Autoreactive T Cells Escape Clonal Deletion in the Thymus by a CD24-Dependent Pathway This information is current as Joseph W. Carl, Jr., Jin-Qing Liu, Pramod S. Joshi, Hani Y. of September 27, 2021. El-Omrani, Lijie Yin, Xincheng Zheng, Caroline C. Whitacre, Yang Liu and Xue-Feng Bai J Immunol 2008; 181:320-328; ; doi: 10.4049/jimmunol.181.1.320 http://www.jimmunol.org/content/181/1/320 Downloaded from References This article cites 54 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/181/1/320.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Autoreactive T Cells Escape Clonal Deletion in the Thymus by a CD24-Dependent Pathway1 Joseph W. Carl, Jr.,* Jin-Qing Liu,* Pramod S. Joshi,* Hani Y. El-Omrani,* Lijie Yin,‡ Xincheng Zheng,§ Caroline C. Whitacre,† Yang Liu,‡ and Xue-Feng Bai2* Despite negative selection in the thymus, significant numbers of autoreactive T cells still escape to the periphery and cause autoimmune diseases when immune regulation goes awry. It is largely unknown how these T cells escape clonal deletion. In this study, we report that CD24 deficiency caused deletion of autoreactive T cells that normally escape negative selection. Restoration of CD24 expression on T cells alone did not prevent autoreactive T cells from deletion; bone marrow chimera experiments suggest that CD24 on radio-resistant stromal cells is necessary for preventing deletion of autoreactive T cells. CD24 deficiency abrogated the development of experimental autoimmune encephalomyelitis in transgenic mice with a TCR specific for a pathogenic autoan- tigen. The role of CD24 in negative selection provides a novel explanation for its control of genetic susceptibility to autoimmune Downloaded from diseases in mice and humans. The Journal of Immunology, 2008, 181: 320–328. t is well established that thymic clonal deletion of autoanti- mental autoimmune encephalomyelitis (EAE)3 induced with my- gen reactive T cells plays a central role in preventing the elin oligodendrocyte glycoprotein (MOG)-peptide immunization. I development of autoimmune diseases, as genetic or experi- Moreover, CD24 polymorphism has emerged as an important ge- mental blockade of this process results in the development of au- netic factor in regulating susceptibility to autoimmune diseases, http://www.jimmunol.org/ toimmune diseases (1–5). Nevertheless, significant numbers of au- including multiple sclerosis (21–23) and systemic lupus erythem- toreactive T cells can be easily detected (6, 7) and expanded (8) atosus in humans (22). To understand how CD24 regulates sus- even in normal individuals. Mice with the Scurfy mutation having ceptibility to autoimmune diseases, we generated CD24-deficient abrogated function of regulatory T cells still succumb to fatal au- mice that express a TCR specific for MOG 35–55, a pathogenic toimmune diseases despite normal negative selection (5, 9). Be- autoantigen in the C57BL6/J mice (2D2ϩCD24Ϫ/Ϫ mice). Surpris- cause the development of autoimmune diseases can be prevented ingly, we observed that 2D2ϩCD24Ϫ/Ϫ mice have atrophic thymi after breeding to TCR transgenic mice (10), some autoreactive T with absent CD4ϩCD8ϩ and CD4ϩCD8Ϫ populations. Transgenic cells must have escaped clonal deletion in Scurfy mice. Although expression of CD24 on thymocytes alone did not prevent T cell lack of expression of self Ags has been largely attributed as a key deletion; bone marrow chimera experiments suggest that CD24 on by guest on September 27, 2021 factor (11–13), we have reported that even T cells specific for P1A, radio-resistant stromal cells is necessary for preventing deletion of a self Ag expressed in thymic medullar epithelial cells (14), can 2D2 T cells. Furthermore, we observed that CD24 also reduced the escape clonal deletion (15). How autoreactive T cells escape clonal efficiency of clonal deletion of viral superantigen (VSAg)-specific deletion may hold a key to understanding the pathogenesis of au- T cells. In contrast, development of T cells specific for OVA was toimmune diseases. unaffected by CD24 deficiency. These data demonstrate a critical CD24 is a glycosyl-phosphatidylinositol-anchored cell surface role for CD24 in escape of autoreactive T cells from thymic clonal glycoprotein with extensive carbohydrate structures attached to a deletion. small protein core (16). CD24 is expressed on various cells in- cluding immature thymocytes and B lymphocytes (17, 18). Al- though immune responses were apparently normal in mice with Materials and Methods targeted mutation of CD24 (17, 19), we have reported (20) that Mice targeted mutation of CD24 abrogates the development of experi- C57BL6 mice were purchased from The Jackson Laboratory. 