Control of Autoimmune Myocarditis and Multiorgan Inflammation by Glucocorticoid-Induced TNF Receptor Family-Related Protein high, This information is current as Foxp3-Expressing CD25 + and CD25− of September 29, 2021. Regulatory T Cells Masahiro Ono, Jun Shimizu, Yoshiki Miyachi and Shimon Sakaguchi Downloaded from J Immunol 2006; 176:4748-4756; ; doi: 10.4049/jimmunol.176.8.4748 http://www.jimmunol.org/content/176/8/4748 http://www.jimmunol.org/ References This article cites 51 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/176/8/4748.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology 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 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Control of Autoimmune Myocarditis and Multiorgan Inflammation by Glucocorticoid-Induced TNF Receptor Family-Related Proteinhigh, Foxp3-Expressing CD25؉ and CD25؊ Regulatory T Cells1 Masahiro Ono,*† Jun Shimizu,‡ Yoshiki Miyachi,† and Shimon Sakaguchi2*§ Anomalies of naturally occurring CD4؉ regulatory T cells (Treg) cause severe autoimmune/inflammatory diseases in humans and rodents. The transcription factor Foxp3 is currently the most specific marker for natural CD4؉ Treg, but it would be useful if other Treg markers, particularly cell surface molecules, could be elucidated. We demonstrate in this study that the vast majority of Foxp3-expressing CD4؉ T cells (whether CD25؉ or CD25؊) show constitutive high-level expression of glucocorticoid-induced Downloaded from TNFR family-related gene/protein (GITR). Transfer of T cell or thymocyte suspensions depleted of GITRhigh cells produces in BALB/c nude mice a wider spectrum and more severe forms of autoimmune diseases than does transfer of similar cell suspensions depleted of CD25؉CD4؉ T cells only. Notably, mice that receive cells depleted of GITRhigh populations develop severe multiorgan inflammation that includes fatal autoimmune myocarditis resembling giant cell myocarditis in humans, accompanying high-titer anti-myosin autoantibodies. Similar transfer of GITRhigh-depleted cells from prediabetic NOD mice to NOD-SCID mice acceler- ates the development of diabetes and induces skeletal muscle myositis and other autoimmune/inflammatory diseases. We conclude http://www.jimmunol.org/ that GITRhigh, Foxp3-expressing natural Treg, containing both CD25؉ and CD25؊ cell populations, contribute to preventing a variety of autoimmune/inflammatory diseases, and depletion of these cells allows the activation of even weak or rare autoreactive T cells yielding widespread severe autoimmune disease. Diseases induced in this way include many which have been suspected of an autoimmune etiology in humans without much evidence. GITRhigh, Foxp3-expressing natural Treg represent a potential target for the treatment and prevention of these diseases. The Journal of Immunology, 2006, 176: 4748–4756. here is accumulating evidence that regulatory T cells type 1 diabetes (T1D) and thyroiditis), inflammatory bowel dis- (Treg),3 in particular naturally occurring CD25ϩCD4ϩ ease, and allergy (such as allergic dermatitis and food allergy) (4, T Treg, play crucial roles in the maintenance of immuno- 5). The disease is so severe that Ͼ80% of the patients, including by guest on September 29, 2021 logic self-tolerance and suppressive control of a variety of patho- those with a diabetes-protective HLA haplotype, develop T1D and logical and physiological immune responses (1–3). Depletion of other autoimmune diseases (5). The cause of IPEX is mutation of ϩ ϩ CD25 CD4 Treg, for example, elicits autoimmune disease in the gene FOXP3, which encodes the forkhead/winged-helix tran- multiple organs such as stomach and thyroid in otherwise normal scription factor Scurfin (5). CD25ϩCD4ϩ natural Treg in mice animals, provokes effective tumor immunity to autologous tumor specifically express Foxp3 (the murine ortholog of human cells, enhances immune responses to invading or cohabiting mi- FOXP3), and ectopic or transgenic expression of Foxp3 can con- crobes, and triggers allergic responses to innocuous environmental vert naive T cells to Treg that phenotypically and functionally substances. The best example of the role of natural Treg in humans ϩ ϩ resemble natural CD25 CD4 Treg (6–8). Foxp3-defective or is a fatal autoimmune/inflammatory disease called IPEX (immune