Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP-690,550) Kamran Ghoreschi, Michael I. Jesson, Xiong Li, Jamie L. Lee, Sarbani Ghosh, Jason W. Alsup, James D. Warner, This information is current as Masao Tanaka, Scott M. Steward-Tharp, Massimo Gadina, of October 1, 2021. Craig J. Thomas, John C. Minnerly, Chad E. Storer, Timothy P. LaBranche, Zaher A. Radi, Martin E. Dowty, Richard D. Head, Debra M. Meyer, Nandini Kishore and John J. O'Shea J Immunol 2011; 186:4234-4243; Prepublished online 7 March 2011; Downloaded from doi: 10.4049/jimmunol.1003668 http://www.jimmunol.org/content/186/7/4234 http://www.jimmunol.org/ References This article cites 79 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/186/7/4234.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP-690,550)

Kamran Ghoreschi,*,1 Michael I. Jesson,†,1 Xiong Li,† Jamie L. Lee,† Sarbani Ghosh,† Jason W. Alsup,† James D. Warner,† Masao Tanaka,‡ Scott M. Steward-Tharp,* Massimo Gadina,‡ Craig J. Thomas,x John C. Minnerly,† Chad E. Storer,† Timothy P. LaBranche,† Zaher A. Radi,† Martin E. Dowty,† Richard D. Head,† Debra M. Meyer,† Nandini Kishore,† and John J. O’Shea*

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we Downloaded from analyzed stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4+ T cells. CP-690,550 inhibited IL-4–dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 IL-17A, IL- 17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-b. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly http://www.jimmunol.org/ improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP- 690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling. The Journal of Immunology, 2011, 186: 4234–4243. by guest on October 1, 2021 ytokines are key mediators of the development and ho- regulate the expression of numerous genes (4). The vital role of meostasis of hematopoietic cells, playing crucial roles in JAK signaling is illustrated best by the circumstances where these C controlling both innate and adaptive immunity (1, 2). kinases are mutated or deleted (5, 6). For instance, although Type I and II cytokine receptors represent a structurally distinct germline deletion of either JAK1 or JAK2 is lethal, mutation of class of integral membrane proteins that lack intrinsic enzymatic JAK3 or TYK2 in humans and mice results in immunodeficiency activity and associate with a family of cytoplasmic protein tyro- (7, 8). TYK2 mainly transmits the signals derived from type I sine kinases known as JAKs. Upon cytokine-induced activation, IFNs and the IL-12 receptor b1 subunit sharing receptors for IL- JAKs phosphorylate the cytoplasmic tail of the receptor, leading to 12 and IL-23 (9), whereas JAK3 has a more discrete function and the recruitment of STATs, which also are phosphorylated by JAKs associates only with the IL-2 receptor common g chain (gc) shared (3). Activated STATs dimerize, translocate to the nucleus, and by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (2). Deficiency of JAK1 leads to nonresponsiveness to type I and type II IFNs, g cytokines, and gp130 subunit-utilizing cytokines (10), *Molecular Immunology and Inflammation Branch, National Institute of Arthritis c and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, whereas JAK2-deficient cells fail to respond to hormone-like MD 20892; †St. Louis Laboratories, Pfizer Global Research and Development, Ches- cytokines, such as , , and GM-CSF terfield, MO 63017; ‡Translational Immunology Section, Office of Science and Tech- nology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, (11). National Institutes of Health, Bethesda, MD 20892; and xNational Institutes of Health JAKs play a critical role in mediating inflammatory immune Chemical Genomics Center, National Human Genome Research Institute, National responses, and their pharmacological modulation represents a Institutes of Health, Bethesda, MD 20892 1 novel approach to the treatment of inflammatory immune- K.G. and M.I.J. contributed equally to this work. mediated diseases. Indeed, the JAK/STAT pathway has gained Received for publication November 3, 2010. Accepted for publication February 1, significant attention as a therapeutic target in inflammation, au- 2011. toimmune disease, hematopoietic disorders, and transplant re- This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases and Pfizer, Inc. jection (12, 13). Several small molecule JAK inhibitors have been Address correspondence and reprint requests to Dr. Kamran Ghoreschi and Dr. John developed and are currently under clinical investigation (14– J. O’Shea, Molecular Immunology and Inflammation Branch, National Institute of 17). Tofacitinib (CP-690,550, formerly tasocitinib) is a selective Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Build- inhibitor of the JAK family with nanomolar potency and a ing 10, Room 13C103, 10 Center Drive, Bethesda, MD 20892. E-mail addresses: [email protected] and [email protected] high degree of kinome selectivity (18–20). In cellular assays, it has demonstrated potent inhibition of gc cytokine signaling Abbreviations used in this article: gc, common g chain; IHC, immunohistochemistry; RA, rheumatoid arthritis; SAA, serum amyloid A; siRNA, small interfering RNA. by blocking IL-2–driven proliferation and functional www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003668 The Journal of Immunology 4235 selectivity over JAK2-dependent GM-CSF–driven proliferation of tiation, cells were stimulated with PMA and ionomycin in the presence of HUO3 cells (18). More recently, CP-690,550 has been shown to brefeldin A for 4 h, fixed in 4% formyl saline, and permeabilized with potently inhibit both JAK3- and JAK1-dependent STAT activation 0.1% saponin buffer prior to intracellular cytokine staining and flow cytometry using a FACSCalibur (BD Biosciences) with FlowJo software with selectivity over JAK2-mediated pathways (21). Results from (Tree Star, Ashland, OR). The following Abs were used: anti–IL-2–allo- a phase II trial of oral CP-690,550 as a monotherapy in patients phycocyanin, –PE, or –FITC, anti–IL-17A–PE, anti–IFN-g–allophyco- with rheumatoid arthritis (RA) showed efficacy, with 70–80% cyanin, –PE, or –FITC, anti–IL-13–PE (all from BD Biosciences), anti–IL- of patients achieving 20% improvement in the American College 17A–allophycocyanin or –FITC, anti–IL-17F–Alexa Fluor 647, anti–IL- 22–PE, and anti–T-bet–Alexa Fluor 647 (all from eBioscience, San Diego, of criteria and an acceptable safety profile CA). To study proliferation, T cells were labeled with 1 mM CFSE (22). CP-690,550 is being evaluated currently in phase III trials (Invitrogen) before culture. in RA and other immune-mediated diseases, including , Mouse arthritis model and LPS model Crohn’s disease, and organ transplant rejection (15, 23) (Clin- icalTrials.gov [http://clinicaltrials.gov/] NCT00615199). Other DBA/1J mice were immunized s.c. with 50 mg of chicken type II collagen JAK inhibitors being studied in the setting of autoimmune disease (Western Institute for Biomedical Research, Salt Lake City, UT) emulsified in CFA (Sigma-Aldrich) and boosted 21 d later with 50 mg of the same Ag include the JAK3 inhibitors VX-509 and WYE-151650, the JAK1/ in IFA (Sigma-Aldrich). In therapeutic efficacy studies, disease was JAK2 inhibitors INCB028050 and INCB018424, and the JAK3/ monitored beginning on day 45, and severity was scored on a scale of 0–3 Spleen tyrosine kinase inhibitor R348 (12, 13, 17, 24, 25) (Clin- for each paw, as described previously (28). Mice were sorted into groups of icalTrials.gov identifiers NCT00902486, NCT00550043, and equivalent severity and dosed orally twice daily with vehicle or 50 mg/kg NCT00789126). CP-690,550 beginning on day 48. For protein and gene expression anal- yses, groups of mice were bled by cardiac puncture and euthanized, and Because CP-690,550 and the other inhibitors of this class target both hind paws were excised and flash-frozen 4 h post-treatment on days Downloaded from more than one JAK, their exact mode of action in the setting of 48, 49, 52, and 55. For histopathology and immunohistochemistry (IHC) inflammatory disease has not been resolved. Autoimmune diseases analyses, fore and rear paws were harvested 4 and 12 h post-treatment on can be driven by CD4+ T cells that produce IFN-g (Th1 cells), day 48 and 4 h post-treatment on days 49 and 55 and fixed in 10% neutral buffered formalin. In studies used to correlate JAK inhibition with efficacy, IL-17 (Th17 cells), or combinations of the two (26). The in- mice were orally administered vehicle or varying twice daily doses of CP- flammatory response is supported by innate immune mechanisms 690,550 on days 22–56, disease was monitored beginning on day 42, and that are also particularly relevant in autoimmunity (27). To begin severity was scored as described. Efficacy was determined using the area to clarify the mechanism of JAK inhibition vis-a`-vis the cognate under the curve of the disease severity time course for each dose. http://www.jimmunol.org/ cytokines that are blocked, we revisited the effects of CP-690,550 DBA/1J or C57BL/6J mice were orally administered vehicle or a 5 mg/kg CP-690,550 and 1 h later injected i.p. with 10 mgofSalmonella typhosa on adaptive and innate immune responses. LPS (Sigma-Aldrich). Plasma was collected before LPS administration as well as 1, 2, and 6 h after LPS injection. Materials and Methods Plasma cytokine determination Mice Plasma cytokines were detected using Luminex-based murine multiplex assays (Millipore, St. Charles, MO) and Luminex 200 instrumentation DBA/1J and C57BL/6J mice were purchased from The Jackson Laboratory (Luminex, Austin, TX) or with Meso Scale Discovery technology (Gai- (Bar Harbor, ME), and STAT1-deficient mice and littermate controls on