2D2 TCR transgenic mice (24) were kindly provided by Dr. V. K. Kuchroo (Harvard Medical School, Boston, MA). CD24Ϫ/Ϫ mice in the C57BL6 background have been described (20, 25). By using Charles River Max-Bax technology *Department of Pathology and Comprehensive Cancer Center and †Department of (Marker-Assisted Accelerated Backcrossing), we have recently produced Virology, Immunology and Molecular Genetics, Ohio State University Medical Cen- CD24Ϫ/Ϫ BALB/c mice. OTII TCR transgenic mice were purchased from ter, Columbus, OH 43210; ‡Department of Surgery and Comprehensive Cancer Cen- § The Jackson Laboratory. Transgenic mice with CD24 expression exclu- ter, University of Michigan, Ann Arbor, MI 48109; and OncoImmune, Inc., Colum- sively on T cells have been described (26, 27). All mice were bred and bus, OH 43212 maintained in a specific pathogen-free animal facility of The Ohio State Received for publication December 19, 2007. Accepted for publication April University. The animal facilities are fully accredited by the American As- 24, 2008. sociation for Accreditation of Laboratory Animal Care. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by grants from National Multiple Sclerosis Society 3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyeli- (RG 3638 to X.-F.B.) and Ohio Department of Development. tis; MOG, myelin oligodendrocyte glycoprotein; WT, wild type; TEC, thymic epi- 2 Address correspondence and reprint requests to Dr. Xue-Feng Bai, Department of thelial cells; DP, double positive; SP, single positive; DC, dendritic cell; VSAg, viral Pathology and Comprehensive Cancer Center, Ohio State University Medical Center, superantigen. 129 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. E-mail address: Xue- [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 321 Abs and flow cytometry PBS in the tail vein at day 0 and again 48 h later. The mice were observed every day and were scored on a scale from 0 to 5 with gradations of 0.5 for The following Abs were used in the experiments according to the manu- intermediate scores: 0, no clinical signs; 1, loss of tail tone; 2, wobbly gait; facturer’s recommendations: unlabeled, FITC-, PE-, PerCp-, allophycocya- 3, hind limb paralysis; 4, moribund; and 5, death. nin- or biotin-labeled anti-CD4 (GK1.4), -CD8␣ (53-6.7), -CD11c (HL3), -CD24 (M1/69), -CD25 (7D4), -CD44 (IM7), -CD45 (30-F11), -CD62L Histology (Mel-14), -CD69 (H1.2F3), V␣2 (B20.1), -V␣3.2 (RR3-16), -V␤3(⌲J25), -V␤5.1/5.2 (MR9-4), -V␤8 (F23.1), -V␤11 (RR3-15), -V␤12 (MR11-1), Mice were sacrificed by inhaling CO2. Spinal cords, cerebellum, and optic and anti-rat IgG2a (RG7/1.30). These Abs were purchased from BD nerves were removed and fixed in 10% formalin/PBS. Paraffin sections Pharmingen or eBioscience. For flow cytometry analysis, cells were incu- were prepared and stained by the histology core facilities of Department of bated with Abs on ice for 30 min followed by extensive washing. Cells Pathology (Ohio State University) for H&E and luxol fast blue (myelin were analyzed on a FACSCalibur cytometer (BD Biosciences). staining). Pathological changes of each spinal cord were evaluated and scored as follows: 0, no changes; 1, focal area involvement; 2, Ͻ5% of Creation of radiation bone marrow chimeras total myelin area involvement; 3, 5–10% of total myelin area involvement; 4, 10–20% of total myelin area involvement; 5, Ͼ20% of total myelin area We prepared bone marrow cells by flushing donor mice femur and tibia involvement. bones with PBS. Recipient mice were lethally irradiated (1000 rads) and reconstituted with 10 ϫ 106 bone marrow cells by i.v. injection. Engraft- ment took place over a 6- to 8-wk period. We used 2D2 TCR transgenic Results mice as our basic model and generated four types of bone marrow chimeras. Thymic clonal deletion of MOG-specific T cells in Chimera 1 (2D2ϩCD24ϩ/ϩ Ͼ CD24ϩ/ϩ mice)-bone marrow cells from 2D2ϩ CD24-deficient mice CD24ϩ/ϩ mice were injected into irradiated CD24ϩ/ϩ mice. In chimera 1 mice, both bone marrow-derived cells and TEC were CD24-positive.