FITC-, PE-, allophycocyanin-orperidin chlorophyll protein – conjugated obtained in presence of medium alone or in the presence of 1 g/ml CCL20, anti-CD3, CD4, CD8, CD25, CD45RA, CD45RO, TCR   , TCR  , 1 g/ml CCL25, or 1 M ionomycin. IFN-  , IL-4, and isotype-matched control mAbs were purchased from BD Biosciences. Anti – IL-17 and -Foxp3 mAbs were obtained from eBioscience. 5 B cell helper activity. 2 10 human B cells, isolated as CD19+ cells by PMA, ionomycin, and brefeldin A were purchased from Sigma-Aldrich. 5 immunomagnetic cell sorting ( 49 ), were stimulated with 10 Th1, Th2, Anti-CCR6 mAb was from R & D Systems. Anti-ROR  t rabbit polyclonal Th17, or Th1/Th17 blasts stimulated with 100 ng/ml anti-CD3 Ab. On day Ab was purchased from Abcam. Anti – T-bet rabbit polyclonal Ab was from 7, supernatants from each cell culture were collected and evaluated by ELISA Santa Cruz Biotechnology. for IgM, IgG, IgA, and IgE content. Flow cytometric detection of granzyme A and evaluation of cell T cell recovery. For isolation of T lymphocytes, epithelial gut layer was cytotoxicity. Granzyme A expression by Th1, Th2, Th17, or Th17/Th1 removed by calcium- and magnesium-free PBS, supplemented with 1 mM clones was assessed according to the manufacturer ’ s instructions (BD Bio- EDTA and 1 mM DTT (Sigma-Aldrich). Fragments were mechanically dis- sciences). Th1, Th2, Th17, and Th1/Th17 cell clones were tested for their rupted with a MEDI machine (BD Biosciences). MCs were then separated ability to kill P815 target cells upon anti-CD3 mAb activation (5 g/ml) by from the other cells by Ficoll-Hypaque gradient centrifugation. PBMC and evaluating the percentage of AnnexinV-binding cells with fl ow cytometry, tonsil mononuclear cell suspensions were also obtained by centrifugation on as described elsewhere ( 52 ). Ficoll-Hypaque gradient. Statistics. Statistical comparison between groups was performed using the Flow cytometry. Analysis of cytokine synthesis at the single-cell level was Mann-Whitney test or the Wilcoxon test, as appropriate. Diff erences were performed as previously reported ( 48 ). Foxp3 fl ow cytometric determination considered as statistically signifi cant when P  0.05. was performed according to the manufacturer ’ s instructions (eBioscience). The experiments reported in this paper were performed with grants from the Confocal microscopy. Detection of ROR  t and T-bet protein in Th17 Associazione Italiana Per La Ricerca Sul Cancro, the Ministero dell ’ Istruzione, and Th1 clones was performed by confocal microscopy, by using a previ- dell ’ Universit à e della Ricerca, the Italian Ministry of Health, and FP6 European ously described technique ( 49 ). Union project INNOCHEM, LSHB-CT 2500-518157. JEM VOL. 204, August 6, 2007 1859 The authors have no confl icting fi nancial interests. 19 . Sutton , C. , C. Brereton , B. Keogh , K.H. Mills , and E.C. Lavell . 2006 . 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Romagnani , P. , L. Maggi , B. Mazzinghi , L. Cosmi , L. Lasagni , F. Liotta , ences between Th1 and Th17 cells and negative regulation of Th1 cell E. Lazzeri , R. Angeli , M. Rotondi , L. Fili , et al . 2005 . CXCR3-mediated diff erentiation by IL-17. J Leukoc Biol . 81 : 1258 – 1268 . opposite eff ects of CXCL10 and CXCL4 on Th1 or Th2 cytokine 44 . Thakker , P. , M.W. Leach , W. Kuang , S.E. Benoit , J.P. Leonard , and production. J. Allergy Clin. Immunol. 116 : 1372 – 1379 . S. Marusic . 2007 . IL-23 is critical in the induction but not in the eff ec- 52 . Krampera , M. , L. Cosmi , R. Angeli , A. Pasini , F. Liotta , A. tor phase of experimental autoimmune encephalomyelitis. J. Immunol. Andreini , V. Santarlasci , B. Mazzinghi , G. Zizzolo , F. Vinante , et al . 178 : 2589 – 2598 . 2006 . Role for interferon-gamma in the immunomodulatory ac- 45 . Gocke , A.R. , P.D. Cravens , L.H. Ben , R.Z. Hussain , S.C. Northrop , tivity of human bone marrow mesenchymal stem cells. Stem Cells . M.K. Racke , and A.E. Lovett-Racke . 2007 . T-bet regulates the 24 : 386 – 398 . JEM VOL. 204, August 6, 2007 1861 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Phenotypic and functional features of human Th17 cells

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Copyright © 2007, The Rockefeller University Press
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ARTICLE Phenotypic and functional features of human Th17 cells 1 1 1 Francesco Annunziato, Lorenzo Cosmi, Veronica Santarlasci, 1 1 1 1 Laura Maggi, Francesco Liotta, Benedetta Mazzinghi, Eliana Parente, 1 1 1 2 Lucia Fil ì , Simona Ferri, Francesca Frosali, Francesco Giudici, 1 1 2 1 Paola Romagnani, Paola Parronchi, Francesco Tonelli, Enrico Maggi, and Sergio Romagnani Center for Research, Transfer and High Education on Chronic, Infl ammatory, Degenerative and Neoplastic Disorders (DENOTHE) and Department of Pathophysiology, University of Florence, Florence 50134, Italy T helper (Th) 17 cells represent a novel subset of CD4 T cells that are protective against extracellular microbes, but are responsible for autoimmune disorders in mice. However, their properties in humans are only partially known. We demonstrate the presence of Th17 cells, some of which produce both interleukin (IL)-17 and interferon (IFN)-  (Th17/Th1), in the gut of patients with Crohn ’ s disease. Both Th17 and Th17/Th1 clones showed selective expression of IL-23R, CCR6, and the transcription factor ROR  t, and they exhibited similar functional features, such as the ability to help B cells, low cytotoxicity, and poor suscepti- bility to regulation by autologous regulatory T cells. Interestingly, these subsets also ex- pressed the Th1-transcription factor T-bet, and stimulation of these cells in the presence of IL-12 down-regulated the expression of ROR  t and the production of IL-17, but in- duced IFN-  . These effects were partially inhibited in presence of IL-23. Similar receptor expression and functional capabilities were observed in freshly derived IL-17 – producing peripheral blood and tonsillar CD4 T cells. The demonstration of selective markers for human Th17 cells may help us to understand their pathogenic role. Moreover, the identifi - cation of a subset of cells sharing features of both Th1 and Th17, which can arise from the modulation of Th17 cells by IL-12, may raise new issues concerning developmental and/or functional relationships between Th17 and Th1. The adaptive eff ector CD4 Th-mediated im- conditions than Th1 or Th2, which have been CORRESPONDENCE Sergio Romagnani: m une response is highly heterogenous, based found to be the early presence at the time of [email