thersburg, MD). Serum amyloid A (SAA) was detected using a murine SAA by guest on October 1, 2021 a 129S6/SvEv background were purchased from Taconic (Hudson, NY). ELISA (Invitrogen) following the manufacturer’s protocol. Use of the animals in these studies was reviewed and approved by the Pfizer Institutional Animal Care and Use Committee or by the Institutional Animal Knockdown of JAK1 and JAK3 with small interfering RNA Care and Use Committee of the National Institute of Arthritis and Mus- culoskeletal and Skin Diseases. The animal care and use program at Pfizer Human CD4+ T cells were purified by negative selection from PBMCs is fully accredited by the Association for Assessment and Accreditation of using magnetic cell separation technology. Cells were transfected with 1.4 Laboratory Animal Care International. Experiments were performed ac- mM SMARTpool small interfering RNA (siRNA) for human JAK1 or cording to the National Institutes of Health guidelines for the use of live JAK3 (Dharmacon, Lafayette, CO) or with a scrambled control using animals. Nucleofector technology (Lonza). Transfected cells were stimulated with 100 ng/ml IL-6 or IL-7 for 15 min, and STAT phosphorylation was JAK inhibitor assessed by intracellular flow cytometry, as described below. CP-690,550 was prepared by Pfizer Research Laboratories or by the Na- RNA isolation and gene expression tional Institutes of Health Chemical Genomics Center and resuspended in 0.5% methylcellulose/0.025% Tween 20 (Sigma-Aldrich, St. Louis, MO) For cultured T cells, total RNA was isolated using the mirVana miRNA for in vivo studies or in DMSO for in vitro use. isolation (Applied Biosystems, Foster City, CA). Frozen paw tissue was powdered in a freezer mill, and total RNA was prepared from each sample T cell purification and differentiation using TRIzol (Invitrogen). Relative gene expression levels were de- CD4+ T cells were purified by negative selection from spleens and lymph termined by quantitative RT-PCR using Taqman Gene Expression primer nodes of C57BL/6J and STAT1-deficient or wild-type 129S6/SvEv mice probe sets and ABI PRISM 7700 or 7900 Taqman systems (Applied Bio- using magnetic cell separation technology (Miltenyi Biotec, Auburn, CA), systems). The comparative threshold cycle method and internal controls + + + 2 ( or b-actin) were used to normalize the expression of tar- and the naive CD4 CD62L CD44 CD25 population was sorted using a FACSAria II (BD Biosciences, San Jose, CA). Naive T cells were acti- get genes. vated by plate-bound anti-CD3/anti-CD28 (10 mg/ml) in fully supple- Western blotting mented RPMI 1640 medium containing 10% FCS, 2 mM glutamine, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, HEPES buffer (all from Invi- Freshly isolated CD4+ T cells were activated with plate-bound anti-CD3/ trogen, Carlsbad, CA), and 2 mM 2-ME (Sigma-Aldrich) or if indicated in anti-CD28 and expanded with IL-2. Cells were washed and rested in fresh serum-free medium (X-VIVO 20; Lonza, Walkersville, MD) for 3–4 d. medium in the presence or absence of CP-690,550 for 30 min before the During T cell activation, cells were treated with the indicated concen- addition of the indicated cytokines (100 ng/ml). After stimulation, cells trations of CP-690,550 dissolved in DMSO. Th1 cells were polarized in the were lysed in Triton X-100 lysis buffer containing protease inhibitors. presence of IL-12 (20 ng/ml) and anti–IL-4 (10 mg/ml), and Th2 cells in Equal amounts of total protein were separated by PAGE, transferred to the presence of IL-4 (10 ng/ml) and anti–IFN-g (10 mg/ml). For Th17 cell nitrocellulose, and blotted with Abs recognizing actin (Millipore), p-AKT, generation, CD4+ T cells were stimulated with the indicated combinations and specific p-STATs (Cell Signaling Technology, MA, or Invitrogen). of IL-6 (20 ng/ml), TGF-b1 (0.5 ng/ml), IL-23 (50 ng/ml), or IL-1b (20 IRDye800- (Rockland, Gilbertsville, PA) and Alexa Fluor 680-labeled ng/ml; all from R&D Systems, Minneapolis, MN) in the presence of anti– (Invitrogen) secondary Abs were used for detection, and specific bands IFN-g Abs (10 mg/ml). TGF-b signaling was neutralized by using anti– were visualized using an Odyssey infrared imaging system (LI-COR Bio- TGF-b1 and anti–TGF-b2 Abs (5 mg/ml; R&D Systems). After differen- sciences, Lincoln, NE). 4236 CP-690,550 CONTROLS T CELL DIFFERENTIATION AND INFLAMMATION

STAT phosphorylation in whole blood Heparinized blood from normal human donors was preincubated with CP- 690,550 for 1 h prior to cytokine stimulation. Nonimmunized DBA/1J mice were orally administered varying doses of CP-690,550, and blood was collected after 1 h. Alternatively, mice immunized to develop collagen- induced arthritis (CIA) were orally administered varying doses of CP- 690,550 twice daily on days 22–56, and blood was collected 1 h after the final dose. Whole blood leukocytes were surface-labeled with FITC- and PE-labeled lineage-specific Abs (BD Biosciences or eBioscience) in advance of cytokine stimulation. CD3 was used to identify human T cells, CD3 and CD8 were used to identify mouse T cell subsets, and CD11b and F4/80 were used to identify mouse monocytes. Blood was stimulated with or without cytokine (100 ng/ml IL-2, IL-4, IL-6, IL-7, IL-15, or IL-21 or 20 ng/ml GM-CSF) for 15–20 min, and activation was stopped by the addition of Lyse/Fix Buffer (BD Biosciences) following the manu- facturer’s protocol. Cells were washed, permeabilized in ice-cold Perm Buffer III (BD Biosciences) for 20–30 min, and stained intracellularly with Alexa Fluor647-conjugated p-STAT–specific mAb (BD Biosciences). Flow cytometric analysis was performed on a FACSCalibur. In human, STAT phosphorylation was assessed in CD3+ T cells; however, in mice, IL- 15– and IL-6–driven STAT activation was examined within CD8+ T cells, because that subpopulation reproducibly yielded greater signal for quan- titative evaluation of inhibition. Alexa Fluor 647 geometric mean channel Downloaded from fluorescence derived from gated populations was used to determine the percentage of control stimulation by comparison of compound-treated and vehicle-treated animals. Plasma from each sample was collected, and CP- FIGURE 1. CP-690,550 inhibits g cytokine signaling in T cells. Equal 690,550 concentration was determined by liquid chromatography/mass c numbers of mouse CD4+ T cells were preincubated with the indicated spectroscopy. concentrations of CP-690,550 before stimulation with or without IL-2 for Histopathology and IHC 15 min. A, Activation of STAT5 and AKT were determined in cell lysates by immunoblotting with phospho-specific Abs. Mouse CD4+ T cells were http://www.jimmunol.org/ Fixed paws were decalcified in Immunocal (Decal Chemical, Tallman, NY) preincubated with or without 0.3 mM CP-690,550 and stimulated with IL- for 7 d and paraffin-embedded. To assess general inflammation, 4-mm 21 for the indicated time periods. B, Activation of STAT3 and STAT1 was sections were stained with H&E, independently examined by two board- certified veterinary pathologists (T.P.L. and Z.A.R.), and scored semi- determined as in A. Human whole blood was preincubated with or without quantitatively, as described previously (29). For monocyte/macrophage CP-690,550 before stimulation with the indicated cytokines for 15 min. C, + IHC, tissue sections were treated with proteinase K (Dako, Carpinteria, The extent of specific STAT phosphorylation within the CD3 T cell CA) and blocked. Incubation with anti-F4/80 mAb (eBioscience) was population was assessed by intracellular flow cytometry using phospho- followed by HRP-conjugated rat-on-mouse micropolymer (Biocare Med- specific Abs. Results are representative of eight separate experiments. ns, ical, Concord, CA), diaminobenzidine detection (Dako), and hema- no cytokine stimulation. toxylin counterstaining. Matched rat IgG was used as a negative control. T cell IHC used Borg high pH retrieval (Biocare Medical) followed by by guest on October 1, 2021 incubation with a rabbit anti-CD3 Ab (Accurate Chemical, Westbury, NY). HRP-conjugated secondary Ab incubation was followed by detection with cytokine receptors in both mouse and human T cells. Because diaminobenzidine and hematoxylin counterstaining. Macrophage and evidence from kinase binding assays has shown that CP-690,550 T cell infiltration were scored semiquantitatively, as described previously also can affect JAK family members other than JAK3 (19), we (30). next asked if the inhibitor also interfered with non-gc signaling in T cells.

