Pathway in the Thymus by a CD24-Dependent Autoreactive T

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

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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 autoantigen. 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 including 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. Chimera Kuchroo and colleagues (24) have produced 2D2 TCR transgenic 2 (2D2ϩCD24Ϫ/Ϫ Ͼ CD24ϩ/ϩ mice)-bone marrow cells from 2D2ϩCD24Ϫ/Ϫ mice, whose TCR recognizes MOG 35–55, a pathogenic epitope in Downloaded from mice were injected into irradiated CD24ϩ/ϩ mice. In chimera 2 mice, TECs the C57BL/6 mice. Because these T cells can develop normally in expressed CD24, while bone marrow-derived dendritic cells (DC) and T cells the mice, we tested whether the autoantigen MOG is expressed were CD24 deficient. Chimera 3 (2D2ϩCD24ϩ/ϩ Ͼ CD24Ϫ/Ϫ mice)-bone marrow cells from 2D2ϩCD24ϩ/ϩ mice were injected into irradiated in the thymus. As shown in Fig. 1A, significant expression of MOG CD24Ϫ/Ϫ mice. In chimera 3 mice, TECs did not express CD24, while bone mRNA was detected in the thymus by real-time PCR. Consistent marrow-derived DC and T cells were CD24-positive. with this finding, other groups also demonstrated MOG mRNA ϩ Ϫ/Ϫ Ͼ Ϫ/Ϫ Chimera 4 (2D2 CD24 CD24 mice)-bone marrow cells from expression in the thymus (28) or TECs (14). Thus, 2D2 T cells http://www.jimmunol.org/ ϩ Ϫ/Ϫ Ϫ/Ϫ 2D2 CD24 mice were injected into irradiated CD24 mice. In chimera have escaped clonal deletion despite expression of MOG in the 4 mice, both bone marrow-derived cells and TECs were CD24 deficient. thymus. Surprisingly, targeted mutation of CD24 caused a massive Real-time RT-PCR reduction of thymic cellularity in the 2D2 transgenic mice but not ϩ/Ϫ Ϫ/Ϫ Total RNA was isolated from thymi or stroma-enriched thymi using the in CD24 or CD24 mice without the 2D2 TCR transgene Trizol method (Invitrogen). The first strand cDNA of each sample was (Fig. 1B). To determine whether the thymic atrophy was related to synthesized using a reverse transcription kit (Invitrogen). Quantitative real age, we examined young mice (18–20 days after birth); we ob- time PCR was performed using an ABI Prism 7900-HT sequence system served similar thymic atrophy in 2D2ϩCD24Ϫ/Ϫ mice (data not (PE Applied Biosystems) with the QuantiTect SYBR Green PCR kit (Qia- ␣␤ gen) in accordance with the manufacturer’s instructions. The following shown). Correspondingly, numbers of -positive 2D2 T cells and ϩ ϩ ϩ Ϫ by guest on September 27, 2021 primers were used: mMOG.F: 5Ј-GCAGCACAGACTGAGAGGAA-3Ј; the CD4 CD8 and CD4 CD8 populations were dramatically mMOG.R: 5Ј-CAGATGATCAAGGCAACCAG-3Ј. Hypoxanthine-gua- reduced in the thymi of the 2D2ϩCD24Ϫ/Ϫ mice compared with nine phosphoribosyltransferase (HPRT).F: 5Ј-AGC CTA AGA TGA GCG that of the 2D2ϩCD24ϩ/ϩ mice (Fig. 1C). CAA GT-3Ј HPRT.R: 5Ј-TTA CTA GGC AGA TGG CCA CA-3Ј. The HPRT gene was amplified and served as endogenous control. PCR was CD24 on thymocytes is insufficient to rescue clonal deletion of ␮ performed under optimal conditions. A total of 1 l of first strand cDNA autoreactive T cells; CD24 on radio-resistant thymic stromal product was amplified with platinum