protected] .it on the development of distinct subsets that antigen presentation to the naive Th of IL-12 are characterized by diff erent profi les of cy- or -4, respectively ( 7 ). Initial fi ndings reported Abbreviations used: Ab, anti- body; CD, Crohn ’ s disease; tokine production. Initially, two polarized forms that, in the presence of Borrelia burgdorferi , IL-17 CIA, collagen-induced arthritis; of Th eff ectors, type 1 (Th1) or type 2 (Th2), could be produced by T cells independent EAE, experimental autoimmune were identifi ed in both mice and humans ( 1, 2 ). of the production of Th1 or Th2 cytokines ( 8 ). encephalomyelitis; PB, periph- Th1 cells produce IFN-  and are mainly de- However, the major breakthrough leading to eral blood; RA, rheumatoid arthritis. voted to protection against intracellular mi- discovery of Th17 lineage came from mouse crobes, whereas Th2 cells produce IL-4, -5, -9, models of autoimmunity. Experimental auto- and -13 and are involved in the protection im mune encephalomyelitis (EAE) and colla- against gastrointestinal nematodes, but are also gen-in duced arthritis (CIA), two prototypical responsible for allergic disorders ( 3, 4 ). A third autoimmune mouse models, have historically type of Th that is able to produce both Th1 been associated with unchecked Th1 responses, and Th2 cytokines, type 0 (Th0), has also been based on studies in which disease development detected ( 5 ). More recently, a novel subset of was ablated by treatment with neutralizing Th, which is distinct from Th1, Th2, and Th0 anti bodies (Ab) specifi c for IL-12p40 or gene- cells and is called Th17, has been described ( 6 ). targeted mice defi cient in the p40 subunit of Th17 cells originate under diff erent polarizing IL-12 ( 9, 10 ). However, with the discovery that a new IL-12 family member, IL-23, shares F. Annunziato and L. Cosmi contributed equally to this paper. the p40 subunit with IL-12 but comprises of JEM © The Rockefeller University Press $15.00 Vol. 204, No. 8, August 6, 2007 1849-1861 www.jem.org/cgi/doi/ 10.1084/jem.20070663 The Journal of Experimental Medicine another subunit, p19, which is diff erent from the p35 subunit synovial fl uids, and synovial biopsies of most rheumatoid of IL-12 ( 11 ), it was found that both EAE and CIA were arthritis (RA) patients, whereas both are absent in osteoar- ablated in mice defi cient in IL-23, but not IL-12 ( 12, 13 ). thritis ( 24, 25 ). IL-17 has also been detected in the sera and Moreover, mice lacking the IL-12R complex also succumbed diseased tissues of patients with systemic lupus erythematosus to EAE ( 14 ), suggesting that IL-23, and not IL-12, is critically ( 26 ), in systemic sclerosis ( 27 ), and in sera and colonic biop- linked to autoimmunity, at least in these models. Although sies of infl ammatory bowel disorders ( 28, 29 ), as well as in the IL-23 appeared to be required for Th17-mediated immuno- aff ected skin of subjects with nickel-induced contact derma- pathology, more recent reports indicate that it is not required titis or psoriasis ( 30 ). However, all of these studies were per- for Th17 commitment, but instead appears to be important formed by assessing the presence of mRNA for IL-17 in for amplifying and/or stabilizing the Th17 phenotype ( 15, 16 ). tissues and/or measuring IL-17 in biological fl uids. Only two The concomitant activity of TGF-  and IL-6 appears to be reports have shown the production of IL-17 by small num- necessary for initiation of Th17 diff erentiation ( 17, 18 ). bers of T cell clones generated from the skin of subjects with Moreover, IL-1  and TNF-  were found to amplify the contact dermatitis ( 30 ) or the synovial membranes and syno- Th17 response induced by TGF-  and IL-6, but could not vial fl uid of subjects with RA ( 31 ); however, no additional substitute for either of these cytokines ( 19 ). Finally, an analy- information on the phenotypic and functional features of sis of transcription-factor expression by IL-17 – producing human IL-17 – producing T cells were provided. CD4 T cells showed that Th17 eff ectors lacked expression In this study, we demonstrate the existence of remark- of T-bet and Hlx in comparison to Th1-polarized cells, and able proportions of Th17 and of IFN-  – producing Th17 of GATA-3 compared with Th2-polarized populations, sup- (Th17/Th1) in the gut of subjects with Crohn ’ s disease (CD). porting and extending previous fi ndings that identifi ed Th17 When expanded in vitro and cloned, Th17 and Th17/Th1 as a product of an eff ector lineage distinct from Th1 and Th2 clones showed selective expression of IL-23R, CCR6, and ( 20, 21 ). The latter fi nding was supported by the recent dem- the transcription factor ROR  t. Both Th17 and Th17/Th1 onstration that the orphan nuclear receptor ROR  t directs clones showed an excellent ability to help B cells, low cyto- the diff erentiation program of mouse Th17 ( 22 ). toxic potential, and reduced susceptibility to suppression by Most of the knowledge of Th17 available so far originates autologous CD4Foxp3 regulatory T (T reg) cells. Similar from studies performed in experimental animal models, receptor expression and functional capabilities were observed whereas very little information on Th17 exists in humans. in IL-17 – producing T cell clones obtained from normal gut, A higher number of IL-17 mRNA – expressing cells were found as well as in freshly derived PB and tonsillar IL-17 – producing by using in situ hybridization in the cerebrospinal fl uid than in CD4 T cells. Interestingly, both Th17 and Th17/Th1 clones peripheral blood (PB) from patients with multiple sclerosis ( 23 ). also expressed the Th1-related transcription factor T-bet, and Moreover both IL-17 and -23p19 are present in the sera, their culturing in the presence of IL-12 down-regulated the Figure 1. Ex vivo identifi cation of human Th17 and Th17/Th1. (A) CD4 T cells from PB of healthy subjects, healthy gut areas of subjects with colon carcinoma, and PB and disease-affected gut areas of subjects with CD were assessed by intracellular staining with fl ow cytometry for their ability to produce IFN-  (black), IL-4 (gray), or IL-17 (white) after stimulation with PMA plus ionomycin. (B) Proportions of IL-17 – producing cells able to also produce IFN-  (black) or IL-4 (gray) among CD4 T cells from PB or gut of subjects with CD. Columns in A and B represent mean values (  the SEM) obtained from 10 different subjects for each group. a versus c, P  0.05; b versus c, P  0.05; d versus e, P  0.05. (C) Representative fl ow cytometric analyses in PB and gut of one subject with CD. 1850 