Results CP-690,550 disrupts non-gc cytokine signaling + CP-690,550 disrupts gc cytokine signaling in CD4 Th cells To investigate the effects of CP-690,550 on JAK3-independent CP-690,550 was designed originally as a JAK3 inhibitor and cytokine receptor signaling, we stimulated CD4+ T cells with therefore was expected to interfere with gc cytokine signaling. As IL-6, a key inflammatory mediator in CIA and RA (34, 35). IL-6 shown in Fig. 1A, IL-2 induced the phosphorylation of STAT5 and signaling involves JAK1 in conjunction with JAK2 (2, 10, 11), and AKT, and CP-690,550 inhibited both events very effectively. Al- in human cells, TYK2 is also a contributor (36, 37). As depicted in though it is well established that STATs are JAK substrates, the Fig. 2A, CP-690,550 inhibited IL-6–driven phosphorylation of ability of CP-690,550 to inhibit AKT phosphorylation argues that STAT3 and STAT1 in mouse CD4+ T cells. Similar results were this pathway is also downstream of JAKs. The CP-690,550–re- obtained in human whole blood T cells (Fig. 2B). In both cases, lated compound PF-956980 also has been shown to inhibit IL-7– STAT1 phosphorylation was more sensitive to CP-690,550 than mediated AKT phosphorylation in human thymocytes (31). These STAT3 phosphorylation. To further examine the role that partic- results indicate that JAK inhibition interferes with both of the ular JAK family members might play in STAT activation, we used major pathways emanating from cytokine receptors. RNA interference (Fig. 2C). Interestingly, inhibiting JAK1 ex- IL-21 is a critical immunoregulatory cytokine with important pression in human CD4+ T cells completely suppressed IL-6– actions on T cells, B cells, and NK cells (32), and it too uses gc mediated STAT1 phosphorylation but had only a partial effect on (33). As expected, CP-690,550 interfered with IL-21 signaling in STAT3 phosphorylation. In these studies, JAK1 inhibition corre- mouse CD4+ T cells, as shown by the inhibition of STAT3 and sponded to .75% reduction in transcript copy number compared STAT1 phosphorylation (Fig. 1B). We also investigated the ability with control siRNA, as assessed by quantitative RT-PCR. JAK1 of CP-690,550 to inhibit gc cytokine signaling in human T cells, siRNA also abrogated IL-7–dependent STAT5 phosphorylation. and as depicted in Fig. 1C, the inhibitor blocked STAT phos- As a control, JAK3 siRNA was included, and although it had no phorylation induced by IL-2, IL-4, IL-7, IL-15, and IL-21 with effect on IL-6 signaling, JAK3 knockdown completely suppressed similar potencies. These results confirmed that CP-690,550 clear- IL-7–mediated STAT5 phosphorylation, as would be expected. ly affects signaling pathways downstream of JAK3-dependent gc These data suggest that at least in T cells both IL-6 activation of The Journal of Immunology 4237

IL-13 (Fig. 3A,3B). As expected, the expression of GATA3 and Th2 cytokines was inhibited by CP-690,550. However, the TCR- mediated proliferation of Th cells was not affected. IL-2 pro- duction was enhanced, consistent with the recognized ability of IL-2 to limit its own production in Th2 and Th1 cells by activating STAT5 (38). It should be noted that adding a strong TCR stimulus (i.e., anti-CD3/anti-CD28) generates cells that produce little IL-4 protein (39). IL-4 mRNA expression was readily detectable in Th2 cells and abolished in the presence of CP-690,550 (data not shown). These findings are consistent with the efficacy of CP-690,550 in preclinical models of Th2-mediated allergic dis- ease (40). Whereas Th2 cells are nonpathogenic in experimental autoim- mune models like CIA, IFN-g–producing Th1 cells and IL-17– producing Th17 cells have been reported to be responsible for destructive arthritis in mice and humans (41). Therefore, we next studied the effects of CP-690,550 on Th1 cell differentiation. The inhibitor potently suppressed the expression of T-bet and the dif- ferentiation of IFN-g–producing Th1 cells without suppressing cell proliferation (Fig. 3A,3C). As in Th2 cells, CP-690,550 also Downloaded from enhanced IL-2 production in Th1 cells. Th1 specification is ini- tiated by IL-12 and STAT4 activation; however, IFN-g amplifies T-bet and IFN-g expression in Th1 cells through STAT1 activation (42). Of note, the inhibition of T-bet and IFN-g expression by CP- FIGURE 2. Inhibition of JAK-mediated IL-6, IFN-g, and IL-12 sig- 690,550 was to the same extent as that seen with IFN-g neutral- naling by CP-690,550. A and B, Mouse CD4+ T cells (A) or human whole izing Ab or STAT1-deficient T cells (Fig. 3D). In view of these http://www.jimmunol.org/ blood CD3+ T cells (B) were preincubated with the indicated concen- data and that presented in Fig. 2D and 2E, we would argue that the trations of CP-690,550 and stimulated with IL-6 for 15 min. Activation of primary mechanism by which CP-690,550 appears to inhibit Th1 STAT3 or STAT1 was determined by phospho-specific Abs and immuno- differentiation is through the inhibition of IFN-g– and IL-12– blotting (A) or flow cytometry (B). C, Cytokine-induced STAT activation mediated STAT1 signaling. after JAK1 or JAK3 siRNA transfection of human CD4+ T cells was assessed by flow cytometry and represents the percentage of STAT phos- CP-690,550 inhibits the generation of IL-23–dependent Th17 phorylation compared with that of control siRNA-transfected cells. Data cells represent the mean 6 SEM from three separate experiments. D and E, + Although Th1 cells were thought originally to be the major me-