Taq polymerase (Invitrogen) and gene-specific primer pairs. Each sample was assayed in triplicate and ex- cells is necessary periments were repeated twice. The relative amounts of mRNA were cal- Because the majority of thymocytes expressed high levels of culated by plotting the Ct (cycle number), and average relative expression was determined by the comparative method (2Ϫ⌬⌬Ct). CD24, we tested the possibility that CD24 expressed on thymocytes may be responsible for the enhanced clonal deletion. We Proliferation assay generated 2D2ϩCD24Ϫ/Ϫ mice that express CD24 under the con- Splenocytes from each strain of TCR transgenic mice (2D2, OTII) with or trol of the proximal lck promoter. The lck promoter is known to be without CD24 deficiency were stimulated with titrated peptide Ags in 96- active in thymocytes, starting at the DN3 stage (29). As shown in well U-bottom plates. In some experiments, we purified CD4 cells from Fig. 2A, essentially all 2D2 TCR-expressing thymocytes in the spleen and lymph nodes and stimulated them with peptide Ags and irra- CD24 transgenic mice expressed high levels of CD24. Neverthe- diated (2000 rad) syngenic APCs. [3H]Thymidine was added into the cul- ture at 48 h and harvested 12 h later. 3H incorporation was measured with less, thymic cellularity (Fig. 2B) and distribution of T cell subsets a scintillation counter. The OVA peptide Ag used in the assay (OVA 323– (Fig. 2C) were unaffected by CD24 expression in thymocytes. 339) was purchased from Sigma-Aldrich. MOG peptide 35–55 (MEVG These results demonstrate that lack of CD24 on thymocytes is not WYRSPFSRVVHLYRNGK) was purchased from Genemed Synthesis. solely responsible for enhanced clonal deletion. Purification of 2D2 T cells from 2D2ϩCD24ϩ/ϩ or 2D2ϩ To determine whether CD24 expressed on bone marrow-derived CD24Ϫ/Ϫ mice or non-bone marrow-derived stromal cells is responsible, we established chimeras using bone marrow from 2D2ϩCD24ϩ/ϩ or 2D2 T cells were purified by negative selection. In brief, spleen and lymph ϩ Ϫ/Ϫ node cells from 2D2 TCR transgenic mice were incubated with a mixture of 2D2 CD24 mice to reconstitute irradiated wild-type (WT) or mAbs (anti-CD8 mAb TIB210, anti-FcR mAb 2.4G2, and anti-CD11c mAb CD24-deficient mice. As shown in Fig. 3A, CD24 expression on N418). After removing the unbound Abs, the cells were incubated with anti- thymocytes indicates a nearly complete replacement of the hema- Ig-coated magnetic beads (Dynal Biotech). A magnet was used to remove the ϩ Ϫ Ϫ topoietic compartment in the chimera mice. In spleens, we de- Ab-coated cells. The remaining cells were CD4 or CD4 CD8 T cells. The ϩ purified 2D2 T cells were used for the proliferation assay. tected intermediate to high levels of CD24 expression on CD11c cells from 2D2ϩCD24ϩ/ϩ Ͼ CD24Ϫ/Ϫ chimeras but not on Induction and assessment of EAE CD11cϩ cells from 2D2ϩCD24Ϫ/Ϫ Ͼ CD24ϩ/ϩ chimeras (Fig. 2D2 TCR transgenic mice (8–12 wk of age) of different CD24 genotypes 3B). Thus, the chimeric mice can be used to evaluate the contri- received 200 ng of pertussis toxin (List Biological Laboratories) in 200 ␮l bution of CD24 in clonal deletion of 2D2 T cells. Mature 2D2 T 322 CD24 AND THYMIC GENERATION OF AUTOREACTIVE T CELLS A B C