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE expression of ROR  t and IL-17 and up-regulated the pro- with colon carcinoma or from PB of subjects with CD, whereas duction of IFN-  , suggesting that, at least in humans, a devel- they were signifi cantly higher in disease-aff ected gut areas of opmental and/or functional relationship between Th17 and the same subjects with CD ( Fig. 1 A ). Comparable propor- Th1 may exist. tions of IL-17 – producing cells were found among CD4 T lymphocytes derived from the disease-aff ected gut areas RESULTS of fi ve subjects suff ering from ulcerative colitis (unpublished Ex vivo demonstration of IL-17 – producing CD4 T cells data). A minor, but consistent, proportion of IL-17 – producing in subjects with CD, and their in vitro expansion and cloning CD4 T cells derived from PB of healthy subjects or The presence of IL-17 – producing cells was evaluated by fl ow healthy gut areas (not depicted), as well as from PB or disease- cytometry on CD4 T cells from PB of 10 healthy individu- aff ected gut areas of subjects with CD ( Fig. 1, B and C ), als, as well as from PB and disease-aff ected areas of 10 subjects also exhibited the ability to produce IFN-  , whereas cells with CD, after their stimulation with PMA and ionomycin. producing both IL-17 and -4 were virtually undetectable. As an additional control, CD4 T cells derived from ap- Notably, proportions of Th17 also producing IFN-  were parently healthy gut areas of 10 subjects who underwent higher among gut T cells than PB CD4 T cells from sub- colectomy because of colon carcinoma were also assessed. jects with CD ( Fig. 1, B and C ). IL-17 – producing CD4 Proportions of Th17 were consistently  1% in PB from T cells were found to be contained within the CD45RO healthy individuals, and only slightly higher among CD4 population, and they all appeared to be TCR    cells (un- T cells derived from apparently healthy gut areas of subjects published data). Figure 2. Cytokine production by in vitro – expanded CD4 T cells from PB and gut and by T cell clones from gut of subjects with CD. (A) CD4 T cells from PB or disease-affected gut areas of 10 subjects with CD were stimulated for 7 d with anti-CD3 plus anti-CD28 mAb and IL-2, and their ability to produce IFN-  (black), IL-4 (gray), or IL-17 (white) was assessed (see Fig. 1 legend). Columns represent the mean values (  the SEM). a versus b, P  0.05. (B) Representative fl ow cytometric analyses on expanded PB or gut CD4 T cells from the same CD subject. (C) A total number of 217 T cell clones (circles) were obtained from CD4 T cells of disease-affected gut areas of two subjects with CD and assessed by fl ow cytometry for intracellular synthesis of IFN-  , IL-4, and IL-17 after stimulation with PMA plus ionomycin. Production of cytokines by each clone was arbitrarily considered as noteworthy when the proportion of producing T cell blasts was  20%. (D) Representative fl ow cytometric analyses of cytokine production for each type of clone. JEM VOL. 204, August 6, 2007 1851 To better investigate the phenotypic and functional fea- tures of IL-17 – producing CD4 T cells, those present in PB or disease-aff ected gut areas of the 10 subjects with CD were expanded in vitro by stimulation for 7 d with a mixture of anti-CD3 and -CD28 mAb in the presence of IL-2. Under these conditions, remarkable proportions of CD4 T-blasts derived from both sources showed the ability to produce IL-17 after their stimulation with PMA plus ionomycin, with those derived from gut being signifi cantly higher ( Fig. 2 A ). Notably, in agreement with the aforementioned ex vivo fi ndings, some of these cells produced both IL-17 and IFN-  . In contrast, virtually no CD4 T-blasts producing both IL-17 and -4 were observed ( Fig. 2 B ). T-blasts from two randomly selected cultures derived from the disease-aff ected gut of subjects with CD were cloned under conditions of limiting dilution and expanded in the presence of alloge- neic irradiated feeder cells and IL-2. 217 CD4 T cell clones were obtained, and the cytokine production (IL-4, IFN-  , and IL-17) by each clone was evaluated by fl ow cytometry after stimulation with PMA plus ionomycin. T cell clones producing IL-17 alone ( n 11), IFN-  alone ( n 72), or Figure 3. IL-23R and -12R  2 expression by Th17 and Th17/Th1 IL-4 alone ( n 7) were classifi ed as Th17, Th1, and Th2, clones and their role in cell proliferation. IL-12R  2 (A) and -23R (B) respectively, whereas those producing both IL-4 and IFN-  , mRNA levels were measured in Th0, Th1, Th2, Th17, and Th17/Th1 clones. but not IL-17 ( n 66), were classifi ed as Th0, and those Columns represent mean values (  the SEM) obtained in 10 clones from each producing both IL-17 and IFN-  ( n 50) or both IL-17 type, except Th2, where they represent 7 clones. (A) d versus a, P  0.05; and -4 ( n 0) were classifi ed as Th17/Th1 or Th17/Th2, d versus b, P ns; d versus c, P  0.05; d versus e, P ns; e versus a, P  0.05; e versus b, P ns; e versus c, P  0.05. (B) i versus f, P  0.01; respectively. 11 clones apparently did not produce any of the i versus g, P  0.05; i versus h, P  0.05; i versus l, P ns; l versus f, three cytokines ( Fig. 2, C and D ). P  0.001; l versus g, P  0.05; l versus h, P  0.05. (C) Proliferative response of Th17 and Th17/Th1 clones after stimulation for 4 d with anti- Th17 and Th17/Th1 clones selectively express IL-23R CD3 plus anti-CD28 Ab without or with the addition of IL-2 (black), IL-12 and CCR6 (gray), IL-15 (white), or IL-23 (striped). Columns represent mean values The expression of IL-12R  2 and IL23-R in Th17 and (  the SEM) of mitogenic indexes (ratio between cpm obtained in the Th17/Th1 clones was fi rst examined and compared with that presence of anti-CD3/28 Ab with the indicated cytokine and cpm obtained found in the other types of T cell clones. IL-12R  2 was ex- with anti-CD3/CD28 Ab alone), obtained by testing seven clones from each type. pressed by all types of clones ( Fig. 3 A ), whereas IL-23R ap- peared to be selectively expressed by Th17 and Th17/Th1 clones ( Fig. 3 B ). However, this receptor was apparently not involved in Th17 or Th17/Th1 cell proliferation because Th17 and Th17/Th1 clones exhibit similar IL-23 did not exert any proliferative activity on both types of functional properties clones (not depicted), even after their stimulation with anti- The ability of Th17 and Th17/Th1 clones to help Ab pro- CD3 plus anti-CD28 Ab ( Fig. 3 C ). In contrast, the addition duction by B cells and to display cytotoxic activity, as well as of IL-2, IL-12, and mainly of IL-15, potentiated