Mouse CD4 T cells were preincubated with CP-690,550 at the indicated by guest on October 1, 2021 concentrations before stimulation with IFN-g for 30 min (D) or IL-12 for diators of immunopathogenesis, it now is recognized increas- 15 min (E). STAT activation was determined as in A. ns, no cytokine ingly that Th17 cells are also important drivers of autoimmunity stimulation. (41, 43, 44). Extensive work indicates that cells that selec- tively produce IL-17A but not other cytokines can arise from naive + STAT1 and gc cytokine activated STAT pathways are critically CD4 T cells in response to specific cytokine stimulation (45). dependent on JAK1. Using the same approach, we were unsuc- IL-23 was thought initially to be important for driving Th17 cessful in our attempts to suppress JAK2 or TYK2 expression differentiation; however, it was argued later that IL-6 in con- using RNA interference. junction with TGF-b was responsible for the initial specification Because CP-690,550 blocked IL-6 signaling, we next studied the of mouse Th17 cells (46–48). In human cells, the requirement for effects of JAK inhibition on IFN-g, a cytokine that also utilizes TGF-b has been less clear, and lately the necessity for TGF-b in JAK1 and JAK2. The inhibitor potently blocked the IFN-g–me- mouse has been called into question (49, 50). We have shown diated phosphorylation of STAT1 in CD4+ T cells (Fig. 2D), recently that Th17 cells can be generated from naive T cells in the further confirming that this inhibitor targets not only JAK3 but absence of TGF-b signaling when using IL-23, IL-1b, and IL-6 also JAK1 and/or JAK2. We then considered the possibility that (51). Such IL-23–induced Th17 cells express a distinct repertoire CP-690,550 might also interfere with IL-12 signaling, which is of transcription factors, receptors, and mediators and are more dependent on JAK2 and TYK2. As shown in Fig. 2E, CP-690,550 pathogenic in vivo. Because IL-2 and IFN-g inhibit Th17 differ- blocked IL-12–driven phosphorylation of STAT1 but showed only entiation (45, 52), it was difficult to predict what the effect of CP- modest suppression of STAT4 activation. Thus, CP-690,550 evi- 690,550 treatment might be on this lineage. We first polarized dently interferes with multiple JAKs and hence multiple cytokine cells in the conventional manner, using TGF-b1 and IL-6. Under signaling pathways in T cells. Given these effects on lineage- these conditions, the addition of CP-690,550 enhanced IL-17A promoting cytokine signaling, we considered the possibility that and IL-2 production (Fig. 4A, top row), consistent with the abil- this JAK inhibitor also would affect the differentiation of Th cells ity of the inhibitor to block feedback inhibition mediated by IL-2 to various fates. (38, 52). Addition of anti–IL-2 had a similar effect to that of the inhibitor (data not shown). Interestingly, the IL-17A–inducing CP-690,550 blocks the differentiation of Th2 and Th1 cells effect of CP-690,550 on Th17 differentiation was strictly de- Differentiation of Th2 cells is mediated by IL-4, a gc cytokine that pendent on the presence of TGF-b1, because neutralizing the bi- signals through JAK3 and JAK1. We therefore expected that CP- ologic activity of this cytokine abolished IL-17A production (Fig. 690,550 would effectively antagonize Th2 specification. Activat- 4A,4B). ing naive Th cells with IL-4 and anti-CD3/anti-CD28 mAb ef- IL-17A–producing cells generated by TGF-b1 and IL-6 can fectively generated GATA3 in cells that expressed high levels of produce the anti-inflammatory cytokine IL-10 and are less path- 4238 CP-690,550 CONTROLS T CELL DIFFERENTIATION AND INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. CP-690,550 inhibits Th2 and Th1 differentiation. Naive FIGURE 4. Modulation of Th17 differentiation by CP-690,550. Sorted + mouse CD4+ T cells were stimulated with anti-CD3/anti-CD28 Abs in the naive CD4 T cells were activated for 3 d with anti-CD3/anti-CD28 Abs in presence or absence of the indicated concentrations of CP-690,550, using the presence of IL-6 in combination with either TGF-b1, IL-23, or TGF-b1 either Th2 polarizing conditions (IL-4 and anti–IFN-g Ab) or Th1 polar- and IL-23. CP-690,550 was added to the cultures at the indicated con- izing conditions (IL-12 and anti–IL-4 Ab). A, On day 3, the expression of centrations. Expression of IL-17A and IL-2 was determined by in- lineage-associated transcription factors GATA3 and T-bet was determined tracellular cytokine staining after stimulation with PMA/ionomycin. The by quantitative RT-PCR. Results were normalized using b-actin transcripts effect of CP-690,550 on IL-17A expression also was studied in conditions and represent relative expression 6 SEM. *p , 0.001. B and C,After where TGF-b1 signaling was blocked by neutralizing Abs. A and B, 3 d in culture, cells were activated with PMA/ionomycin. Th cell differ- Representative flow cytometry plots (A) and summarized data of three to entiation and proliferation were assessed by intracellular cytokine staining four separate experiments are shown (B). C, Expression of Rorc, Ahr, and and CFSE dilution. Th2 cell polarization was evaluated by IL-13 expres- Il23r in cells activated as in A was determined by quantitative RT-PCR. sion (B), and Th1 cell polarization was evaluated by IFN-g expression (C). Results were normalized using b-actin transcripts and represent fold in- + 6 Data are representative of three separate experiments. Naive CD4 T cells crease of expression (mean SEM) compared with that of Th0 conditions. from wild-type (WT) or STAT1-deficient (STAT1 KO) mice were activated with IL-12 and anti–IL-4 Ab for 3 d in the presence or absence of either CP-690,550 or anti–IFN-g neutralizing Abs. D, Intracellular staining of ogenic upon adoptive transfer than those generated in the pre- PMA/ionomycin-activated cells shows IFN-g and T-bet expression. Data sence of IL-23 (53). We also have shown recently that IL-17A– are representative of three to four separate experiments with similar producing cells generated in the absence of TGF-b1 are more results. pathogenic than those generated in the presence of this immuno- The Journal of Immunology 4239 regulatory cytokine (51). We therefore examined the effects of T cells found at sites of autoimmune lesions express both Rorgt CP-690,550 on Th17 cells induced in the absence of TGF-b and T-bet (55). Importantly, Th17 cells generated in the absence of and found that in sharp contrast to its effects on IL-17A pro- TGF-b also express both Rorgt and T-bet (51), and CP-690,550 duction induced by IL-6 and TGF-b1 the JAK inhibitor dramat- blocked T-bet expression in these cells (Fig. 5C). Thus, although ically suppressed the differentiation of Th17 cells generated CP-690,550 can enhance IL-17 production in TGF-b1–induced with IL-6 and IL-23 (Fig. 4A,4B). Under these conditions, neu- Th17 cells, it suppresses IL-17 production in pathogenic IL-23– tralizing TGF-b1 did not affect the action of CP-690,550. In induced Th17 cells and also inhibits expression of Rorgt, T-bet, contrast, when TGF-b1 was added to the IL-6/IL-23 condition, and IL-23R. the JAK inhibitor enhanced IL-17A expression (Fig. 4B). In- terestingly, Rorc expression was inhibited in all of the conditions CP-690,550 rapidly suppresses CIA, inflammatory biomarkers, in the presence of CP-690,550, even if increased IL-17A expres- and STAT1-dependent gene expression sion was observed (Fig. 4C). CP-690,550 also blocked the ex- Therapies that selectively target T cell activation or differentiation pression of Ahr, which is induced primarily in the presence of can block CIA when used prophylactically during TGF-b1 (51). As shown previously, Il23r expression was induced but may be less effective in established disease where innate im- dramatically by IL-6 and IL-23 in the absence of TGF-b1 (51). mune mechanisms are also prominent (56). The efficacy of anti- Importantly, CP-690,550 completely abrogated the expression of TNF therapies in RA further underscores the role of innate cells IL-23R, which strictly depends on STAT3 activation (51) (Fig. in chronic arthritis. Therefore, we examined whether CP-690,550 4C). could influence the course of established arthritis. Mice that had As shown previously, Th17 cells generated with IL-6, IL-1b, and developed symptoms of arthritis by day 45 after collagen immu- either TGF-b1 or IL-23 produce not only IL-17A but also IL-17F nization were treated with CP-690,550 beginning on day 48. As Downloaded from and IL-22, all of which can contribute to the pathogenicity of these shown in Fig. 6A, a significant reduction in arthritis was apparent cells (51). As shown in Fig. 5A, CP-690,550 effectively blocked within 48 h (day 50) of initiating treatment, and mice with con- the expression of IL-17A, IL-17F, and IL-22 when Th17 cells tinuous inhibitor treatment improved throughout the study. Sig- were generated in the absence of TGF-b1. In contrast, the JAK nificant expression of inflammatory mediators was noted in the inhibitor did not affect IL-17A or IL-17F expression when Th17 plasma of vehicle-treated mice on day 48. Strikingly, many of cells were induced in the presence of TGF-b1, but IL-22 pro- these markers were reduced within 4 h of initial CP-690,550 ad- http://www.jimmunol.org/ duction was affected. IL-21 is another key cytokine produced by ministration (Fig. 6B), suggesting a surprisingly rapid mode of both Th17 cells and follicular Th cells. Of note, its production was action. It should be noted that in these studies we were unable to blocked efficiently by CP-690,550 regardless of how the Th17 detect IL-17 in plasma. From our experience with the mouse CIA cells were generated (Fig. 5B). model, IL-17 is more readily detectable earlier in disease pro- Increasingly, it has been recognized that Th cells that arise in the gression prior to the development of arthritic symptoms (21). setting of autoimmunity can produce both IL-17A and IFN-g; this Because CP-690,550 potently suppressed STAT1 activation in particularly in human IL-17–producing cells (54, 55). Similarly, T cells in response to IL-6, IFN-g, and IL-12, we sought to de- termine if the inhibitor also would reduce the expression of ca- by guest on October 1, 2021 nonical STAT1 target genes (57). Interestingly, the prominent ex- pression of many STAT1-responsive genes was evident in mice with arthritis, and the expression of these genes was suppressed rapidly by CP-690,550, as measured in the inflamed joints (Fig. 6C). Continuous CP-690,550 treatment further suppressed the expression of STAT1-responsive genes to near normal levels as disease resolved (data not shown). To ensure that the observed transcript suppression was not the result of alterations of tissue-infiltrating cells, we examined the inflammatory infiltration in paw tissues from mice treated by the same regimen. As shown in Fig. 6D using histopathology as well as macrophage and T cell IHC assessment of joint tissue, there was no decrease in inflammatory cell infiltrates within the first 24 h of CP-690,550 treatment. These results confirmed the rapid suppression of STAT1 signaling pathways and demonstrated that the beneficial effects of JAK inhibition were not due to leukocyte depletion. Consistent with CP-690,550 effects on the arthritis se- verity score, histopathology and IHC assessment did, however, reveal significantly reduced inflammation after 7 d of treatment (Fig. 6D,6E).

FIGURE 5. CP-690,550 inhibits the differentiation of IL-23–induced Efficacy in mouse CIA correlates with inhibition of both JAK1 Th17 cells and associated cytokines. Sorted naive CD4+ T cells were ac- and JAK3 tivated in serum-free media in the absence or presence of CP-690,550 with To assess the relative JAK inhibition by CP-690,550 in vivo, we anti-CD3/anti-CD28 Abs and the combination of IL-6, IL-1b, and either measured STAT phosphorylation in ex vivo cytokine-stimulated TGF-b1 or IL-23. A, After 4 d of culture, cells were restimulated with PMA/ionomycin, and the expression of IL-17A, IL-17F, and IL-22 was whole blood from mice, which had been treated orally with the assessed by flow cytometry. B and C, Expression of Il21 (B) and Tbx21 (C) inhibitor. For these experiments, IL-6 signaling through STAT1 was was determined by quantitative RT-PCR. Results were normalized using used as a measure of JAK1/JAK2 activity, whereas IL-15 and GM- b-actin transcripts and represent relative expression (mean 6 SEM). B, CSF stimulation of STAT5 were used to assess JAK1/JAK3 and *p , 0.001; C,*p , 0.01. JAK2 signaling pathways, respectively. As shown in Fig. 7A, mice 4240 CP-690,550 CONTROLS T CELL DIFFERENTIATION AND INFLAMMATION Downloaded from http://www.jimmunol.org/

