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 diseases in mice and humans.

It is well established that thymic clonal deletion of autoantigen reactive T cells plays a central role in preventing the development of autoimmune diseases, as genetic or experimental blockade of this process results in the development of autoimmune diseases (1, 2, 3, 4, 5). Nevertheless, significant numbers of autoreactive T cells can be easily detected (6, 7) and expanded (8) even in normal individuals. Mice with the Scurfy mutation having abrogated function of regulatory T cells still succumb to fatal autoimmune diseases despite normal negative selection (5, 9). Because the development of autoimmune diseases can be prevented after breeding to TCR transgenic mice (10), some autoreactive T cells must have escaped clonal deletion in Scurfy mice. Although lack of expression of self Ags has been largely attributed as a key factor (11, 12, 13), we have reported that even T cells specific for P1A, a self Ag expressed in thymic medullar epithelial cells (14), can escape clonal deletion (15). How autoreactive T cells escape clonal deletion may hold a key to understanding the pathogenesis of autoimmune diseases.

CD24 is a glycosyl-phosphatidylinositol-anchored cell surface glycoprotein with extensive carbohydrate structures attached to a small protein core (16). CD24 is expressed on various cells including immature thymocytes and B lymphocytes (17, 18). Although immune responses were apparently normal in mice with targeted mutation of CD24 (17, 19), we have reported (20) that targeted mutation of CD24 abrogates the development of experimental autoimmune encephalomyelitis (EAE)3 induced with myelin oligodendrocyte glycoprotein (MOG)-peptide immunization. Moreover, CD24 polymorphism has emerged as an important genetic factor in regulating susceptibility to autoimmune diseases, including multiple sclerosis (21, 22, 23) and systemic lupus erythematosus in humans (22). To understand how CD24 regulates susceptibility to autoimmune diseases, we generated CD24-deficient mice that express a TCR specific for MOG 35–55, a pathogenic autoantigen in the C57BL6/J mice (2D2+CD24−/− mice). Surprisingly, we observed that 2D2+CD24−/− mice have atrophic thymi with absent CD4+CD8+ and CD4+CD8 populations. Transgenic expression of CD24 on thymocytes alone did not prevent T cell deletion; bone marrow chimera experiments suggest that CD24 on radio-resistant stromal cells is necessary for preventing deletion of 2D2 T cells. Furthermore, we observed that CD24 also reduced the efficiency of clonal deletion of viral superantigen (VSAg)-specific T cells. In contrast, development of T cells specific for OVA was unaffected by CD24 deficiency. These data demonstrate a critical role for CD24 in escape of autoreactive T cells from thymic clonal deletion.

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). CD24−/− mice in the C57BL6 background have been described (20, 25). By using Charles River Max-Bax technology (Marker-Assisted Accelerated Backcrossing), we have recently produced CD24−/− BALB/c mice. OTII TCR transgenic mice were purchased from The Jackson Laboratory. Transgenic mice with CD24 expression exclusively on T cells have been described (26, 27). All mice were bred and maintained in a specific pathogen-free animal facility of The Ohio State University. The animal facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.

The following Abs were used in the experiments according to the manufacturer’s recommendations: unlabeled, FITC-, PE-, PerCp-, allophycocyanin- or biotin-labeled anti-CD4 (GK1.4), -CD8α (53-6.7), -CD11c (HL3), -CD24 (M1/69), -CD25 (7D4), -CD44 (IM7), -CD45 (30-F11), -CD62L (Mel-14), -CD69 (H1.2F3), Vα2 (B20.1), -Vα3.2 (RR3-16), -Vβ3 (ΚJ25), -Vβ5.1/5.2 (MR9-4), -Vβ8 (F23.1), -Vβ11 (RR3-15), -Vβ12 (MR11-1), and anti-rat IgG2a (RG7/1.30). These Abs were purchased from BD Pharmingen or eBioscience. For flow cytometry analysis, cells were incubated with Abs on ice for 30 min followed by extensive washing. Cells were analyzed on a FACSCalibur cytometer (BD Biosciences).