the anti- their susceptibility to the suppressive activity of an autologous CD3/28 Ab induced proliferation of both Th17 and Th17/ CD4CD25Foxp3 T cell clone were then evaluated and Th1 clones ( Fig. 3 C ). Among chemokine R, a high expres- compared with the same activities assessed on Th1 and Th2 sion of CXCR4 and CXCR6 by Th17 and Th17/Th1 clones clones. Both Th17 and Th17/Th1 clones showed the ability was shared with Th1 or Th2 clones (unpublished data). Sig- to induce B cell production of IgM, IgG, and IgA, but not of nifi cantly higher levels of CCR4 ( Fig. 4 A ) and CCR5 ( Fig. IgE, with the latter appearing to be a selective property of Th2 4 B ) were found in Th17 clones in comparison with all other clones ( Fig. 5 A ). In regard to the cytotoxic potential, Th17, types of clones, whereas Th17 and Th17/Th1 apparently Th17/Th1, and, as expected, Th2 clones exhibited granzyme lacked CXCR3-A, CXCR3-B, CCR3, CCR8 and CCR9 A expression at levels signifi cantly lower than those of Th1 (not depicted). In contrast, CCR6 was selectively expressed clones, with the granzyme levels of Th17/Th1 clones higher by both Th17 and Th17/Th1, but not by Th1, Th2 or Th0, than those present in Th17 and Th2 clones ( Fig. 5 B ). More- clones ( Fig. 4 C ). CCR6 expressed by Th17 clones was func- over, the cytotoxic ability of Th17 and Th17/Th1 clones tionally active inasmuch as its ligand, CCL20, induced calcium was signifi cantly lower than that of Th1 clones ( Fig. 5 C ). infl ux in Th17, but not in Th1, clones, whereas the CCR9 li- Finally, the ability of Th17 and Th17/Th1 clones to prolifer- gand, CCL25, did not exert any eff ect ( Fig. 4 D ). ate in response to allogeneic stimulation in the presence of 1852 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE an autologous CD4CD25 T reg cell clone was assessed. This clone expressed Foxp3, did not produce cytokines in response to stimulation with PMA plus ionomycin ( Fig. 6 A ), and showed the ability to suppress the proliferative response of autologous CD25 T cells, as well as that of Th1 or Th2 clones ( Fig. 6 B ). In contrast, Th17 and Th17/Th1 exhibited a signifi cantly lower susceptibility to the suppressive activity of the same autologous T reg clone ( Fig. 6 B ). IL-12 down-regulates ROR  t and IL-17 expression, but up-regulates T-bet and IFN-  expression, in Th17 clones We asked what was the relationship between Th17 and Th17/Th1, as both types of cells were already found ex vivo, and thus the latter could not result from a possible artifact caused by T cell culturing in vitro. To this end, we fi rst as- sessed the expression of T-bet, which is a transcription factor that has been found to be selective for Th1 in both mice and humans ( 32 ), and of ROR  t, which is a transcription factor that has been found to be selective for mouse Th17 ( 21 ). As control, the expression of GATA-3, which is a transcription factor selective for both mouse and human Th2 ( 32 ), was also assessed. As expected, Th2 clones selectively expressed GATA-3 and Th1 clones selectively expressed T-bet. In con- trast, both Th17 and Th17/Th1 clones were found to ex- press either ROR  t or T-bet ( Fig. 7, A – C ). The presence of ROR  t and T-bet proteins in Th17 clones was then assessed by confocal microscopy. Whereas ROR  t was expressed in Th17, but not in Th1, clones ( Fig. 7 D ), T-bet protein could be detected in both Th1 and Th17 clones ( Fig. 7 E ). Therefore, we asked whether Th17/Th1 could derive from each of the single Th1 or Th17 subset under particular environmental conditions. To answer this question, Th1 and Th17 clones were stimulated with anti-CD3 plus anti-CD28 Ab and IL-2 in the absence or presence of IL-12, and the ex- pression of ROR  t and T-bet, as well as the cytokine pro- duction profi le, of these clones were evaluated after 7 d of culture. IL-12 down-regulated ROR  t and up-regulated T-bet mRNA levels ( Fig. 8 A ), and induced a signifi cant increase of IFN-  and a reduction of IL-17, as shown by measuring cytokine mRNA levels ( Fig. 8 B ), cytokine con- centrations into cell-free supernatants by ELISA ( Fig. 8 C ), or the proportions of IFN-  – and IL-17 – producing cells by fl ow cytometry after stimulation with PMA plus ionomycin ( Fig. 8, D and E ). We fi nally asked whether the IL-12 – induced eff ects on cytokine production on Th17 could be infl uenced by the presence of IL-23. To this end, three Th17 and three Th1 clones were stimulated for 7 d with anti- CD3/28 Ab and IL-2 in presence of IL-12, -23, or -12 plus -23. Figure 4. Selective expression of functionally active CCR6 by Th17 The addition of IL-12 strongly increased the concentrations and Th17/Th1 clones. CCR4 (A), CCR5 (B), and CCR6 (C) mRNA levels in Th0, Th1, Th2, Th17, and Th17/Th1 clones. Columns represent mean values (  the SEM) obtained in 10 clones from each type, except Th2, where they represent 7 clones. (A) d versus a, P  0.005; d versus b, P  0.05; d versus c, P  0.001; d versus e, P ns; e versus a, P  0.05; d versus c, P  0.05; d versus e, P  0.005; e versus a, P ns; e versus b, e versus b, P  0.05; e versus c, P  0.05. (D) Cells from one Th17 and one P ns; e versus c, P ns. (B) d versus a, P  0.001; d versus b, P  0.05; Th1 clone were incubated with Fluo-4 and stimulated with medium alone, d versus c, P  0.05; d versus e, P  0.01; e versus a, P  0.05; e versus b, the CCR6 ligand, CCL20, the CCR9 ligand, CCL25, or ionomycin. Similar P ns; e versus c, P ns. (C) d versus a, P  0.0001; d versus b, P  0.001; results were obtained with other two Th17 and three Th17/Th1 clones. JEM VOL. 204, August 6, 2007 1853 Figure 5. B cell helper ability and cytotoxic potential by Th17 and Th17/Th1 clones. (A) Measurement by ELISA of IgM, IgG, IgA, and IgE levels in supernatants of B cells cocultured for 7 d with autologous anti-CD3 Ab-stimulated Th1, Th2, Th17, or Th17/Th1 clones. Columns represent mean values (  the SEM) of fi ve different clones from each group. b versus a, P  0.05; c versus a, P  0.05; b versus c, P ns. (B) Granzyme A expression by different types of clones, as assessed by fl ow cytometry. Columns represent mean values (  the SEM) obtained with seven different clones of each type. c versus a, P  0.005; c versus b, P ns; c versus d, P  0.05; d versus a, P  0.01; d versus b, P ns. (C) Cytotoxic activity by different types of clones, as assessed by detection of AnnexinV binding on target cells P815 with fl ow cytometry. Columns represent mean values (  the SEM) obtained with six different clones from each group. c versus a, P  0.01; c versus b, P ns; c versus d, P  0.05; d versus a, P  0.05; d