FIGURE 7. CP-690,550 blocks innate JAK signaling in vivo. Mice were administered single doses of CP-690,550, and whole blood was collected

after 1 h (∼Cmax) and stimulated ex vivo with the indicated cytokines. A, STAT phosphorylation expressed as the percentage of control cells (from nontreated mice) is plotted against plasma drug exposure in the same

sample. Each data point represents an individual mouse from one of four by guest on October 1, 2021

separate experiments. EC50 corresponded to 470, 273, and 6656 nM for JAK1/JAK2, JAK1/JAK3, and JAK2 inhibition, respectively. B, JAK in- hibition after chronic dosing in mice induced with CIA. Data represent the mean 6 SEM JAK inhibition or mean area under the curve for efficacy from one of two separate studies involving five mice at each dose. C, FIGURE 6. Rapid amelioration of established arthritis and inflammation Plasma cytokine levels were assessed 1, 2, and 6 h after a 10 mg i.p. ad- by CP-690,550. Mice with fully developed CIA were orally administered ministration of LPS to DBA/1J mice pretreated for 1 h with or without 5 vehicle or 50 mg/kg CP-690,550 twice daily beginning on day 48 post- mg/kg CP-690,550. Time courses of TNF, IL-6, IL-12p70, IL-10, IFN-g, immunization (arrow). A, Data represent the mean 6 SEM severity score and IL-1b production in plasma are shown. Data represent plasma cytokine from eight mice in each group. B, Plasma concentrations of SAA, G-CSF, concentrations (mean 6 SEM) from one of two separate studies with IL-6, CXCL1 (KC), CCL2 (MCP-1), and CXCL10 (IP-10) were measured similar results involving eight mice at each dose. 4 h after dosing on day 48. C, Relative expression of STAT1-induced genes in paw tissue 4 h after dosing on day 48. Quantitative RT-PCR results were normalized to cyclophilin and represent mean 6 SEM relative expression The same method was used to evaluate CP-690,550 inhibition from nondiseased animals (normal) as well as vehicle- and CP-690,550– of gc cytokine and STAT1-dependent signaling pathways in the treated mice with CIA. D, Cellular infiltration of inflamed paw tissue in context of inflammatory disease. Cytokine signaling was exam- mice treated as in A was assessed by histology and IHC at the indicated ined in mice that had been treated orally with various doses of CP- time points. Data represent the mean 6 SEM score for all four paws from 690,550 for 5 wk as treatment for CIA (Fig. 7B), and the results eight mice per treatment group. *p , 0.01, **p , 0.001. E, Representative histology (H&E) and F4/80 or CD3 IHC of paw tissue sections collected revealed that the inhibition of both JAK1/JAK2 and JAK1/JAK3 7 d after the onset of treatment with vehicle or CP-690,550. Scale bars, pathways correlated with efficacy, although there was little or no 200 mm. Isg15, IFN-stimulated gene 15; Mx1, myxovirus resistance pro- inhibition of JAK2 at therapeutic doses. These results suggested tein 1; Oas1a,29–59 oligoadenylate synthase 1A; Usp18, ubiquitin-specific that the anti-inflammatory activity of CP-690,550 is mediated by peptidase 18. its potent inhibition of both JAK1 and JAK3 activity. CP-690,550 rapidly blocks innate responses in vivo administered a single oral dose of CP-690,550 had similar in- The rapid suppression of inflammatory cytokines and STAT1- hibition of JAK1/JAK2 and JAK1/JAK3 pathways and signifi- dependent gene expression observed in mice with CIA after CP- cantly reduced suppression of the JAK2 pathway, confirming that 690,550 treatment suggested that in addition to suppressing in vivo CP-690,550 administration inhibits cytokine receptor sig- T cell function the JAK inhibitor also might be affecting innate naling pathways activating STAT1 to a similar extent as gc cyto- immune responses. To investigate this possibility, we examined the kine signaling pathways. effect of CP-690,550 on the acute response to LPS in vivo, a model The Journal of Immunology 4241 known to be dependent upon IFN-g and STAT1 (58, 59). We found suppression as demonstrated by the effect of anti–IFN-g neutral- that a single dose of the JAK inhibitor suppressed TNF and IL-6 izing Abs. The consequences of CP-690,550 treatment on Th1 production along with other inflammatory cytokines (Fig. 7C), differentiation and STAT1 signaling also could explain the effi- confirming a rapid anti-inflammatory mode of action. Inter- cacy of the inhibitor in a mouse graft-versus-host disease model, estingly, the production of IL-10 was enhanced by the treatment, where Th1 responses were limited by CP-690,550 without affect- consistent with the reported STAT1-mediated repression of this ing cell proliferation (66). anti-inflammatory cytokine (60). Collectively, these data indicated Although blocking Th1 responses can be highly efficient in graft- that the immunosuppressive effects of CP-690,550 appear to be versus-host disease and transplant rejection, this mechanism alone mediated by the blockade of innate as well as adaptive immune likely would be less successful in autoimmune diseases in which responses. Th17 cells also play a major role. Thus, using inhibitors that target not only JAK3 but also JAK1 or JAK2 and subsequently affect the Discussion differentiation of Th1 as well as Th17 cells could be of benefit in CP-690,550 is being studied currently in a variety of autoimmune autoimmune settings. The generation of Th17 cells is regulated by diseases. In this study, we show that the inhibitor blocks signaling multiple factors. Although IL-6 and TGF-b1 can efficiently induce by JAK3-dependent gc cytokine receptors as well as by other IL-17 production, IL-6 together with IL-23 and IL-1b, in the cytokine receptors that signal through JAK1. Accordingly, we absence of TGF-b1, also can induce IL-17 in naive Th cells (50, found that CP-690,550 interfered with Th1 and Th2 differentiation 51, 67). Indeed, we have shown recently that Th17 cells generated and also impaired the production of inflammatory Th17 cells in the absence of TGF-b are more pathogenic in vivo than those generated in response to IL-1b, IL-6, and IL-23. In contrast, the generated in the presence of this cytokine (51). Moreover, we have JAK inhibitor enhanced the production of IL-17A in cells cultured found that the balance between STAT3 and STAT5 activation can Downloaded from with IL-6 and TGF-b1. These effects were associated with the have opposing regulatory effects on IL-17 expression (68). Results amelioration of murine arthritis, which correlated with the reduced from the present studies demonstrate that CP-690,550, most likely expression of STAT1-dependent genes. Furthermore, CP-690,550 by inhibiting STAT5, increases IL-17 expression when Th17 cells also suppressed cytokine production in a sepsis model, suggesting are generated with TGF-b and IL-6. In contrast, in the absence of that the mechanism of action of this drug involves blocking the TGF-b signaling, CP-690,550 blocked IL-17 expression. action of cytokines during innate and adaptive responses. Although the regulation of IL-17A and IL-17F expression is http://www.jimmunol.org/ Despite its advanced stage of clinical development, the mode of more complex (68), the expression of IL-23R and IL-22 are action by which CP-690,550 exerts efficacy in RA and other au- strictly dependent on STAT3 activation (51). We show in these toimmune settings remains unresolved (15, 22, 23). In contrast to studies that CP-690,550 interferes with IL-23 action by blocking its activity against isolated kinases, CP-690,550 demonstrates the upregulation of its receptor and subsequent IL-17 induction. functional specificity for JAK1 and JAK3 over other JAK family Moreover, CP-690,550 inhibited IL-23R expression under either members in cells, although the basis of this apparent discrepancy Th17 condition. Similarly, the JAK inhibitor abrogated STAT3- has not been determined (18, 21). Because many of the cytokines mediated IL-22 and IL-21 expression in Th17 cells and also involved in RA and other autoimmune diseases signal through inhibited RORgt and T-bet expression. Thus, CP-690,550 potently by guest on October 1, 2021 receptors associated with JAKs, the question arises as to how the suppresses the generation of pathogenic Th17 cells with an IL- effects of CP-690,550 relate to the apparent efficacy of the drug in 23/STAT3 signature. Inhibitory effects on Th17-associated cyto- the setting of autoimmune disease. A central component of the kines also have been suggested for the JAK1/JAK2 inhibitor pathophysiology of RA and psoriasis is the action of autoreactive INCB028050 (25). T cells and the inflammatory cytokines that act upon them (61– This mode of action of CP-690,550 may be of interest in a 63). As was expected, CP-690,550 potently inhibited gc cytokine number of autoimmune diseases where interfering with IL-23 signaling pathways in the current studies by targeting JAK1 and signaling attenuates disease (41, 69–72). Thus, it may very well JAK3 in T cells. Similar results have been observed in JAK1- and be that a clinically important action of CP-690,550 is to block the JAK3-deficient cells (2, 10) and with JAK1-selective inhibitors combined actions of IL-23. (M.I. Jesson, unpublished observations), suggesting that the block- In contrast, IL-6 has wide-ranging biological activities in vari- ade of either or both of these kinases can modulate gc cytokine ous target cells. In addition to promoting Th17 differentiation, receptor signals. A recent study also has demonstrated that a se- it regulates immune responses, the acute phase reaction, hema- lective JAK3 inhibitor, WYE-151650, is effective in CIA (24). topoiesis, and bone metabolism (73, 74). IL-6–deficient mice are Neither the clinical efficacy of CP-690,550 nor the potential protected from experimental autoimmune diseases such as CIA efficacy of other JAK inhibitors is likely to be explained by the (34). Additionally, elevated serum IL-6 levels have been observed inhibition of gc cytokine receptor signaling alone. By such a in patients with inflammatory diseases such as RA and Crohn’s mechanism, the differentiation of naive T cells to Th2 effector disease, and , a humanized anti–IL-6R Ab that blocks cells would be inhibited, but Th2 cells are likely not relevant to the IL-6 signaling, has shown clinical efficacy in these indications, pathogenesis of CIA in mice or RA and psoriasis in humans (41, ameliorating inflammation and normalizing acute phase protein 62). Surprisingly, CP-690,550 also prevented Th1 differentiation. levels (75). Our data indicate that CP-690,550 interferes with the Although previous observations have indicated that cellular JAK3 production of IL-6 and also blocks IL-6 signaling, which could be deficiency or inhibition of JAK3 can suppress Th1 differentiation explained by the effects of the inhibitor on JAK1 and/or JAK2. (64), our data suggest a different mechanism, because CP-690,550 Thus, an additional mechanism underlying CP-690,550 efficacy in suppressed the expression of the Th1-associated transcription RA is likely mediated through effects on IL-6. factor T-bet. Th1 differentiation is driven by IL-12 and IFN-g and We were surprised by the rapid effects of CP-690,550 on by the activation of STAT1 and T-bet (42, 65). Our results indicate established disease in the mouse CIA model. Indeed, effects of the that CP-690,550 has only a modest effect on IL-12–induced inhibitor were observable within hours of initiating treatment. STAT4 activation while profoundly inhibiting STAT1 activation in Despite the inhibitory consequences of CP-690,550 on Th cell T cells induced by either IL-12 or IFN-g. Indeed, the inhibition of differentiation, it seemed unlikely that this could induce such rapid IFN-g signaling alone likely could account for the observed Th1 effects in vivo. Rather, the rapid suppression of inflammatory 4242 CP-690,550 CONTROLS T CELL DIFFERENTIATION AND INFLAMMATION responses suggested that the blockade of innate immune mecha- patients with autosomal severe combined immune deficiency (SCID). Nature 377: 65–68. nisms might represent part of the salutatory effects of JAK in- 6. Levine, R. L., A. Pardanani, A. Tefferi, and D. G. Gilliland. 