FIGURE 1. CD24 inhibits thymic deletion of MOG-speciﬁc T cells. A, MOG Ag is expressed in the thymi of C57BL6 mice. A standard 40-cycle real-time PCR was used to detect MOG mRNA expression in the thymus. Data shown represent three experiments with similar results. B, Thymocyte

cellularity. Each triangle represents the value from a single mouse. Thick lines represent median numbers of each group of mice. Student’s t test Downloaded from was used for the comparison. C, Impact of CD24 deﬁciency on the development of 2D2 TCR transgenic T cells. Thymocytes from 2D2ϩCD24Ϫ/Ϫ and 2D2ϩCD24ϩ/ϩ mice were stained for V␣3.2, V␤11, CD4, and CD8. The thymi from 2D2ϩCD24Ϫ/Ϫ mice had dramatically reduced V␣3.2ϩ V␤11ϩ populations and failed to generate CD4ϩCD8ϩ and CD4ϩCD8Ϫ T cells compared with that of 2D2ϩCD24ϩ/ϩ mice. Data represent at least ﬁve experiments with similar results.

ϩ ϩ/ϩ cells (CD4-single positive (SP)) were generated in 2D2 CD24 is not sufﬁcient for rescuing 2D2 T cell deletion. Thus, these http://www.jimmunol.org/ Ͼ CD24ϩ/ϩ chimeras but not in 2D2ϩCD24Ϫ/Ϫ Ͼ CD24Ϫ/Ϫ chi- bone marrow chimera data suggest that CD24 expression on meras (Fig. 3, C and D, left and right panels). CD24 expression on radio-resistant stromal cells inhibits deletion of transgenic T the radio-resistant stromal cells (Fig. 3, C and D, middle left cells at the CD4ϩCD8ϩ stage. panel), but not on bone marrow-derived cells (Fig. 3, C and D, Because TECs, especially medullary epithelial cells, are the middle right panel), rescued transgenic TCR␣␤ϩ cells at the major nonhematopoietic cells that are involved in negative se- CD4ϩCD8ϩ stage; however, because no mature CD4ϩ 2D2 T lection (14, 30–32), we analyzed expression of CD24 on TECs. cells were generated (Fig. 3D, middle left), it is likely that As shown in Fig. 4A, about 50% of CD45-negative thymic stro- CD24 expression on radio-resistant thymic stromal cells alone mal cells express CD24; almost 100% of medullar epithelial by guest on September 27, 2021 AB

FIGURE 2. Restoration of CD24 on 2D2 T cells in CD24-deficient mice does not prevent thymic deletion. We have bred 2D2ϩCD24Ϫ/Ϫ mice with TCD24TGCD24Ϫ/Ϫ mice (mice with CD24 expression on T cells only) and produced double transgenic mice with CD24 deficiency (2D2.TCD24TGCD24Ϫ/Ϫ mice). A, Phenotypes of three different mice identified by flow cytometry. Single-cell suspensions of thymocytes were stained for CD24 and V␣3.2 markers followed by flow cytometry analysis. Data shown were gated on V␣3.2-positive thymocytes. B, Thymocyte numbers in mice with different genotypes. No significant difference was observed between 2D2ϩCD24Ϫ/Ϫ mice with 2D2. C TCD24TGCD24Ϫ/Ϫ mice. C, Thymocytes from 2D2.TCD24TGCD24Ϫ/Ϫ mice show a phenotype similar to that seen in 2D2ϩCD24Ϫ/Ϫ mice. The Journal of Immunology 323

FIGURE 3. CD24 on radio-resis- AB tant thymic stromal cells is necessary but not sufficient for the generation of mature CD4 T cells specific for MOG peptide. CD24Ϫ/Ϫ and CD24ϩ/ϩ mice were lethally irradiated (1000 Rad) and reconstituted with 2D2ϩCD24Ϫ/Ϫ or 2D2ϩCD24ϩ/ϩ donor bone marrow cells. A, CD24 expression on the thymocytes from different bone mar- CD24 row chimeric mice. B, CD24 expres- ϩ sion on splenic CD11c cells from C different bone marrow chimeras. Splenocytes were digested with colla- genase IV, and the resulting mononu- clear cells were stained for CD11c and CD24 followed by flow cytometry analysis. Data shown were gated on CD11c-positive cells. C, Genera- tion of 2D2 T cells in the thymi of Downloaded from bone marrow chimeras. Thymocytes D were stained for V␣3.2, V␤11, CD4, and CD8 followed by flow cytometry analysis. D, 2D2 T cell subsets in bone marrow chimeras. Data shown were gated on V␣3.2ϩV␤11ϩ thymo-

cytes. The result shown represents http://www.jimmunol.org/ three independent experiments with similar results.

cells (B7ϩ) (33) express CD24. These data are consistent with CD24 deficiency does not alter development of T cells specific a role of CD24 on radio-resistant TECs. To determine whether for a foreign Ag CD24 deficiency affects expression of MOG Ag in the thymus, we compared MOG mRNA expression in the thymus and en- As a comparison to the development of autoreactive transgenic T riched-thymic stromal cells. As shown in Fig. 4B, similar levels cells, we also studied the development of transgenic T cells spe- by guest on September 27, 2021 of MOG-transcripts were detected in WT and CD24-deficient cific for OVA in WT and CD24-deficient hosts. As shown in Fig. thymi, regardless of whether RNA from whole thymus extract 5, the TCR distribution (Fig. 5A), thymic cellularity (Fig. 5B), and ϩ ϩ/ϩ or from enriched-stromal cells were compared. As such, the subset distribution (Fig. 5C) are similar in OTII CD24 and ϩ Ϫ Ϫ function of CD24 is unlikely through regulation of peripheral OTII CD24 / transgenic mice. Moreover, the OTII T cells in Ag expression in the thymus. the periphery are fully functional (Fig. 5D). These results