versus b, P ns. of IFN-  into cell-free supernatants of Th17 clones, and even proportions of Th17 clonal cells that acquired the ability to more into those of Th1 clones ( Figs. 8 C and 9 A ), whereas produce IFN-  in response to IL-12, and their degree of the addition of IL-23 alone did not exhibit any eff ect on proliferation ( Fig. 9 B ). IFN-  production by either Th17 or Th1 clones ( Fig. 9 A ). Receptor expression and functional properties of Th17 However, the addition of IL-23 partially reduced the IL-12 – induced increase in the production of IFN-  ( Fig. 9 A ), the clones derived from CD patients are shared by Th17 clones from normal gut and by freshly derived Th17 cells Because the majority of experiments were performed on T cell clones derived from disease-aff ected areas of gut from patients with CD, we asked whether the observed pheno- typic and functional features of Th17 cells were particular to the disease or whether they were shared by T cell clones derived from normal gut. As shown in Fig. 10 A , Th17 and Th17/Th1 clones derived under the same experimental conditions from normal gut showed nearly selective CCR6, IL-23R, and ROR  t expression. Despite exhibiting IL-23R, even these clones did not proliferate in response to IL-23, both in the absence or presence of anti-CD3/CD28 Ab (un- published data). Moreover, they displayed excellent B cell helper ability for IgM, IgG, and IgA, but not IgE, production and reduced cytolytic capacity, as Th17 clones derived from diseased areas of subjects with CD (unpublished data). We fi nally asked whether the phenotypic and functional features of Th17 clones resulted from an artifact introduced by the cloning procedures or they were also shared by freshly derived Th17 cells. The ex vivo existence of T cells able to produce both IL-17 and IFN-  , in addition to those able to produce IL-17 alone, has already been shown in Fig. 1 . How- ever, in subsequent experiments, we assessed the presence of Figure 6. Low susceptibility of Th17 and Th17/Th1 clones to the Th17 cells in human PB and tonsil CD4 T cell suspensions suppressive activity of an autologous T reg cell clone. (A) Foxp3 and by taking advantage of the observation performed on Th17 CD25 expression by the T reg cell clone (left) and its inability to produce clones of their apparently selective expression of CCR6. A IL-4 and IFN-  in response to PMA plus ionomycin (right), as detected by small, but consistently detectable, number of CCR6 CD4 fl ow cytometry. (B) Suppression by the T reg cell clone of the proliferative T cells was observed in both human PB and tonsils by fl ow response of autologous CD4CD25 T cells stimulated with allogeneic cytometry. CCR6 and CCR6 CD4 T cells were then irradiated PBMC, as well as of an autologous Th1 or Th2, but not Th17 or separated by immunomagnetic cell sorting and assessed for Th17/Th1, clone. Columns represent mean values ( ± the SEM) of percent- age of inhibition of the proliferative response in triplicate determinations. their ability to produce IL-17 and IFN-  in response to 1854 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE Figure 7. Expression of T-bet and ROR  t by Th17 and Th17/Th1 clones. ROR  t (A), T-bet (B), and GATA-3 (C) mRNA levels were measured in Th0, Th1, Th2, Th17, and Th17/Th1 clones. Columns represent mean values (  the SEM) in 10 clones from each type, except Th2, where they represent 7 clones. (A) d versus a, P  0.0001; d versus b, P  0.005; d versus c, P  0.05; d versus e, P ns; e versus a, P  0.001; e versus b, P  0.01; e versus c, P  0.05. (B) d versus a, P ns; d versus b, P ns; d versus c, P  0.05; d versus e, P ns; e versus a, P ns; e versus b, P ns; e versus c, P ns; b versus c, P  0.05. (C) d versus a, P ns; d versus b, P ns; d versus c, P  0.05; e versus a, P  0.001; e versus b, P ns; e versus c, P  0.001; b versus c, P  0.001. ROR  t (D) and T-bet (E) protein expression, as assessed by confocal microscopy. Data from one Th17 and one Th1 clone are reported. Similar pictures were obtained in other two Th17 and two Th1 clones. Bars, 10 m. TO-PRO-3 (red) was used to counterstain the nuclei. stimulation with PMA plus ionomycin. Cell sorting enabled least in mice, Th17 cells arise as a part of mucosal host defense a strong enrichment for CCR6 T cells (consistently  90%), and their major role seems to be protection against infections whereas virtually no CCR6 cells were detected in the sustained by extracellular bacteria ( 33 – 35 ), but under certain CCR6-depleted CD4 T cell population (unpublished data). circumstances they can also be involved in the pathogenesis Notably, all T cells producing IL-17 alone or IL-17 plus IFN-  of chronic infl ammatory disorders, including some models of in response to PMA plus ionomycin were contained within autoimmune diseases ( 9 – 14 ). Notably, in these models, IFN- the CCR6-enriched population, whereas CCR6-depleted T produced by Th1 cells, which are crucial for the protection cells could be induced to produce IFN-  , but not IL-17 ( Fig. against intracellular bacteria, does not appear to be patho- 10 B , top). Moreover, IL-23R and ROR  t mRNA levels in genic, but instead appears protective, as inhibition of IFN- CCR6-enriched and -depleted T cells were measured. Levels signaling enhances the development of pathogenic Th17 and of both IL-23R and ROR  t mRNA were found to be sig- exacerbates autoimmunity ( 20 ). Even the neutralization of IL-4 nifi cantly higher in the CCR6-enriched than in the CCR6- produced by Th2 is critical in neutralizing the development of depleted population from either PB or tonsils ( Fig. 10 B , IL-17; however, neither IFN-  nor IL-4 seem to be eff ective bottom). Again, despite the presence of IL-23R, even freshly on already established Th17 ( 20 ). derived IL-17 – producing CD4 T cells did not proliferate Currently, very little is known about human Th17. Some in response to IL-23, both in the absence or presence of anti- studies have shown that the presence of IL-17 mRNA or IL-17 CD3/CD28 mAbs (unpublished data). protein in tissues or biological fl uids of subjects with diff erent autoimmune disorders ( 23 – 27 ) or other chronic infl amma- DISCUSSION tory diseases ( 28 – 30 ). The results of this study provide evi- Several studies demonstrate the existence in experimental an- dence of the existence of increased numbers of CD4 T cells imal models of a novel subset of Th eff ectors that are distinct producing IL-17 in the disease-aff ected gut areas of subjects from the classic Th1 and Th2, and that have been named with CD, compared with either PB or apparently healthy