2007. Role of JAK2 hibition. This led us to examine the efficacy of the JAK inhibitor in in the pathogenesis and therapy of myeloproliferative disorders. Nat. Rev. the sepsis model. Importantly, we found that CP-690,550 had no 7: 673–683. 7. Leonard, W. J., and J. J. O’Shea. 1998. Jaks and STATs: biological implications. direct effect on TLR4 signaling in vitro, because we did not observe Annu. Rev. Immunol. 16: 293–322. the inhibition of LPS-induced TNF or IL-6 production from human 8. Shimoda, K., K. Kato, K. Aoki, T. Matsuda, A. Miyamoto, M. Shibamori, PBMCs (data not shown). Rather, the suppression of acute TNF M. Yamashita, A. Numata, K. Takase, S. Kobayashi, et al. 2000. Tyk2 plays a restricted role in IFN alpha signaling, although it is required for IL-12- responses in vivo after LPS administration is more consistent with mediated T cell function. Immunity 13: 561–571. the inhibition of IFN-g signaling by the blockade of JAK1, be- 9. Rosenzweig, S. D., and S. M. Holland. 2005. Defects in the -gamma cause both STAT1-deficient and IFN-gR–deficient mice are re- and interleukin-12 pathways. Immunol. Rev. 203: 38–47. 10. Rodig, S. J., M. A. Meraz, J. M. White, P. A. Lampe, J. K. Riley, C. D. Arthur, sistant to LPS-induced endotoxemic shock (58, 59,). In contrast, K. L. King, K. C. Sheehan, L. Yin, D. Pennica, et al. 1998. Disruption of the Jak1 IFN-g priming of macrophages has been shown to enhance both gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine- induced biologic responses. Cell 93: 373–383. LPS-stimulated TNF production in vivo (76) and STAT1 expres- 11. Parganas, E., D. Wang, D. Stravopodis, D. J. Topham, J. C. Marine, S. Teglund, sion (77), and it has been suggested that IFN-g activation of E. F. Vanin, S. Bodner, O. R. Colamonici, J. M. van Deursen, et al. 1998. Jak2 is STAT1 may alter signaling pathways downstream of anti- essential for signaling through a variety of cytokine receptors. Cell 93: 385–395. 12. Pesu, M., A. Laurence, N. Kishore, S. H. Zwillich, G. Chan, and J. J. O’Shea. inflammatory cytokines such as IL-10 or TGF-b, leading to an- 2008. Therapeutic targeting of Janus kinases. Immunol. Rev. 223: 132–142. tagonism of their suppressive function (78). If this were the case, 13. Ghoreschi, K., A. Laurence, and J. J. O’Shea. 2009. Selectivity and therapeutic then CP-690,550 suppression of STAT1-responsive genes could inhibition of kinases: to be or not to be? Nat. Immunol. 10: 356–360. 14. Pardanani, A. 2008. JAK2 inhibitor therapy in myeloproliferative disorders: override the effect of priming. IL-10 responses to LPS are en- rationale, preclinical studies and ongoing clinical trials. Leukemia 22: 23–30. Downloaded from hanced in mice made deficient for IFN-a/b/g or STAT1 (60), 15. van Gurp, E., W. Weimar, R. Gaston, D. Brennan, R. Mendez, J. Pirsch, S. Swan, suggesting that STAT1 is a negative regulator of IL-10 gene ex- M. D. Pescovitz, G. Ni, C. Wang, et al. 2008. Phase 1 dose-escalation study of CP-690 550 in stable renal allograft recipients: preliminary findings of safety, pression. Our observations were consistent with this hypothesis, tolerability, effects on subsets and . Am. J. because we observed enhanced IL-10 levels in LPS-treated mice Transplant. 8: 1711–1718. 16. Santos, F. P., H. M. Kantarjian, N. Jain, T. Manshouri, D. A. Thomas, G. Garcia- given the JAK inhibitor. Another possible contribution to CP- Manero, D. Kennedy, Z. Estrov, J. Cortes, and S. Verstovsek. 2010. Phase 2 study of 690,550 suppression of LPS responses in vivo could involve the CEP-701, an orally available JAK2 inhibitor, in patients with primary or post- blockade of IL-15 signaling, because both IL-15 deficiency and polycythemia vera/essential thrombocythemia myelofibrosis. Blood 115: 1131–1136. http://www.jimmunol.org/ 17. Verstovsek, S., H. Kantarjian, R. A. Mesa, A. D. Pardanani, J. Cortes-Franco, anti–IL-15 neutralizing Ab have been shown to suppress LPS- D. A. Thomas, Z. Estrov, J. S. Fridman, E. C. Bradley, S. Erickson-Viitanen, induced endotoxemia in vivo (79). Although there is no doubt et al. 2010. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in that IL-15 signaling is inhibited potently by CP-690,550, this myelofibrosis. N. Engl. J. Med. 363: 1117–1127. 18. Changelian, P. S., M. E. Flanagan, D. J. Ball, C. R. Kent, K. S. Magnuson, mechanism cannot explain completely the results from the current W. H. Martin, B. J. Rizzuti, P. S. Sawyer, B. D. Perry, W. H. Brissette, et al. study, because the blockade of IL-15 signaling would not be 2003. Prevention of organ allograft rejection by a specific 3 in- expected to affect IL-10 in this model. hibitor. Science 302: 875–878. 19. Karaman, M. W., S. Herrgard, D. K. Treiber, P. Gallant, C. E. Atteridge, The simultaneous control of signaling pathways involved in B. T. Campbell, K. W. Chan, P. Ciceri, M. I. Davis, P. T. Edeen, et al. 2008. A innate and adaptive immune responses by CP-690,550 may explain quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26: 127–132. by guest on October 1, 2021 why this JAK inhibitor has produced rapid clinical improvement in 20. Changelian, P. S., D. Moshinsky, C. F. Kuhn, M. E. Flanagan, M. J. Munchhof, T. M. Harris, D. A. Whipple, J. L. Doty, J. Sun, C. R. Kent, et al. 2008. The RA patients who previously have failed other disease-modifying specificity of JAK3 kinase inhibitors. Blood 111: 2155–2157. antirheumatic drug therapies or TNF antagonists (22). On the 21. Meyer, D. M., M. I. Jesson, X. Li, M. M. Elrick, C. L. Funckes-Shippy, J. D. Warner, C. J. Gross, M. E. Dowty, S. K. Ramaiah, J. L. Hirsch, et al. 2010. basis of the present data, it appears that the efficacy of CP-690,550 Anti-inflammatory activity and reductions mediated by the JAK1/JAK3 is likely based on its ability to block multiple cytokines and break inhibitor, CP-690,550, in rat adjuvant-induced arthritis. J. Inflamm. (Lond.) 7: 41. the cycle of inflammation. Clearly, it will be important to try to 22. Kremer, J. M., B. J. Bloom, F. C. Breedveld, J. H. Coombs, M. P. Fletcher, D. Gruben, S. Krishnaswami, R. Burgos-Vargas, B. Wilkinson, C. A. Zerbini, understand which critical cytokines are blocked in humans un- and S. H. Zwillich. 2009. The safety and efficacy of a JAK inhibitor in patients dergoing JAK inhibitor treatment and the extent to which signal- with active rheumatoid arthritis: Results of a double-blind, placebo-controlled ing is abrogated. As such, our findings have implications for the phase IIa trial of three dosage levels of CP-690,550 versus placebo. Arthritis Rheum. 60: 1895–1905. possible utility of CP-690,550 in a wide variety of inflammatory 23. Boy, M. G., C. Wang, B. E. Wilkinson, V. F. Chow, A. T. Clucas, J. G. Krueger, disorders. A. S. Gaweco, S. H. Zwillich, P. S. Changelian, and G. Chan. 2009. Double-blind, placebo-controlled, dose-escalation study to evaluate the pharmacologic effect of CP-690,550 in patients with psoriasis. J. Invest. Dermatol. 129: 2299–2302. Disclosures 24. Lin, T. H., M. Hegen, E. Quadros, C. L. Nickerson-Nutter, K. C. Appell, The National Institutes of Health (NIH) has a patent related to JAKs as novel A. G. Cole, Y. Shao, S. Tam, M. Ohlmeyer, B. Wang, et al. 2010. Selective functional inhibition of JAK-3 is sufficient for efficacy in collagen-induced ar- immunomodulatory agents, and J.J.O. and the NIH have a Collaborative thritis in mice. Arthritis Rheum. 62: 2283–2293. Research Agreement and Development Award with Pfizer. M.I.J., C.E.S., 25. Fridman, J. S., P. A. Scherle, R. Collins, T. C. Burn, Y. Li, J. Li, T.P.L, Z.A.R., M.E.D., R.D.H., and D.M.M are current Pfizer employees. M. B. Covington, B. Thomas, P. Collier, M. F. Favata, et al. 2010. Selective X.L., J.L.L., S.G., J.W.A., J.D.W., J.C.M., and N.K have been employed inhibition of JAK1 and JAK2 is efficacious in rodent models of arthritis: pre- clinical characterization of INCB028050. J. Immunol. 184: 5298–5307. by Pfizer within the last 5 years. M.I.J., T.P.L., Z.A.R., M.E.D., R.D.H., 26. Damsker, J. M., A. M. Hansen, and R. R. Caspi. 2010. Th1 and Th17 cells: D.D.M., and N.K. have stock or equity interests. adversaries and collaborators. Ann. N. Y. Acad. Sci. 1183: 211–221. 27. Lang, K. S., A. Burow, M. Kurrer, P. A. Lang, and M. Recher. 2007. The role of the innate immune response in autoimmune disease. J. Autoimmun. 29: 206–212. References 28. Milici, A. J., E. M. Kudlacz, L. Audoly, S. Zwillich, and P. Changelian. 2008. 1. Baker, S. J., S. G. Rane, and E. P. Reddy. 2007. Hematopoietic cytokine receptor Cartilage preservation by inhibition of in two rodent models of signaling. Oncogene 26: 6724–6737. rheumatoid arthritis. Arthritis Res. Ther. 10: R14. 2. Ghoreschi, K., A. Laurence, and J. J. O’Shea. 2009. Janus kinases in immune 29. Bendele, A., T. McAbee, G. Sennello, J. Frazier, E. Chlipala, and D. McCabe. 1999. Efficacy of sustained blood levels of interleukin-1 receptor antagonist in cell signaling. Immunol. Rev. 228: 273–287. animal models of arthritis: comparison of efficacy in animal models with human 3. Levy, D. E., and J. E. Darnell, Jr. 2002. Stats: transcriptional control and bi- clinical data. Arthritis Rheum. 42: 498–506. ological impact. Nat. Rev. Mol. Cell Biol. 3: 651–662. 30. LaBranche, T. P., C. L. Hickman-Brecks, D. M. Meyer, C. E. Storer, M. I. Jesson, 4. Shuai, K., and B. Liu. 2003. Regulation of JAK-STAT signalling in the immune K. M. Shevlin, F. A. Happa, R. A. Barve, D. J. Weiss, J. C. Minnerly, et al. 2010. system. Nat. Rev. Immunol. 3: 900–911. Characterization of the KRN cell transfer model of rheumatoid arthritis (KRN- 5. Macchi, P., A. Villa, S. Giliani, M. G. Sacco, A. Frattini, F. Porta, A. G. Ugazio, CTM), a chronic yet synchronized version of the K/BxN mouse. Am. J. Pathol. J. A. Johnston, F. Candotti, J. J. O’Shea, et al. 1995. Mutations of Jak-3 gene in 177: 1388–1396. The Journal of Immunology 4243