A B

FIGURE 4. Expression of CD24 and MOG in thymic stromal cells. A, Thymi from CD24Ϫ/Ϫ and CD24ϩ/ϩ C57BL6 mice were digested with col- lagenase, and cell suspensions were enriched for stromal cells and were then stained for CD45, CD24, and B7-1 or B7-2 markers. Data shown were gated on CD45Ϫ cells. B, Real- time PCR was used to examine MOG gene expression in total thymocytes and enriched thymic stromal cells. Three experiments were performed with similar results. 324 CD24 AND THYMIC GENERATION OF AUTOREACTIVE T CELLS A B

FIGURE 5. CD24 expression is not required for the thymic generation of CD4 T cells speciﬁc for OVA. OTII TCR transgenic mice were bred with CD24Ϫ/Ϫ mice for two generations. The resulting OTIIϩCD24Ϫ/Ϫ mice were compared with WT mice for thymocyte development. A, CD24 expression in the thymocytes of OTII TCR transgenic mice with different genotypes. B, Summary of thymocyte C D numbers in mice of different genotypes. C, Flow cytometry analysis of the thymi of OTII TCR transgenic mice with or without CD24. OTIIϩ CD24ϩ/ϩ and OTIIϩCD24Ϫ/Ϫ mice

revealed similar generation of OTII T Downloaded from cells. D, Splenocytes from OTIIϩ CD24ϩ/ϩ and OTIIϩCD24Ϫ/Ϫ mice show similar proliferation in response to OVA peptide. Data presented in A, C, and D represent ﬁve independent experiments with similar results. http://www.jimmunol.org/

demonstrate that CD24 does not have a general effect on the de- function and pathogenicity of 2D2 T cells developed in the velopment of all transgenic T cells. Because the development of presence or absence of the CD24 gene. In spleens of 2D2ϩ the OTII T cells requires positive selection, our data emphasize CD24ϩ/ϩ mice, the V␣3.2ϩV␤11ϩ population of cells are that the CD24 gene does not regulate positive selection. mainly CD4ϩ, while the V␣3.2ϩV␤11ϩ cells from 2D2ϩ by guest on September 27, 2021 CD24Ϫ/Ϫ mice do not express CD4 (Fig. 7A); thus, no mature CD24 deficiency increases the efficiency of clonal deletion of 2D2 T cells (CD4ϩV␣3.2ϩV␤11ϩ) are present in the spleens of VSAg-reactive T cells in the thymus 2D2ϩCD24Ϫ/Ϫ mice (Fig. 7B). Immunostaining of the V␣3.2ϩ ϩ ϩ Ϫ/Ϫ The VSAgs react with the variable regions of the TCR ␤-chain V␤11 T cells from 2D2 CD24 mice suggests that the ma- (V␤). Because T cells expressing any given V␤ can be monitored jority of the transgenic T cells are of a naive phenotype, as is by flow cytometry, quantification for the frequencies of these T reflected by high expression of CD62L and low or no expres- cells is a useful assay available to evaluate clonal deletion in mice sion of CD25 and CD69 (Fig. 7C). TCR expression levels on ϩ Ϫ Ϫ without a transgenic TCR. BALB/c mice have integrations of 2D2 T cells from 2D2 CD24 / mice were down-regulated ϩ ϩ ϩ mouse mammary tumor provirus types 6, 8, and 9. As a result, the compared with 2D2 T cells from 2D2 CD24 / mice (Fig. majority of T cells expressing V␤3, V␤5, V␤11, and V␤12 have 7C). Thus, it is likely that TCR-positive T cells in the peripheral been deleted (34). We, therefore, generated CD24Ϫ/Ϫ BALB/c lymphoid organs of 2D2ϩCD24Ϫ/Ϫ mice were anergic. Func- mice by five generations of marker-assisted backcross to achieve tional analysis suggests that splenocytes from 2D2ϩCD24Ϫ/Ϫ nearly 100% of the BALB/c genome. The CD24ϩ/Ϫ BALB/c mice mice failed to respond to MOG-peptide stimulation, whereas were intercrossed, and the CD24ϩ/ϩ and CD24Ϫ/Ϫ littermates splenocytes from 2D2ϩCD24ϩ/ϩ mice had vigorous prolifera- were compared for the V␤3, 5, 8, 11, and 12 to determine whether tive responses to MOG peptide even at low concentrations (Fig. CD24 deficiency increased the efficacy of clonal deletion. As 8A). This difference is not attributable to the costimulatory ac- shown in Fig. 6A, VSAgs-reactive T cells among CD4 or CD8 SP tivity of CD24 on APC as purified T cells from WT and ϩ ϩ Ϫ Ϫ thymocytes from CD24 / or CD24 / BALB/c mice were quan- CD24Ϫ/Ϫ 2D2 transgenic mice exhibit the same difference titated. We observed significantly reduced frequencies of V␤3-, 5-, when WT APC were used (Fig. 8B). 11-, and 12-positive CD4 SP thymocytes (Fig. 6B); V␤3- and 12- 2D2 T cells are encephalitogenic, as the injection of pertussis positive CD8 SP thymocytes were also significantly reduced (Fig. toxin induces EAE in 2D2 TCR transgenic mice (24). We, there- 6B). These data demonstrate that CD24 deficiency increased the fore, used this model to determine the immunological consequence efficiency of clonal deletion of VSAg-reactive T cells. Therefore, of CD24-mediated escape of autoreactive T cells. As shown in Fig. the function of CD24 is not limited to MOG-specific autoreactive 9A, two doses of pertussis toxin at days 0 and 2 induced severe T cells. EAE symptoms in 7 of 10 2D2ϩCD24ϩ/ϩ mice, with disease onset at about day 10, peaked at about day 18, and persisted over the CD24 deficiency abrogated pertussis toxin-induced observation period. In contrast, only 1 of 10 2D2ϩCD24Ϫ/Ϫ mice experimental autoimmune encephalomyelitis that received the same dose of pertussis toxin developed EAE. To understand the immunological consequence of CD24-medi- Histologic analysis further revealed that inflammatory cells infil- ated clonal deletion for autoimmune diseases, we compared the trated the cerebellum and spinal cords of 2D2ϩCD24ϩ/ϩ mice and The Journal of Immunology 325