gut Th17 because of their ability to produce IL-17. These cells areas. Based on this fi nding, IL-17 – producing CD4 T cells represent a distinct lineage that originates mainly in the pres- derived from the gut of CD subjects were expanded in vitro ence of TGF-  and IL-6 and need the presence of IL-23 for and cloned to get an amount of these cells suitable for pheno- their expansion and/or maintenance. IL-23 is a member of typic and functional investigations. the IL-12 cytokine family, which shares the p40 subunit with The fi rst information produced by our study was that a IL-12 and diff ers in the expression of p19 instead of p35. At remarkable proportion of IL-17 – producing CD4 T cells JEM VOL. 204, August 6, 2007 1855 Figure 8. Effects of IL-12 on ROR  t and T-bet expression and on IFN-  and IL-17 production by Th17 clones. (A) Decreased ROR  t and increased T-bet mRNA expression in fi ve Th17 clones after stimulation for 7 d with anti-CD3 plus anti-CD28 Ab and IL-2, in the absence (black) or presence (gray) of IL-12. a versus b, P  0.05; c versus d, P  0.05. (B) Decreased IL-17 and increased IFN-  mRNA levels in Th17 clones cultured under the condi- tions described in A. Columns represent mean values (  the SEM). a versus b, P  0.01; c versus d, P  0.01. (C) Reduced levels of IL-17 and increased levels of IFN-  in supernatants of Th17 clones, as measured by ELISA. a versus b, P  0.05; c versus d, P  0.001. (D) Decreased proportions of IL-17 – producing and increased proportions of IFN-  – producing cells in response to PMA plus ionomycin by Th17 clones. a versus b, P  0.05; c versus d, P  0.05. (E) Flow cytometric analysis of IL-17 and IFN-  production by one representative Th17 clone. share the ability to produce IFN-  , whereas CD4 T cells gut mucosa and to get Th17 clones, is apparently at variance producing both IL-17 and -4 were never observed. The cells with recent results showing that IL-2 constrains generation producing both IL-17 and IFN-  , which we named Th17/ of mouse Th17 cells ( 36 ). The reason for such a discrepancy Th1, were not the result of an in vitro artifact, as about one is presently not clear. Among chemokine R, both Th17 and third of ex vivo – derived IL-17 – producing CD4 T cells Th17/Th1 clones apparently lacked CXCR3-A, CXCR3-B, already showed a double IL-17IFN-   phenotype. When CCR3, CCR8, and CCR9, but exhibited high levels of examined at clonal level, Th17 and Th17/Th1 shared dif- CXCR4 and CXCR6. Th17 clones also expressed signif- ferent phenotypic properties from the other types of clones, icantly higher levels of CCR4 and CCR5 than the other some of which appearing to be selective. For example, only types of clones, and both Th17 and Th17/Th1 selectively Th17 and Th17/Th1 clones exhibited the expression of expressed CCR6. CCR4 expression has been associated with IL-23R. Interestingly, the IL-23R did not apparently play any the ability of cells to traffi c into peripheral tissues ( 37 ). CCR6 role in the expansion of these cell subsets, because they did has been found to be expressed by B cells, DCs, and memory, not proliferate in response to IL-23 alone, and IL-23 did not but not naive, T cells. However, its ligand, MIP-3  /CCL20 potentiate their anti-CD3/CD28 Ab – induced prolifera- does not exert chemotactic activity on B cells, but only on tion. Thus, it is probable that IL-23 is not important for hu- memory T cells ( 38 ). Interestingly, however, CCR6 expres- man Th17 expansion, but rather for their survival and/or sion by memory T cells is lost after their prolonged TCR maintenance. In contrast, under the same conditions, anti- triggering ( 39 ). Thus, the selective expression of CCR6 by CD3/28 – stimulated Th17 and Th17/Th1 clones proliferated Th17 and Th17/Th1, but not by Th1, Th2, or Th0, clones in response to IL-2, -12, or -15. The ability of IL-2 to pro- may mean that Th17 and Th17/Th1 are the only memory mote the proliferation of human Th17, a fi nding that was T cells that continue to express CCR6 even after prolonged also supported by the observation that IL-2 was the growth antigen activation, thereby maintaining the possibility of factor used in this study to expand Th17 from both PB or recruitment in response to MIP-3  /CCL20. This fi nding may 1856 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE portant role of IL-17 – producing cells in the maintenance of the infl ammatory processes in autoimmune disorders. The receptor expression and functional capabilities ob- served in T cell clones derived from the aff ected areas of gut from subjects with CD were neither related to their patho- logical source nor to some in vitro artifact referable to the cloning procedure. Indeed, similar receptor expression and function were found in T cell clones derived from healthy gut areas of subjects who underwent colectomy because of colon carcinoma. More importantly, the existence of Th17 and Th17/Th1 cells, as well as their expression of CCR6, IL-23R, and ROR  t was also observed in freshly derived CD4 T cells from gut, PB, and tonsil. In this respect, it is of note that sorting of CCR6 cells allowed to obtain popu- lations strongly enriched in IL-17 – producing cells, suggesting that this marker may be useful to get high numbers of Th17 cells and, therefore, of potentially great help in clarifying their pathophysiologic role in humans. Of note, during the revision process of this paper, a study was published ( 40 ) that also reports preferential expression of CCR6 and ROR  t by human PB Th17 cells. Another important fi nding emerging from this study was the apparent discrepancy with some results reported in mice in regard to the relationship between Th17 and Th1. In a fi rst proposed mouse model, it was suggested that the early diff erentiation of Th1 and Th17 from naive CD4+ T cell precursors was shared, and thus Th1 and Th17 diverged contingent upon the selective availability of IL-12 or -23 Figure 9. IL-23 partially inhibits the increase of IFN-  production acting on a common “ Th1 precursor ” or “ pre-Th1 ” inter- and the proliferation induced by IL-12 on Th17 clones. (A) Detection mediate that coexpressed IL-12R and -23R ( 41 ). However, of IFN-  into cell-free supernatants of Th17 and Th1 clones cultured for 7 d with anti-CD3/CD28 and IL-2 (black), plus IL-12 (gray), -23 (white), in a subsequently proposed mouse model, which was based or -12 plus -23 (striped). Columns represent mean values (  the SEM) on the demonstration that the diff erentiation into Th17 de- obtained in three different Th17 and in three different Th1 clones. a versus b, pends upon TGF-  and