31. Johnson, S. E., N. Shah, A. A. Bajer, and T. W. LeBien. 2008. IL-7 activates the 56. Drexler, S. K., P. L. Kong, J. Wales, and B. M. Foxwell. 2008. Cell signalling in phosphatidylinositol 3-kinase/AKT pathway in normal human thymocytes but macrophages, the principal innate immune effector cells of rheumatoid arthritis. not normal human B cell precursors. J. Immunol. 180: 8109–8117. Arthritis Res. Ther. 10: 216. 32. Ettinger, R., S. Kuchen, and P. E. Lipsky. 2008. Interleukin 21 as a target of 57. Robertson, G., M. Hirst, M. Bainbridge, M. Bilenky, Y. Zhao, T. Zeng, intervention in autoimmune disease. Ann. Rheum. Dis. 67(Suppl. 3): iii83–iii86. G. Euskirchen, B. Bernier, R. Varhol, A. Delaney, et al. 2007. Genome-wide 33. Parrish-Novak, J., S. R. Dillon, A. Nelson, A. Hammond, C. Sprecher, profiles of STAT1 DNA association using chromatin immunoprecipitation and J. A. Gross, J. Johnston, K. Madden, W. Xu, J. West, et al. 2000. Interleukin 21 massively parallel sequencing. Nat. Methods 4: 651–657. and its receptor are involved in NK cell expansion and regulation of lymphocyte 58. Car, B. D., V. M. Eng, B. Schnyder, L. Ozmen, S. Huang, P. Gallay, D. Heumann, function. Nature 408: 57–63. M. Aguet, and B. Ryffel. 1994. receptor deficient mice are 34. Alonzi, T., E. Fattori, D. Lazzaro, P. Costa, L. Probert, G. Kollias, F. De resistant to endotoxic shock. J. Exp. Med. 179: 1437–1444. Benedetti, V. Poli, and G. Ciliberto. 1998. is required for the de- 59. Kamezaki, K., K. Shimoda, A. Numata, T. Matsuda, K. Nakayama, and velopment of collagen-induced arthritis. J. Exp. Med. 187: 461–468. M. Harada. 2004. The role of Tyk2, Stat1 and Stat4 in LPS-induced endotoxin 35. Smolen, J. S., A. Beaulieu, A. Rubbert-Roth, C. Ramos-Remus, J. Rovensky, signals. Int. Immunol. 16: 1173–1179. E. Alecock, T. Woodworth, R. Alten; OPTION Investigators. 2008. Effect of 60. VanDeusen, J. B., M. H. Shah, B. Becknell, B. W. Blaser, A. K. Ferketich, interleukin-6 receptor inhibition with tocilizumab in patients with rheumatoid G. J. Nuovo, B. M. Ahmer, J. Durbin, and M. A. Caligiuri. 2006. STAT-1- arthritis (OPTION study): a double-blind, placebo-controlled, randomised trial. mediated repression of monocyte interleukin-10 gene expression in vivo. Eur. Lancet 371: 987–997. J. Immunol. 36: 623–630. 36. Minegishi, Y., M. Saito, T. Morio, K. Watanabe, K. Agematsu, S. Tsuchiya, 61. McInnes, I. B., and G. Schett. 2007. Cytokines in the pathogenesis of rheumatoid H. Takada, T. Hara, N. Kawamura, T. Ariga, et al. 2006. Human tyrosine kinase arthritis. Nat. Rev. Immunol. 7: 429–442. 2 deficiency reveals its requisite roles in multiple cytokine signals involved in 62. Ghoreschi, K., P. Thomas, S. Breit, M. Dugas, R. Mailhammer, W. van Eden, innate and acquired immunity. Immunity 25: 745–755. R. van der Zee, T. Biedermann, J. Prinz, M. Mack, et al. 2003. Interleukin-4 37. Watford, W. T., and J. J. O’Shea. 2006. Human tyk2 kinase deficiency: another therapy of psoriasis induces Th2 responses and improves human autoimmune primary immunodeficiency syndrome. Immunity 25: 695–697. disease. Nat. Med. 9: 40–46. 38. Villarino, A. V., C. M. Tato, J. S. Stumhofer, Z. Yao, Y. K. Cui, L. Hennighausen, 63. Wilson, N. J., K. Boniface, J. R. Chan, B. S. McKenzie, W. M. Blumenschein, J. J. O’Shea, and C. A. Hunter. 2007. Helper T cell IL-2 production is limited by J. D. Mattson, B. Basham, K. Smith, T. Chen, F. Morel, et al. 2007. De- negative feedback and STAT-dependent cytokine signals. J. Exp. Med. 204: 65–71. velopment, cytokine profile and function of human -producing 39. Tao, X., S. Constant, P. Jorritsma, and K. Bottomly. 1997. Strength of TCR helper T cells. Nat. Immunol. 8: 950–957. Downloaded from signal determines the costimulatory requirements for Th1 and Th2 CD4+ T cell 64. Shi, M., T. H. Lin, K. C. Appell, and L. J. Berg. 2008. Janus-kinase-3-dependent differentiation. J. Immunol. 159: 5956–5963. signals induce chromatin remodeling at the Ifng locus during T helper 1 cell 40. Kudlacz, E., M. Conklyn, C. Andresen, C. Whitney-Pickett, and P. Changelian. differentiation. Immunity 28: 763–773. 2008. The JAK-3 inhibitor CP-690550 is a potent anti-inflammatory agent in 65. Schulz, E. G., L. Mariani, A. Radbruch, and T. Ho¨fer. 2009. Sequential polari- a murine model of pulmonary eosinophilia. Eur. J. Pharmacol. 582: 154–161. zation and imprinting of type 1 T helper by interferon-gamma and 41. van den Berg, W. B., and P. Miossec. 2009. IL-17 as a future therapeutic target interleukin-12. Immunity 30: 673–683. for rheumatoid arthritis. Nat. Rev. Rheumatol. 5: 549–553. 66. Park, H. B., K. Oh, N. Garmaa, M. W. Seo, O. J. Byoun, H. Y. Lee, and

