The Journal of Immunology IL-23 Drives Pathogenic IL-17-Producing CD8؉ T Cells1 Bogoljub Ciric,2,3* Mohamed El-behi,3* Rosalyn Cabrera,† Guang-Xian Zhang,* and Abdolmohamad Rostami* ,IL-17-producing CD8؉ T cells (Tc17) appear to play a role in a range of conditions, such as autoimmunity and cancer. Thus far Tc17 cells have been only marginally studied, resulting in a paucity of data on their biology and function. We demonstrate that Tc17 and Th17 cells share similar developmental characteristics, including the previously unknown promoting effect of IL-21 on Tc17 cell differentiation and IL-23-dependent expression of IL-22. Both STAT1 and STAT4 are required for optimal devel- opment of Tc17 cells and maximal secretion of cytokines. Tc17 cells are cytotoxic, and they can be either pathogenic or non- pathogenic upon adoptive transfer in the model of autoimmune diabetes. Tc17 cells treated with TGF-␤1 plus IL-6 are not diabetogenic, whereas IL-23-treated cells potently induce the disease. IL-17A and IL-17F are necessary but not sufficient for diabetes induction by Tc17 cells. Tc17 cells treated with TGF-␤1 plus IL-6 or IL-23 likely differ in pathogenicity due to their disparate capacity to attract other immune cells and initiate inflammation. The Journal of Immunology, 2009, 182: 5296–5305. he involvement of IL-17-producing CD8ϩ T cells (Tc17) that tumors, by secreting large quantities of TGF-␤1, induce de- in various conditions, such as infection, cancer, and au- velopment of IL-17Aϩ T cells, which in turn promote tumor sur- T toimmune inflammation, has been documented in both vival in an IL-17A-dependent manner (12). Tajima et al. demon- humans and mice; however, unlike Th17 cells, Tc17 cells have strated IL-6-driven spontaneous expansion of Tc17 cells, which received only marginal attention. The basic factors that determine induced colitis in their mouse model (13). In multiple sclerosis a development of Th17 and Tc17 cells appear to be identical. Acti- high proportion (70–80%) of Tc17 and Th17 cells among brain- ϩ vation of mouse CD8 T cells in the presence of TGF-␤1 and IL-6 infiltrating T cells have been found in active lesions (14). In ex- results in expression of retinoic acid-related orphan receptor perimental autoimmune uveitis, immunization with peptide inter- (ROR)␥t4 and development of Tc17 cells (1–4), analogous to photoreceptor retinoid-binding protein (IRBP)1–20 induced Th17 cells (5, 6). Th17 cells secrete IL-21, which in an autocrine autoreactive Tc17 and Th17 cells (15). Mixed populations of these manner promotes their development (7, 8). The presence of IL-21 cells induced experimental autoimmune uveitis when transferred mRNA in Tc17 cells has been reported recently (9), but the effect into naive mice, but the uveitogenic potential of Tc17 cells alone of IL-21 on the development of Tc17 cells has not been described. has not been addressed. Caruso et al. showed that CD8ϩ and ␥ ϩ Cytokines known to antagonize development of Th17 cells, IFN- CD4 T cells in gastric mucosa of helicobacter pylori-infected (2), IL-2, IL-4, IL-12 (10), and IL-27 (4), appear to have an in- individuals produce IL-17A (10) in an IL-23- and STAT3-depen- hibitory effect on Tc17 cells as well. IL-23 plays a central role in dent manner. In a contact hypersensitivity model, allergen sensi- the biology of Th17 cells, while its effect on Tc17 cells has been tization induced the development of CD8ϩ T cell subpopulations less well studied. The comparable stimulatory effects of IL-23 ␥ ϩ ϩ ϩ that produce IFN- or IL-17A (16). In another study, most CD8 on IL-17A secretion by both CD8 and CD4 T cells have been and CD4ϩ T cell clones derived from lesional psoriatic skin ex- observed (10, 11). ϩ pressed IL-17A, suggesting that skin-infiltrating IL-17A T cells Tc17 cells are largely found in lung and digestive mucosa, par- contribute to disease pathogenesis (17). Collectively, these reports alleling distribution of Th17 cells, with Tc17 cells being less abun- demonstrate that Tc17 cells play a role in a variety of diseases and dant (1). Tumor-bearing mice have greater numbers of Th17 and homeostatic mechanisms. Tc17 cells that are particularly abundant in tumors (2). It appears Type 1 diabetes (T1D) is caused by the autoimmune destruction of insulin-producing islet ␤-cells of the pancreas (18). In newly ϩ *Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107; diagnosed patients with T1D, CD8 T cells represent a significant † and Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN portion of pancreas infiltrating cells (19), and islet Ag-specific 55905 CD8ϩ T cells are present in their peripheral blood (20). Studies in Received for publication January 7, 2009. Accepted for publication February 20, ϩ 2009. a NOD