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FIGURE 6. Frequencies of VSAg-reactive T cells are reduced in CD24Ϫ/Ϫ BALB/c mice. CD24ϩ/Ϫ BALB/c mice were bred with CD24ϩ/Ϫ BALB/c mice, and CD24ϩ/ϩ BALB/c and CD24Ϫ/Ϫ BALB/c mice were generated. Thymocytes from mice (4–5 wk old) were stained for CD4, CD8, and a single V␤. Frequencies of each type of V␤ϩ cells (V␤3, 5, 8, 11, 12) were quantitated. A total of 200,000 cells were harvested for each sample. A, A representative ϩ Ϫ Ϫ ϩ ﬂow cytometry proﬁle of CD4 CD8 and CD4 CD8 thymocytes from one pair of mice is shown. B, Summary of frequencies of VSAg-reactive http://www.jimmunol.org/ CD4ϩCD8Ϫ and CD4ϪCD8ϩ thymocytes. Each triangle represents the number from a single mouse. A closed triangle represents the number from a CD24ϩ/ϩ BALB/c mouse; an open triangle represents the number from a CD24Ϫ/Ϫ BALB/c mouse. Student’s t test was used for the comparison.

caused demyelination, while the cerebellum and spinal cords of nerves of 2D2ϩCD24Ϫ/Ϫ mice (Fig. 9D). Therefore, CD24-medi- 2D2ϩCD24Ϫ/Ϫ mice were largely devoid of inﬂammation and de- ated escape of clonal deletion preserved autopathogenic T cells myelination (Fig. 9, B and C). We also detected severe inﬂamma- that can potentially cause autoimmune diseases under the condi- ϩ ϩ/ϩ

tion in the optic nerves of 2D2 CD24 mice but not in optic tion of inﬂammatory insults. by guest on September 27, 2021