IL-6 rather than upon IL-23, it was P  0.05; b versus c, P  0.05; d versus e, P  0.05; e versus f, P  0.05. suggested that Th1 and Th17 subsets were not overlapping (B) Flow cytometric analysis of IL-17 and IFN-  production by one and represented distinct lineages ( 19, 20 ). In this study, we representative Th17 clone, and of its proliferation, as assessed by CFDA- do not provide any evidence for the mechanisms responsi- SE content. Similar results were obtained in other two Th17 clones. ble for the diff erentiation of human Th17, as well as for the question of whether in humans, as in mice, Th17 and Th1 have important implications for the long-term maintenance represent distinct lineages. However, we not only demon- of Th17 infl ux, supporting their important pathogenic role strated the ex vivo existence of CD4 T cells able to pro- into the infl amed tissues. duce both IL-17 and IFN-  but we were also able to induce Human Th17 and Th17/Th1 clones also shared some Th17 clones to produce IFN-  in addition to IL-17 after peculiar functional properties. We found that both types of their culturing in the presence of IL-12. The possible rela- clones were able, as Th1 clones, to provide B cell help for the tionship between human Th17 and Th1 was also supported production of IgM, IgG, and IgA, but not IgE, Ab. In contrast, by the demonstration that Th17 clones expressed not only as Th2 clones, they showed poor granzyme A expression and ROR  t, a fi nding that is in agreement with the results re- low cytotoxic capability, thus displaying an intermediate func- ported in mice ( 21 ), but also remarkable amounts of the tional pattern in regard to B cell helper and cytotoxic activity Th1 transcription factor T-bet, and that amounts of both between Th1 and Th2 clones. However, the most impressive T-bet and ROR  t were comparable in human Th17 and functional feature of both Th17 and Th17/Th1 clones was Th17/Th1 clones. their lower susceptibility, in comparison with both Th1 and Interestingly, the ability of IL-12 to induce Th17 cells to Th2 clones, to the suppressive activity of an autologous T reg produce IFN-  , in addition to IL-17 associated with the up- cell clone derived from circulating CD4CD25highFoxp3 regulation of T-bet and the down-regulation of both ROR  t cells. Although the mechanism for the higher resistance of expression and IL-17 production in Th17 clones. This means Th17 and Th17/Th1 to the action of T reg cells is still un- that in humans, even established Th17 clones, independent clear, this fi nding can provide additional support for an im- of whether they have a distinct or common origin with Th1, JEM VOL. 204, August 6, 2007 1857 Figure 10. Th17 clones derived from normal gut, as well as freshly derived IL-17 – producing CD4 T cells, exhibit similar features to gut CD-derived Th17 clones. (A) CCR6, IL-23R, and ROR  t mRNA expression by Th1, Th17, and Th17/Th1 clones derived from normal gut of subjects who underwent colectomy because of colon carcinoma. Columns represent mean values (  the SEM) obtained from fi ve clones of each type. (B) Detection of IL-17 and IFN-  production by CCR6-depleted and CCR6-enriched CD4 T cell populations from PB or tonsil (top). Measurement of IL-23R and ROR  t mRNA levels in CCR6-depleted (black columns) and CCR6-enriched (red columns) CD4 T cells from PB (left) or tonsils (right). Columns represent mean values  the SEM were obtained in three different donors of PB or tonsil. ported in studies performed in mice ( 42, 43 ). Second, T-bet are responsive to IL-12 and are still suffi ciently fl exible to ac- has been found to be required for optimal IL-17 production quire the ability to produce IFN-  . These fi ndings strongly in the presence of IL-23 ( 44 ). Finally, therapeutic admin- suggest that in human Th17 both ROR  t and T-bet can istration of a small interfering RNA specifi c for T-bet sig- play an important modulatory role. The possibility that T-bet – nifi cantly improved the clinical course of established EAE dependent signaling was responsible for the increased pro- by limiting the diff erentiation of autoreactive Th1 and in- duction of IFN-  by our T cell clones was not directly explored hibiting pathogenic Th17 via regulation of IL-23R ( 45 ). in this study, and thus remains unproved. However, it has re- These fi ndings suggest that even in mouse models T-bet and cently been shown that ectopic T-bet expression in mouse ROR  t may play distinct, yet complementary, roles in the Th17 cells can promote IFN-  secretion and decrease IL-17 development of Th17 cells. In this study, the IL-12 – induced production, suggesting that even in mice the Th17 pheno- IFN-  production by Th17 clones was partially inhibited by type is not stable and can give rise to IFN-  production in the contemporaneous presence of IL-23. Because a similar vitro via T-bet – mediated signaling ( 42 ). In this respect, it inhibitory eff ect by IL-23 on the IL-12 – induced IFN-  pro- should be taken into account that other recent data in mice, duction could also be observed in Th1 clones, which do not again, raise the question of the developmental relationship express IL-23R, it is reasonable to suggest that it was mediated between Th1 and Th17. First, the existence of a dual popula- by the ability of IL-23 to bind both IL-23R and IL-12R  1 tion of IL-17- and IFN-  – producing cells has also been re- 1858 HUMAN T H 17 CELLS | Annunziato et al. ARTICLE Immunomagnetic cell sorting. Separation of CCR6 from CCR6 PB via the p40 chain shared between IL-12 and -23, which there- or tonsillar CD4 T cells was performed by immunomagnetic cell sorting, fore display binding competition. Because IFN-  is a well as previously described ( 49 ). known inducer of IL-12R  2 ( 46 ), it can strongly infl uence the susceptibility of Th17 to the activity of IL-12, thus favor- T cell expansion and cloning. Gut-derived and PB T lymphocytes were ing the shifting of these cells toward the Th1 phenotype. This expanded by stimulation for 10 d with 5 g/ml anti-CD3 plus 5 g/ml anti- fi nding may also account for the reported inhibitory activity CD28 mAb (BD Biosciences) and 20 UI/ml IL-2 (Eurocetus). On day 10, T cell blasts were cloned under limiting dilution (0.3 cell/well), as previously of IFN-  on the development of mouse Th17, as well as for reported ( 50 ). T reg clones were generated from CD4CD25Foxp3 T its protective eff ects ( 47 ). lymphocytes obtained from PBMC of one subject with CD. To this end, DCs In conclusion, our data provide the fi rst detailed pheno- were generated from CD14+ cells cultured for 5 d with IL-4 