ϩ ϩ -deficient mice fail to develop CD25 CD4 Treg, leading to the dysregulation, polyendocrinopathy, enteropathy, X-linked syn- occurrence of fatal inflammatory disease (7). Foxp3 thus appears drome), which accompanies severe autoimmune disease (such as to be a master control gene for the development and function of naturally occurring Treg, and is currently the most specific molec- *Department of Experimental Pathology, Institute for Frontier Medical Sciences, ular marker for this T cell subpopulation (6–9). A critical question, Kyoto University, Kyoto, Japan; †Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; ‡Tokyo Metropolitan Institute for Ger- then, in elucidating the cause and mechanism of human autoim- ontology, Itabashi-ku, Tokyo, Japan; and §Core Research for Evolutional Science and mune diseases is how FOXP3 deficiency causes such fatal auto- Technology, Japan Science and Technology Agency, Kawaguchi, Japan immune/inflammatory diseases, and whether anomaly of FOXP3- Received for publication December 12, 2005. Accepted for publication January 26, 2006. expressing Treg can be a cause of other human inflammatory The costs of publication of this article were defrayed in part by the payment of page diseases including those where etiology is unknown. charges. This article must therefore be hereby marked advertisement in accordance It has been shown that CD25, the IL-2R ␣-chain, is not only a with 18 U.S.C. Section 1734 solely to indicate this fact. useful molecular marker for natural CD4ϩ Treg but also an essen- 1 This work was supported by grants-in-aid from the Ministry of Education, Sports tial molecule for their development and function as an indispens- and Culture of Japan. able component of the high-affinity IL-2R. For example, IL-2-, 2 Address correspondence and reprint requests to Dr. Shimon Sakaguchi, Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, CD25-, or CD122 (IL-2R ␤-chain)-deficient mice develop fatal 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address: autoimmune/inflammatory disease similar to Foxp3 deficiency [email protected] (10–12). Neutralization of circulating IL-2 for a limited period 3 Abbreviations used in this paper: Treg, regulatory T cell; IPEX, immune dysregu- ϩ ϩ lation, polyendocrinopathy, enteropathy, X-linked syndrome; T1D, type 1 diabetes; selectively reduces the number of CD25 CD4 Treg and elicits GITR, glucocorticoid-induced TNFR family-related protein; C, complement. autoimmune disease similar to the one produced by depletion of Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 4749 ϩ ϩ CD25 CD4 Treg (13). In contrast, there is accumulating evi- purified by the MACS system (Miltenyi Biotec), using biotinylated anti- dence for the existence of regulatory activity in the CD25ϪCD4ϩ CD4, anti-CD8, or anti-CD19, respectively, with PE-labeled streptavidin T cell population. For example, CD25ϪCD45RBlowCD4ϩ T cells and anti-PE microbeads. Other T cell subpopulations were prepared as Ϫ ϩ ϩ described previously (19). For assessing GITR expression on the residual in rodents and CD25 CD45RO CD4 T cells in humans contain cells after anti-GITR (DTA-1) or anti-CD25 (7D4)ϩC (complement)-treat- cells expressing Foxp3/FOXP3 at a low level (6, 9, 14). ment or C-treatment alone, they were incubated again with DTA-1 and CD25ϪCD4ϩ T cells bear an autoimmune-suppressive activity in 7D4, washed, and stained with rabbit polyclonal biotinylated anti-murine various animal models of autoimmune disease, although the activ- GITR Ab. ϩ ϩ ity is generally weaker than that of natural CD25 CD4 Treg Quantitation of mRNA expression by real-time RT-PCR (15–17). It must be determined then to what degree CD25ϪCD4ϩ Treg contribute to the maintenance of natural self-tolerance and Quantitation of Foxp3 and HPRT mRNA by real-time PCR was described previously (6). how severe the autoimmune diseases will be, in particular what spectrum of autoimmune/inflammatory disease will develop, when Histopathology ϩ Ϫ both CD25 and CD25 Foxp3-expressing Treg are depleted from Myocarditis and other autoimmune diseases were histologically evaluated the normal immune system. and graded as described previously (21–23). CD25ϩCD4ϩ Treg express GITR (glucocorticoid-induced TNFR family related gene/protein) at higher levels than other T Immunohistochemistry