FIGURE 7. Phenotype analysis of 2D2 T cells in the peripheral lymphoid organs of mice with or without CD24. A, Phenotypes of 2D2 T cells in the peripheral lymphoid organs. Splenocytes were stained for different cell surface markers and flow cytometry was used to analyze the stained splenocytes. Data represent at least five experiments with similar results. The V␣3.2ϩV␤11ϩ cells in the peripheral lymphoid organs of 2D2ϩCD24Ϫ/Ϫ mice are mainly CD4ϪCD8Ϫ. B, The peripheral lymphoid organs (spleen) of 2D2ϩCD24Ϫ/Ϫ mice contain no mature 2D2 (CD4ϩV␣3.2ϩV␤11ϩ) T cells. Significant, but reduced, numbers of V␣3.2ϩV␤11ϩ cells were detected in the spleens of 2D2ϩCD24Ϫ/Ϫ mice. Splenocytes were stained for different cell surface markers and numbers of each subset of cells were calculated. Each triangle denotes the number from one single mouse. Thick lines represent median numbers. Student’s t test was used for statistical analysis. C,V␣3.2ϩV␤11ϩ cells in the peripheral lymphoid organs of 2D2ϩCD24Ϫ/Ϫ mice are of a naive phenotype with down-regulated TCR expression. Cell suspensions of spleen were stained for V␣3.2, V␤11, and one of the other cell surface markers. Data shown were gated on V␣3.2ϩV␤11ϩ cells. Three independent experiments were done with similar results. 326 CD24 AND THYMIC GENERATION OF AUTOREACTIVE T CELLS

A Discussion Clonal deletion of autoreactive T cells in the thymus has been established as a central mechanism of immune tolerance (2, 33, 35). Studies have demonstrated that clonal deletion requires expression of self Ags in the thymus, particularly by the peripheral Ag-expressing cells as well as costimulatory molecules, such as B7-1 and B7-2 (4, 36). Nevertheless, clonal deletion is incomplete as significant numbers of autoreactive T cells can be easily detected and expanded even in normal individuals (6, 7). When immune regulation goes awry, as in cases of the germline mutation of B FoxP3 in Scurfy mice (37) and IPEX patients (38, 39), lethal autoimmune attacks ensue. An interesting issue is whether the escape of autoreactive T cells is merely due to a failure in expressing autoantigens or costimulatory molecules, or due to active mechanisms that allow escape of some autoreactive T cells. In this study, we demonstrate that CD24 actively inhibits clonal deletion, as CD24-deficient mice exhibit much more efficient clonal deletion

compared with WT mice. This is a general phenomenon as the Downloaded from enhancement can be observed with transgenic T cells specific for autoantigen MOG and polyclonal T cells specific for VSAgs. Im- FIGURE 8. V␣3.2ϩV␤11ϩ cells in the peripheral lymphoid organs of portantly, CD24 deficiency enhanced clonal deletion without af- 2D2ϩCD24Ϫ/Ϫ mice failed to proliferate in response to their cognate pep- fecting self-Ag expression (Fig. 4). These data have established tide Ag. Splenocytes from mice with different genotypes were cultured in that an active suppressive mechanism exists to enable escape of 3

the presence of titrated MOG peptide. [ H]Tritium assay was used to quan- autoreactive T cells from negative selection. The biological ben- http://www.jimmunol.org/ tify DNA synthesis in response to MOG peptide stimulation. A, Spleno- efits of the CD24-mediated prevention of thymic negative selec- ϩ ϩ/ϩ cytes from 2D2 CD24 mice proliferated vigorously to MOG peptide tion of autoreactive clones are unclear; considering the majority of ϩ Ϫ/Ϫ stimulation, while splenocytes from 2D2 CD24 mice failed to respond tumor Ags are self Ags (40, 41), it is conceivable to speculate that to MOG peptide. B, Purified 2D2 T cells from 2D2ϩCD24Ϫ/Ϫ mice failed ϩ Ϫ Ϫ ϩ CD24-mediated protection of self-reactive T cells may be required to respond to MOG Ag. 2D2 T cells from 2D2 CD24 / and 2D2 CD24ϩ/ϩ mice were purified from spleens by negative selection using for the immune system to preserve antitumor immunity. Dynabeads. Equal numbers of purified 2D2 T cells were then used as Another interesting issue is the cellular mechanisms by which responders, while irradiated splenocytes were used as APCs. Because the CD24 suppresses clonal deletion. Recent studies (30, 31) have re- two lines representing purified T cells only are overlapping, only three lines vealed that both CD4ϩCD8ϩ double positive (DP) as well as semi- can be seen in this figure. Data presented in A and B represent three inde- mature SP thymocytes are targets of negative selection. Medullary by guest on September 27, 2021 pendent experiments with similar results. AB