and GM-CSF. typic and functional characterization of human Th17 isolated CD4CD25 or CD25high T cells were obtained from PBMC by cell sort- from the disease-aff ected gut mucosa of subjects with CD, ing. CD4CD25high cells were cultured with autologous DCs (1:1 ratio) and as well as from normal tissues, showing that Th17 display 100 U/ml IL-2 for 10 d and cloned under limiting dilution (0.3 cell/well). distinct functional properties from Th1 or Th2 cells and identi- RNA isolation, cDNA synthesis, and real-time quantitative RT- fying IL-23R, CCR6, and ROR  t as Th17 – specifi c markers. PCR. Total RNA was extracted by using the RNeasy Micro kit (QIAGEN) In addition, we describe a new subset of IFN-  – producing and treated with DNase I to eliminate possible genomic DNA contamina- Th17 sharing features with both Th1 and Th17, which has tion. Taq-Man RT-PCR was performed, as described elsewhere ( 51 ). Prim- not been previously reported in mice. This novel subset ex- ers and probes used were purchased from Applied Biosystems. ists in vivo in humans and can be induced in vitro by stim- ulating Th17 in presence of IL-12, thus raising new issues Proliferation assay. 5 10 total T cell blasts from each clone were stimu- lated with 2 g/ml anti-CD3 plus 2 g/ml anti-CD28 mAb (BD Bio- on the Th17 developmental and/or functional relationship sciences), in the absence or presence of diff erent cytokines (rIL-2, 50 IU/ml; with Th1. rIL-12, 2.5 ng/ml; rIL-15, 7 ng/ml; rIL-23, 20 ng/ml). In T cell suppression experiments, 2 10 T cell blasts from Th1, Th2, Th17, or Th1/Th17 clones MATERIALS AND METHODS were cultured with 4 10 irradiated (9,000 rad) T cell-depleted allogeneic Subjects. Small bowel specimens were obtained from the apparently healthy 4 high PBMC, with or without 2 10 T cell blasts from a CD4+CD25 Foxp3+ areas of 10 subjects who underwent surgery because of colon cancer, the T clone. Cultures were pulsed for the last 8 h with 0.5 Ci (0.0185 MBq) disease-aff ected areas of 10 subjects with CD, and the disease-aff ected areas of H-TdR (GE Healthcare), harvested, and radionuclide uptake was mea- of 5 subjects with ulcerative colitis. PB samples were obtained from the 10 sured by scintillation counting. In some experiments, cells were labeled with subjects with CD and from 13 healthy donors. Tonsillar specimens were ob- CFDA-SE (Invitrogen), as previously described ( 52 ). tained from three children, who were subjected to tonsillectomy because they were aff ected by chronic tonsillitis. The procedures followed in the study Calcium infl ux. 5 10 cells from a Th1 and a Th17 clone were incu- were in accordance with the ethical standards of the Regional Committee on bated in complete medium in the presence of 2.5 M Fluo-4 AM (Invitro- Human Experimentation. gen) for 30 min in the dark at room temperature. After dye loading, cells were washed twice in complete medium and incubated at 37 ° C in the pres- Reagents. The medium used was RPMI 1640 (Seromed) supplemented ence of 5% CO for an additional 30 min. Cells were analyzed by a BD-LS- with 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 2 RII fl ow cytometer using the Diva software (Becton Dickinson). The basal 10 M 2-mercaptoethanol (2-ME; all from Invitrogen), and 10% FCS. fl uorescence intensity was assessed before the stimulus. The stimulation was FITC-, PE-, allophycocyanin-orperidin chlorophyll protein – conjugated obtained in presence of medium alone or in the presence of 1 g/ml CCL20, anti-CD3, CD4, CD8, CD25, CD45RA, CD45RO, TCR   , TCR  , 1 g/ml CCL25, or 1 M ionomycin. IFN-  , IL-4, and isotype-matched control mAbs were purchased from BD Biosciences. Anti – IL-17 and -Foxp3 mAbs were obtained from eBioscience. 5 B cell helper activity. 2 10 human B cells, isolated as CD19+ cells by PMA, ionomycin, and brefeldin A were purchased from Sigma-Aldrich. 5 immunomagnetic cell sorting ( 49 ), were stimulated with 10 Th1, Th2, Anti-CCR6 mAb was from R & D Systems. Anti-ROR  t rabbit polyclonal Th17, or Th1/Th17 blasts stimulated with 100 ng/ml anti-CD3 Ab. On day Ab was purchased from Abcam. Anti – T-bet rabbit polyclonal Ab was from 7, supernatants from each cell culture were collected and evaluated by ELISA Santa Cruz Biotechnology. for IgM, IgG, IgA, and IgE content. Flow cytometric detection of granzyme A and evaluation of cell T cell recovery. For isolation of T lymphocytes, epithelial gut layer was cytotoxicity. Granzyme A expression by Th1, Th2, Th17, or Th17/Th1 removed by calcium- and magnesium-free PBS, supplemented with 1 mM clones was assessed according to the manufacturer ’ s instructions (BD Bio- EDTA and 1 mM DTT (Sigma-Aldrich). Fragments were mechanically dis- sciences). Th1, Th2, Th17, and Th1/Th17 cell clones were tested for their rupted with a MEDI machine (BD Biosciences). MCs were then separated ability to kill P815 target cells upon anti-CD3 mAb activation (5 g/ml) by from the other cells by Ficoll-Hypaque gradient centrifugation. PBMC and evaluating the percentage of AnnexinV-binding cells with fl ow cytometry, tonsil mononuclear cell suspensions were also obtained by centrifugation on as described elsewhere ( 52 ). Ficoll-Hypaque gradient. Statistics. Statistical comparison between groups was performed using the Flow cytometry. Analysis of cytokine synthesis at the single-cell level was Mann-Whitney test or the Wilcoxon test, as appropriate. Diff erences were performed as previously reported ( 48 ). Foxp3 fl ow cytometric determination considered as statistically signifi cant when P  0.05. was performed according to the manufacturer ’ s instructions (eBioscience). The experiments reported in this paper were performed with grants from the Confocal microscopy. Detection of ROR  t and T-bet protein in Th17 Associazione Italiana Per La Ricerca Sul Cancro, the Ministero dell ’ Istruzione, and Th1 clones was performed by confocal microscopy, by using a previ- dell ’ Universit à e della Ricerca, the Italian Ministry of Health, and FP6 European ously described technique ( 49 ). Union project INNOCHEM, LSHB-CT 2500-518157. JEM VOL. 204, August 6, 2007 1859 The authors have no confl icting fi nancial interests. 19 . Sutton , C. , C. Brereton , B. Keogh , K.H. Mills , and E.C. Lavell . 2006 . 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Stem Cells . M.K. Racke , and A.E. Lovett-Racke . 2007 . T-bet regulates the 24 : 386 – 398 . JEM VOL. 204, August 6, 2007 1861

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The Journal of Experimental Medicine – Pubmed Central

Published: Aug 6, 2007

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