mouse model of T1D have indicated that CD8 T cells ␤ The costs of publication of this article were defrayed in part by the payment of page inflict damage to islet -cells both at the early stage in diabetes charges. This article must therefore be hereby marked advertisement in accordance development and at the final effector phase (21–23). Other models with 18 U.S.C. Section 1734 solely to indicate this fact. of diabetes employ mouse strains with transgenic expression of 1 This work was supported by grants from the National Multiple Sclerosis Society (to Ags governed by insulin promoter. In RIP-mOVA mice expression B.C.) and the National Institutes of Health (to A.M.R.). of membrane-bound chicken OVA in pancreatic islet ␤-cells is 2 Address correspondence and reprint requests to Dr. Bogoljub Ciric, Department of Neurology, Thomas Jefferson University, 300 Jefferson Hospital for Neuro- controlled by rat insulin promoter (24). Transfer of in vitro-acti- ϩ science Building, 900 Walnut Street, Philadelphia, PA 19107. E-mail address: vated OT-I CD8 T cells, specific for the OVA-derived peptide [email protected] SIINFEKL in the context of H-2Kb, into RIP-mOVA mice induces 3 B.C. and M.E.-b. contributed equally to this work. rapid onset of diabetes (24–26). 4 Abbreviations used in this paper: ROR, retinoic acid-related orphan receptor; EAE, We show herein that Tc17 and Th17 cells are similar in their experimental autoimmune encephalomyelitis; T1D, type 1 diabetes; WT, wild type. development, including the previously unknown promoting effect Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 of IL-21 on Tc17 cell development, as well as IL-23-dependent www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900036 The Journal of Immunology 5297 FIGURE 1. Effect of various cytokines on development of Tc17 cells. Isolated CD8ϩ T cells from naive C57BL/6 mice were activated with anti-CD3 and anti-CD28 Abs in the presence of various cytokines and neutralizing Abs (shown at the top of plots). Seventy-two hours after activation, cells were stimulated for 4 h with PMA and ionomycin in the presence of GolgiPlug, stained, and analyzed by flow cytometry for IL-17A and IFN-␥ expression. Data are representative of three experiments. IL-22 expression. Tc17 cells are cytolitic and can have either a izing conditions (no cytokines added) or in Th1 conditions (5 ng/ml IL-12, pathogenic or nonpathogenic phenotype in vivo, depending on ex- 5 ng/ml IL-2) or in Th17 conditions (2 ng/ml TGF-␤1, 20 ng/ml IL-6). posure to IL-23. Furthermore, the diabetogenic potential of Tc17 Where indicated in figure legends, cultures were supplemented with anti- mouse IFN-␥ (5 ␮g/ml), anti-mouse IL-4 (5 ␮g/ml), IL-1␤ (10 ng/ml), cells is dependent on IL-17A and IL-17F. ϩ TNF-␣ (10 ng/ml), or IL-27 (10 ng/ml). CD8 cells from spleens of OT-I mice were purified using anti-CD8 microbeads (Miltenyi Biotec) and stim- Materials and Methods ulated at a ratio of 1:5 with irradiated (3000 rad) splenocytes and 1 ␮g/ml Mice SIINFEKL peptide (OVA257–264) in either Tc1 or Tc17 supporting condi- tions. Seventy-two hours after stimulation, cells were used for flow cyto- C57BL/6J, OT-I transgenic (C57BL/6-Tg(TcraTcrb)1100Mjb/J), and RIP- metric analysis or RNA extraction and supernatants were used for cytokine mOVA (C57BL/6-Tg(Ins2-TFRC/OVA)296Wehi/WehiJ) mice and T-bet-, measurement by ELISA. STAT4- and IFN-␥-deficient mice were purchased from The Jackson Lab- oratory. STAT1-deficient mice on a C57BL/6 background were purchased from Taconic. Experimental procedures were approved by the Institutional Flow cytometry Animal Care and Use Committee of Thomas Jefferson University. For all intracellular staining, cells were stimulated for 4 h with PMA Cell preparation and culture (50 ng/ml) and ionomycin (500 ng/ml; Sigma-Aldrich) and treated with 6 CD4ϩ or CD8ϩ T cells were enriched from spleen mononuclear cells by GolgiPlug (1 ␮g per 1 ϫ 10 cells; BD Pharmingen). In the staining magnetic microbead cell sorting (Miltenyi Biotec). Cells were typically procedure, Fc receptors on cells were first blocked with anti-CD16/32 cultured in RPMI 1640 supplemented with 10% FBS (Invitrogen), 2 mM Ab (2.4G2; BD Pharmingen), and surface and intracellular staining with L-glutamine, 1 mM Na pyruvate, 1ϫ nonessential amino acids, 100 ␮g/ml Abs was performed following the manufacturer’s instructions for stain- penicillin, 100 ␮g/ml streptomycin, and 0.5 ␮M 2-ME. T cells were stim- ing using Fix & Perm reagents (Caltag Laboratories). Data were ac- ulated with anti-CD3 (1 ␮g/ml) and anti-CD28 (1 ␮g/ml) Abs in 24-well quired on a FACSAria (BD Biosciences) and analyzed with FlowJo plates (2 ml of media containing 1.5 ϫ 106 cells/well) either in nonpolar- software (Tree Star).