FIGURE 9. CD24 deficiency prevents EAE in 2D2 transgenic mice. We induced EAE in mice with different genotypes by pertussis toxin injection (200 ng i.v. on days 0 and 2). A, 2D2ϩCD24ϩ/ϩ mice developed severe EAE while 2D2ϩCD24Ϫ/Ϫ mice show no signs of EAE, with the sole exception of one 2D2ϩCD24Ϫ/Ϫ mouse which became severely dis- abled later. B, H&E and fast blue stainings of cerebellum and spinal cords revealed severe inflammation and demyelination in 2D2ϩCD24ϩ/ϩ C D but not in 2D2ϩCD24Ϫ/Ϫ mice. Blown up sections are shown to the left. C, Summary of histology scores in each group. The median score for 2D2ϩCD24ϩ/ϩ mice was 3.0 and only one 2D2ϩCD24Ϫ/Ϫ mouse reached a score of 3.0. D, H&E staining of the optic nerves revealed 2D2ϩ CD24ϩ/ϩ but not 2D2ϩCD24Ϫ/Ϫ mice have inflammation in response to pertussis toxin (bottom row shows the blown up images). The Journal of Immunology 327

TECs can synthesize peripheral Ags and, thereby, are predicted to 8. Ota, K., M. Matsui, E. L. Milford, G. A. Mackin, H. L. Weiner, and D. A. Hafler. play a central role in negative selection (4, 14). However, there is 1990. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 346: 183–187. also argument that other TECs can also mediate negative selection 9. Godfrey, V. L., J. E. Wilkinson, and L. B. Russell. 1991. X-linked lymphore- (31). In addition, considerable evidence exists that peripheral Ags ticular disease in the scurfy (sf) mutant mouse. Am. J. Pathol. 138: 1379–1387. can come from the blood, captured and presented by DC to im- 10. Zahorsky-Reeves, J. L., and J. E. Wilkinson. 2001. The murine mutation scurfy (sf) results in an antigen-dependent lymphoproliferative disease with altered T mature thymocytes (42, 43). Although our results have demon- cell sensitivity. Eur. J. Immunol. 31: 196–204. strated the importance of radio-resistant TEC in rescuing DP thy- 11. Kuchroo, V. K., A. C. Anderson, H. Waldner, M. Munder, E. Bettelli, and L. B. Nicholson. 2002. T cell response in experimental autoimmune encephalo- mocytes, CD4 SP thymocytes are not rescued in bone marrow myelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and chimeric mice with CD24 expression only on TEC (Fig. 3). Thus, regulating the autopathogenic T cell repertoire. Annu. Rev. Immunol. 20: it is conceivable to speculate that CD24 on DC can rescue imma- 101–123. 12. Klein, L., M. Klugmann, K. A. Nave, V. K. Tuohy, and B. Kyewski. 2000. ture SP thymocytes. Additional experiments are required to prove Shaping of the autoreactive T-cell repertoire by a splice variant of self protein this point. Taken together, we propose that CD24 on TEC and expressed in thymic epithelial cells. Nat. Med. 6: 56–61. perhaps on DC transmit an inhibitory signal in immature thymo- 13. 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gression of multiple sclerosis. Proc. Natl. Acad. Sci. USA 100: 15041–15046. by guest on September 27, 2021 humans (21, 22). Our previous studies have demonstrated several 22. Wang, L., S. Lin, K. W. Rammohan, Z. Liu, J. Q. Liu, R. H. Liu, N. Guinther, mechanisms by which CD24 facilitates autoimmune diseases, in- J. Lima, Q. Zhou, T. Wang, et al. 2007. A dinucleotide deletion in CD24 confers cluding expansion of autoreactive T cells in the target organ (25) protection against autoimmune diseases. PLoS Genet. 3: e49. 23. Otaegui, D., A. Saenz, P. Camano, L. Blazquez, M. Goicoechea, and regulation of homeostatic proliferation (53, 54). Our study J. Ruiz-Martinez, J. Olaskoaga, J. A. Emparanza, and A. Lopez de Munain. 2006. presented here suggests a novel mechanism by which CD24 me- CD24 V/V is an allele associated with the risk of developing multiple sclerosis diates pathogenesis of autoimmune diseases, namely the escape of in the Spanish population. Mult. Scler. 12: 511–514. 