The Journal of Immunology IL-17– and IFN-g–Secreting Foxp3 + T Cells Infiltrate the Target Tissue in Experimental Autoimmunity Marianna Esposito,* ,1 Francesca Ruffini,* Alessandra Bergami,* Livia Garzetti,* Giovanna Borsellino, † Luca Battistini, † Gianvito Martino, ‡ and Roberto Furlan* CD4 + Foxp3 + regulatory T cells (Tregs) have been considered crucial in controlling immune system homeostasis, and their de- rangement is often associated to autoimmunity. Tregs identification is, however, difficult because most markers, including CD25 and Foxp3, are shared by recently activated T cells. We show in this paper that CD4 + Foxp3 + T cells are generated in peripheral lymphoid organs on immunization and readily accumulate in the target organ of an autoimmune reaction, together with classical inflammatory cells, constituting up to 50% of infiltrating CD4 + T cells. Most CD4 + Foxp3 + T cells are, however, CD25 2 and express proinflammatory cytokines such as IL-17 and IFN-g, questioning their suppressive nature. Moreover, in vitro CD4 + T lymphocytes from naive and autoimmune mice, stimulated to differentiate into Th1, Th2, Th17, and induced Tregs, display early mixed expression of lineage-specific markers. These results clearly point to an unprecedented plasticity of naive CD4 + T cells, that integrating inflammatory signals may change their fate from the initial lineage commitment to a different functional phenotype. The Journal of Immunology, 2010, 185: 7467–7473. T ight regulation of the immune system is essential to ensure that immune activation is predominantly protective rather than autoimmune. There are several mechanisms to ensure immune tolerance to autoantigens including active suppression of effector T cells (Teffs) in the periphery. This function is mediated by populations of T cells known as regulatory T cells (Tregs) (1, 2). The most important subsets of Tregs are thought to be thymically derived naturally occurring CD4 + CD25 hi Foxp3 + Tregs ( n Tregs) (3, 4) and peripheral CD4 + induced Tregs ( i Tregs), which differentiate from naive precursors during activation in the presence of ap- propriate cytokines, such as TGF-b (5, 6). In this way, n Tregs can be activated, and i Tregs generated, to perform suppressive effects at sites expressing their cognate Ags, namely, in the periphery to maintain homeostasis and in the target organ and draining lymph nodes to limit tissue injury during inflammation. Tregs are essential in preventing the development of auto- immune diseases in both humans (7) and mice (8), and abnormali- ties of number, function, and expression of FOXP3/Foxp3 in these populations have been identified in chronic inflammatory diseases, such as relapsing-remitting experimental autoimmune encepha- lomyelitis (RR-EAE), an induced murine model of human mul- tiple sclerosis (MS) (9). The cause(s) of the impaired suppressive function of Tregs in MS patients has not been elucidated as yet. It could be that genetic or environmental factors, or both, limit FOXP3/Foxp3 expression, leading to an altered balance between functional encephalitogenic Th cells and Tregs in MS, as well as in the experimental autoimmune encephalomyelitis (EAE) model (10). Dysfunction of CD4 + CD25 hi Tregs described in MS (11, 12) might have an impact on susceptibility to disease or the clinical phenotype (in particular, relapsing-remitting MS) and course (13, 14). One of the confounding factors in the interpretation of pre- viously published works has been that the CD4 + CD25 hi T cell compartment of patients with MS contains the Treg population, as well as a potential contaminant of activated conventional CD4 + T cells expressing CD25. As a result, identification of Tregs in patients with MS solely based on high expression of CD25 is not ideal. Recent studies using more specific markers of Tregs, such as low expression of CD127 (CD4 + CD25 hi CD127 lo Tregs) confirms that the circulating Treg population in patients with MS retains suppressive function (15), implying that Tregs can lose their function in the CNS. The actual suppressive function of Tregs within the MS CNS is still a matter of debate (16). Assuming that in RR-EAE the im- mune system is actively attempting to control disease progression by recruitment of Tregs to the sites of inflammation (10), ongoing autoimmunity is likely to represent defective function of Tregs per se or an alteration of phenotype to either an anergic state or proinflammatory lineage by the CNS milieu (10, 17). This latter position is strengthened by the demonstration that Tregs lose Foxp3 expression under inflammatory conditions and can be in- duced to express proinflammatory cytokines, such as IL-17 (IL- 17 + Foxp3 + ) and IFN-g (IFN-g + Foxp3 + ) (18–20). In particular, TGF-b– i Tregs readily lose Foxp3 and acquire IL-17 expression in Th17-polarizing conditions (19, 21). This is mirrored by accu- mulating evidence that different T cell lineages may also be un- stable and capable of being induced to produce cytokines and *Clinical Neuroimmunology Unit and ‡ Neuroimmunology Unit, Division of Neuro- science, Institute of Experimental Neurology, San Raffaele Scientific Institute, Milan; and † Neuroimmunology Unit, European Brain Research Institute, Santa Lucia Foun- dation, Rome, Italy 1 Current address: Medical Research Council Centre for Transplantation and National Institute for Health Research Comprehensive Biomedical Research Centre, King’s College, Guy’s Hospital, London, United Kingdom. Received for publication May 11, 2010. Accepted for publication October 12, 2010. This work was supported by the Italian Foundation for Multiple Sclerosis (to R.F., G.M., and G.B.) and the Italian Ministry of Health (to G.M.). Address correspondence and reprint requests to Dr. Roberto Furlan, Clinical Neuro- immunology Unit, Institute of Experimental Neurology, San Raffaele Scientific In- stitute, Via Olgettina 58, 20132 Milan, Italy. E-mail address: furlan.roberto@hsr.it The online version of this article contains supplemental material. Abbreviations used in this paper: AU, arbitrary units; CLN, cervical lymph nodes; DLN, axillary and inguinal draining lymph nodes; EAE, experimental autoimmune encephalomyelitis; GITR, glucocorticoid-induced tumor necrosis factor receptor; i Treg, induced regulatory T cell; MFI, mean fluorescence intensity; n Treg, naturally occurring regulatory T cell; PLP, proteolipid protein; Pre-clin., preclinical phase of the disease; Rec., recovery from the disease; Rel., relapse of the disease; RR-EAE, relapsing-remitting experimental autoimmune encephalomyelitis; SPL, spleen; Teff, effector T cell; Treg, regulatory T cell. 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