We prepared bone marrow cells by flushing donor mice femur and tibia bones with PBS. Recipient mice were lethally irradiated (1000 rads) and reconstituted with 10 × 106 bone marrow cells by i.v. injection. Engraftment took place over a 6- to 8-wk period. We used 2D2 TCR transgenic mice as our basic model and generated four types of bone marrow chimeras. Chimera 1 (2D2+CD24+/+ > CD24+/+ mice)-bone marrow cells from 2D2+CD24+/+ mice were injected into irradiated CD24+/+ mice. In chimera 1 mice, both bone marrow-derived cells and TEC were CD24-positive. Chimera 2 (2D2+CD24−/− > CD24+/+ mice)-bone marrow cells from 2D2+CD24−/− mice were injected into irradiated CD24+/+ mice. In chimera 2 mice, TECs expressed CD24, while bone marrow-derived dendritic cells (DC) and T cells were CD24 deficient. Chimera 3 (2D2+CD24+/+ > CD24−/− mice)-bone marrow cells from 2D2+CD24+/+ mice were injected into irradiated CD24−/− mice. In chimera 3 mice, TECs did not express CD24, while bone marrow-derived DC and T cells were CD24-positive.

Chimera 4 (2D2+CD24−/− > CD24−/− mice)-bone marrow cells from 2D2+CD24−/− mice were injected into irradiated CD24−/− mice. In chimera 4 mice, both bone marrow-derived cells and TECs were CD24 deficient.

Total RNA was isolated from thymi or stroma-enriched thymi using the Trizol method (Invitrogen). The first strand cDNA of each sample was synthesized using a reverse transcription kit (Invitrogen). Quantitative real time PCR was performed using an ABI Prism 7900-HT sequence system (PE Applied Biosystems) with the QuantiTect SYBR Green PCR kit (Qiagen) in accordance with the manufacturer’s instructions. The following primers were used: mMOG.F: 5′-GCAGCACAGACTGAGAGGAA-3′; mMOG.R: 5′-CAGATGATCAAGGCAACCAG-3′. Hypoxanthine-guanine phosphoribosyltransferase (HPRT).F: 5′-AGC CTA AGA TGA GCG CAA GT-3′ HPRT.R: 5′-TTA CTA GGC AGA TGG CCA CA-3′. The HPRT gene was amplified and served as endogenous control. PCR was performed under optimal conditions. A total of 1 μl of first strand cDNA product was amplified with platinum Taq polymerase (Invitrogen) and gene-specific primer pairs. Each sample was assayed in triplicate and experiments were repeated twice. The relative amounts of mRNA were calculated by plotting the Ct (cycle number), and average relative expression was determined by the comparative method (2−ΔΔCt).

Splenocytes from each strain of TCR transgenic mice (2D2, OTII) with or without CD24 deficiency were stimulated with titrated peptide Ags in 96-well U-bottom plates. In some experiments, we purified CD4 cells from spleen and lymph nodes and stimulated them with peptide Ags and irradiated (2000 rad) syngenic APCs. [3H]Thymidine was added into the culture at 48 h and harvested 12 h later. 3H incorporation was measured with a scintillation counter. The OVA peptide Ag used in the assay (OVA 323–339) was purchased from Sigma-Aldrich. MOG peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK) was purchased from Genemed Synthesis.

2D2 T cells were purified by negative selection. In brief, spleen and lymph node cells from 2D2 TCR transgenic mice were incubated with a mixture of mAbs (anti-CD8 mAb TIB210, anti-FcR mAb 2.4G2, and anti-CD11c mAb N418). After removing the unbound Abs, the cells were incubated with anti-Ig-coated magnetic beads (Dynal Biotech). A magnet was used to remove the Ab-coated cells. The remaining cells were CD4+ or CD4CD8 T cells. The purified 2D2 T cells were used for the proliferation assay.

2D2 TCR transgenic mice (8–12 wk of age) of different CD24 genotypes received 200 ng of pertussis toxin (List Biological Laboratories) in 200 μl 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 intermediate scores: 0, no clinical signs; 1, loss of tail tone; 2, wobbly gait; 3, hind limb paralysis; 4, moribund; and 5, death.

Mice were sacrificed by inhaling CO2. Spinal cords, cerebellum, and optic nerves were removed and fixed in 10% formalin/PBS. Paraffin sections were prepared and stained by the histology core facilities of Department of Pathology (Ohio State University) for H&E and luxol fast blue (myelin staining). Pathological changes of each spinal cord were evaluated and scored as follows: 0, no changes; 1, focal area involvement; 2, <5% of total myelin area involvement; 3, 5–10% of total myelin area involvement; 4, 10–20% of total myelin area involvement; 5, >20% of total myelin area involvement.