42. Lighvani, A. A., D. M. Frucht, D. Jankovic, H. Yamane, J. Aliberti, D. S. Lee. 2010. CP-690550, a , suppresses CD4+ T-cell- http://www.jimmunol.org/ B. D. Hissong, B. V. Nguyen, M. Gadina, A. Sher, W. E. Paul, and J. J. O’Shea. mediated acute graft-versus-host disease by inhibiting the interferon-g pathway. 2001. T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid Transplantation 90: 825–835. cells. Proc. Natl. Acad. Sci. USA 98: 15137–15142. 67. Chung, Y., S. H. Chang, G. J. Martinez, X. O. Yang, R. Nurieva, H. S. Kang, 43. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham, L. Ma, S. S. Watowich, A. M. Jetten, Q. Tian, and C. Dong. 2009. Critical J. D. Sedgwick, T. McClanahan, R. A. Kastelein, and D. J. Cua. 2005. IL-23 regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity drives a pathogenic T cell population that induces autoimmune inflammation. J. 30: 576–587. Exp. Med. 201: 233–240. 68. Yang, X.-P., K. Ghoreschi, S. M. Steward-Tharp, J. Rodriguez-Canales, J. Zhu, 44. Komiyama, Y., S. Nakae, T. Matsuki, A. Nambu, H. Ishigame, S. Kakuta, J. R. Grainger, K. Hirahara, H.-W. Sun, L. Wei, G. Vahedi, et al. 2011. Opposing K. Sudo, and Y. Iwakura. 2006. IL-17 plays an important role in the development regulation of the locus encoding IL-17 through direct, reciprocal actions of of experimental autoimmune encephalomyelitis. J. Immunol. 177: 566–573. STAT3 and STAT5. Nat. Immunol. 12: 247–254. 45. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy, 69. Murphy, C. A., C. L. Langrish, Y. Chen, W. Blumenschein, T. McClanahan,

K. M. Murphy, and C. T. Weaver. 2005. Interleukin 17-producing CD4+ effector R. A. Kastelein, J. D. Sedgwick, and D. J. Cua. 2003. Divergent pro- and by guest on October 1, 2021 T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J. Nat. Immunol. 6: 1123–1132. Exp. Med. 198: 1951–1957. 46. Aggarwal, S., N. Ghilardi, M. H. Xie, F. J. de Sauvage, and A. L. Gurney. 2003. 70. Leonardi, C. L., A. B. Kimball, K. A. Papp, N. Yeilding, C. Guzzo, Y. Wang, Interleukin-23 promotes a distinct CD4 T cell activation state characterized by S. Li, L. T. Dooley, K. B. Gordon; PHOENIX 1 study investigators. 2008. Ef- the production of interleukin-17. J. Biol. Chem. 278: 1910–1914. ficacy and safety of , a human interleukin-12/23 monoclonal anti- 47. Mangan, P. R., L. E. Harrington, D. B. O’Quinn, W. S. Helms, D. C. Bullard, body, in patients with psoriasis: 76-week results from a randomised, double- C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, and C. T. Weaver. 2006. blind, placebo-controlled trial (PHOENIX 1). Lancet 371: 1665–1674. Transforming growth factor-beta induces development of the T(H)17 lineage. 71. Tato, C. M., and D. J. Cua. 2008. Reconciling id, ego, and superego within in- Nature 441: 231–234. terleukin-23. Immunol. Rev. 226: 103–111. 48. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger. 72. McGeachy, M. J., Y. Chen, C. M. Tato, A. Laurence, B. Joyce-Shaikh, 2006. TGFbeta in the context of an inflammatory cytokine milieu supports de W. M. Blumenschein, T. K. McClanahan, J. J. O’Shea, and D. J. Cua. 2009. The novo differentiation of IL-17-producing T cells. Immunity 24: 179–189. receptor is essential for the terminal differentiation of interleukin 49. Acosta-Rodriguez, E. V., G. Napolitani, A. Lanzavecchia, and F. Sallusto. 2007. 17-producing effector T helper cells in vivo. Nat. Immunol. 10: 314–324. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for 73. Naugler, W. E., and M. Karin. 2008. The wolf in sheep’s clothing: the role of the differentiation of interleukin 17-producing human T helper cells. Nat. interleukin-6 in immunity, inflammation and cancer. Trends Mol. Med. 14: 109– Immunol. 8: 942–949. 119. 50. Das, J., G. Ren, L. Zhang, A. I. Roberts, X. Zhao, A. L. Bothwell, L. Van Kaer, 74. Fonseca, J. E., M. J. Santos, H. Canha˜o, and E. Choy. 2009. Interleukin-6 as Y. Shi, and G. Das. 2009. Transforming growth factor beta is dispensable for the a key player in systemic inflammation and joint destruction. Autoimmun. Rev. 8: molecular orchestration of Th17 cell differentiation. J. Exp. Med. 206: 2407–2416. 538–542. 51. Ghoreschi, K., A. Laurence, X. P. Yang, C. M. Tato, M. J. McGeachy, J. E. Konkel, 75. Ohsugi, Y., and T. Kishimoto. 2008. The recombinant humanized anti-IL-6 re- H. L. Ramos, L. Wei, T. S. Davidson, N. Bouladoux, et al. 2010. Generation of ceptor tocilizumab, an innovative drug for the treatment of rheumatoid pathogenic T(H)17 cells in the absence of TGF-b signalling. Nature 467: 967–971. arthritis. Expert Opin. Biol. Ther. 8: 669–681. 52. Laurence, A., C. M. Tato, T. S. Davidson, Y. Kanno, Z. Chen, Z. Yao, R. B. Blank, 76. Williams, J. G., G. J. Jurkovich, G. B. Hahnel, and R. V. Maier. 1992. Macro- F. Meylan, R. Siegel, L. Hennighausen, et al. 2007. Interleukin-2 signaling via phage priming by interferon gamma: a selective process with potentially harmful STAT5 constrains T helper 17 cell generation. Immunity 26: 371–381. effects. J. Leukoc. Biol. 52: 579–584. 53. McGeachy, M. J., K. S. Bak-Jensen, Y. Chen, C. M. Tato, W. Blumenschein, 77. Hu, X., K. H. Park-Min, H. H. Ho, and L. B. Ivashkiv. 2005. IFN-gamma-primed T. McClanahan, and D. J. Cua. 2007. TGF-beta and IL-6 drive the production of macrophages exhibit increased CCR2-dependent migration and altered IFN- IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat. gamma responses mediated by Stat1. J. Immunol. 175: 3637–3647. Immunol. 8: 1390–1397. 78. Hu, X., and L. B. Ivashkiv. 2009. Cross-regulation of signaling pathways by 54. Annunziato, F., L. Cosmi, F. Liotta, E. Maggi, and S. Romagnani. 2009. Type 17 interferon-gamma: implications for immune responses and autoimmune dis- T helper cells-origins, features and possible roles in rheumatic disease. Nat. Rev. eases. Immunity 31: 539–550. Rheumatol. 5: 325–331. 79. Fehniger, T. A., H. Yu, M. A. Cooper, K. Suzuki, M. H. Shah, and 55. Kebir, H., I. Ifergan, J. I. Alvarez, M. Bernard, J. Poirier, N. Arbour, P. Duquette, M. A. Caligiuri. 2000. Cutting edge: IL-15 costimulates the generalized and A. Prat. 2009. Preferential recruitment of interferon-gamma-expressing Shwartzman reaction and innate immune IFN-gamma production in vivo. J. TH17 cells in multiple sclerosis. Ann. Neurol. 66: 390–402. Immunol. 164: 1643–1647.