24. Bettelli, E., M. Pagany, H. L. Weiner, C. Linington, R. A. Sobel, and autoreactive T cells from clonal deletion. V. K. Kuchroo. 2003. Myelin oligodendrocyte glycoprotein-specific T cell re- ceptor transgenic mice develop spontaneous autoimmune optic neuritis. J. Exp. Acknowledgments Med. 197: 1073–1081. 25. Bai, X. F., O. Li, Q. Zhou, H. Zhang, P. S. Joshi, X. Zheng, Y. Liu, Y. Wang, and We thank Dr. V. K. Kuchroo (Harvard Medical School, Boston, MA) for P. Zheng. 2004. CD24 controls expansion and persistence of autoreactive T cells providing us 2D2 TCR-transgenic mice. in the central nervous system during experimental autoimmune encephalomyelitis. J. Exp. Med. 200: 447–458. Disclosures 26. Zhou, Q., Y. Wu, P. J. Nielsen, and Y. Liu. 1997. Homotypic interaction of the heat-stable antigen is not responsible for its co-stimulatory activity for T cell The authors have no financial conflict of interest. clonal expansion. Eur. J. Immunol. 27: 2524–2528. 27. Zhou, Q., Y. Guo, and Y. Liu. 1998. Regulation of the stability of heat-stable References antigen mRNA by interplay between two novel cis elements in the 3Ј untranslated region. Mol. Cell. Biol. 18: 815–826. 1. Sebzda, E., S. Mariathasan, T. Ohteki, R. Jones, M. F. Bachmann, and P. S. Ohashi. 1999. Selection of the T cell repertoire. Annu. Rev. Immunol. 17: 28. Delarasse, C., P. Daubas, L. T. Mars, C. Vizler, T. Litzenburger, A. Iglesias, 829–874. J. Bauer, B. Della Gaspera, A. Schubart, L. Decker, et al. 2003. Myelin/oligo- 2. Ohashi, P. S. 2003. Negative selection and autoimmunity. Curr. Opin. Immunol. dendrocyte glycoprotein-deficient (MOG-deficient) mice reveal lack of immune 15: 668–676. tolerance to MOG in wild-type mice. J. Clin. Invest. 112: 544–553. 3. Anderton, S. M., and D. C. Wraith. 2002. Selection and fine-tuning of the auto- 29. Shimizu, C., H. Kawamoto, M. Yamashita, M. Kimura, E. Kondou, Y. Kaneko, immune T-cell repertoire. Nat. Rev. Immunol. 2: 487–498. S. Okada, T. Tokuhisa, M. Yokoyama, M. Taniguchi, et al. 2001. Progression of 4. Anderson, M. S., E. S. Venanzi, L. Klein, Z. Chen, S. P. Berzins, S. J. Turley, T cell lineage restriction in the earliest subpopulation of murine adult thymus H. von Boehmer, R. Bronson, A. Dierich, C. Benoist, and D. Mathis. 2002. visualized by the expression of lck proximal promoter activity. Int. Immunol. 13: Projection of an immunological self shadow within the thymus by the aire pro- 105–117. tein. Science 298: 1395–1401. 30. Sprent, J., and H. Kishimoto. 2002. The thymus and negative selection. Immunol. 5. Chen, Z., C. Benoist, and D. Mathis. 2005. How defects in central tolerance Rev. 185: 126–135. impinge on a deficiency in regulatory T cells. Proc. Natl. Acad. Sci. USA 102: 31. von Boehmer, H., and P. Kisielow. 2006. Negative selection of the T-cell rep- 14735–14740. ertoire: where and when does it occur? Immunol. Rev. 209: 284–289. 6. Sun, J. B., T. Olsson, W. Z. Wang, B. G. Xiao, V. Kostulas, S. Fredrikson, 32. Hanahan, D. 1998. Peripheral-antigen-expressing cells in thymic medulla: factors H. P. Ekre, and H. Link. 1991. Autoreactive T and B cells responding to myelin in self-tolerance and autoimmunity. Curr. Opin. Immunol. 10: 656–662. proteolipid protein in multiple sclerosis and controls. Eur. J. Immunol. 21: 33. Kyewski, B., and L. Klein. 2006. A central role for central tolerance. Annu. Rev. 1461–1468. Immunol. 24: 571–606. 7. Liblau, R., E. Tournier-Lasserve, J. Maciazek, G. Dumas, O. Siffert, G. Hashim, 34. Abe, R., M. Foo-Phillips, and R. J. Hodes. 1991. Genetic analysis of the Mls and M. A. Bach. 1991. T cell response to myelin basic protein epitopes in mul- system: formal Mls typing of the commonly used inbred strains. Immunogenetics tiple sclerosis patients and healthy subjects. Eur. J. Immunol. 21: 1391–1395. 33: 62–73. 328 CD24 AND THYMIC GENERATION OF AUTOREACTIVE T CELLS

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