Kuchroo and colleagues (24) have produced 2D2 TCR transgenic mice, whose TCR recognizes MOG 35–55, a pathogenic epitope in the C57BL/6 mice. Because these T cells can develop normally in the mice, we tested whether the autoantigen MOG is expressed in the thymus. As shown in Fig. 1,A, significant expression of MOG mRNA was detected in the thymus by real-time PCR. Consistent with this finding, other groups also demonstrated MOG mRNA expression in the thymus (28) or TECs (14). Thus, 2D2 T cells have escaped clonal deletion despite expression of MOG in the thymus. Surprisingly, targeted mutation of CD24 caused a massive reduction of thymic cellularity in the 2D2 transgenic mice but not in CD24+/− or CD24−/− mice without the 2D2 TCR transgene (Fig. 1,B). To determine whether the thymic atrophy was related to age, we examined young mice (18–20 days after birth); we observed similar thymic atrophy in 2D2+CD24−/− mice (data not shown). Correspondingly, numbers of αβ-positive 2D2 T cells and the CD4+CD8+ and CD4+CD8 populations were dramatically reduced in the thymi of the 2D2+CD24−/− mice compared with that of the 2D2+CD24+/+ mice (Fig. 1 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 was used for the comparison. C, Impact of CD24 deficiency on the development of 2D2 TCR transgenic T cells. Thymocytes from 2D2+CD24−/− and 2D2+CD24+/+ mice were stained for Vα3.2, Vβ11, CD4, and CD8. The thymi from 2D2+CD24−/− mice had dramatically reduced Vα3.2+Vβ11+ populations and failed to generate CD4+CD8+ and CD4+CD8 T cells compared with that of 2D2+CD24+/+ mice. Data represent at least five experiments with similar results.

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 was used for the comparison. C, Impact of CD24 deficiency on the development of 2D2 TCR transgenic T cells. Thymocytes from 2D2+CD24−/− and 2D2+CD24+/+ mice were stained for Vα3.2, Vβ11, CD4, and CD8. The thymi from 2D2+CD24−/− mice had dramatically reduced Vα3.2+Vβ11+ populations and failed to generate CD4+CD8+ and CD4+CD8 T cells compared with that of 2D2+CD24+/+ mice. Data represent at least five experiments with similar results.

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Because the majority of thymocytes expressed high levels of CD24, we tested the possibility that CD24 expressed on thymocytes may be responsible for the enhanced clonal deletion. We generated 2D2+CD24−/− mice that express CD24 under the control of the proximal lck promoter. The lck promoter is known to be active in thymocytes, starting at the DN3 stage (29). As shown in Fig. 2,A, essentially all 2D2 TCR-expressing thymocytes in the CD24 transgenic mice expressed high levels of CD24. Nevertheless, thymic cellularity (Fig. 2,B) and distribution of T cell subsets (Fig. 2 C) were unaffected by CD24 expression in thymocytes. These results demonstrate that lack of CD24 on thymocytes is not solely responsible for enhanced clonal deletion.

FIGURE 2.

Restoration of CD24 on 2D2 T cells in CD24-deficient mice does not prevent thymic deletion. We have bred 2D2+CD24−/− mice with TCD24TGCD24−/− mice (mice with CD24 expression on T cells only) and produced double transgenic mice with CD24 deficiency (2D2.TCD24TGCD24−/− mice). A, Phenotypes of three different mice identified by flow cytometry. Single-cell suspensions of thymocytes were stained for CD24 and Vα3.2 markers followed by flow cytometry analysis. Data shown were gated on Vα3.2-positive thymocytes. B, Thymocyte numbers in mice with different genotypes. No significant difference was observed between 2D2+CD24−/− mice with 2D2.TCD24TGCD24−/− mice. C, Thymocytes from 2D2.TCD24TGCD24−/− mice show a phenotype similar to that seen in 2D2+CD24−/− mice.

FIGURE 2.

Restoration of CD24 on 2D2 T cells in CD24-deficient mice does not prevent thymic deletion. We have bred 2D2+CD24−/− mice with TCD24TGCD24−/− mice (mice with CD24 expression on T cells only) and produced double transgenic mice with CD24 deficiency (2D2.TCD24TGCD24−/− mice). A, Phenotypes of three different mice identified by flow cytometry. Single-cell suspensions of thymocytes were stained for CD24 and Vα3.2 markers followed by flow cytometry analysis. Data shown were gated on Vα3.2-positive thymocytes. B, Thymocyte numbers in mice with different genotypes. No significant difference was observed between 2D2+CD24−/− mice with 2D2.TCD24TGCD24−/− mice. C, Thymocytes from 2D2.TCD24TGCD24−/− mice show a phenotype similar to that seen in 2D2+CD24−/− mice.

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To determine whether CD24 expressed on bone marrow-derived or non-bone marrow-derived stromal cells is responsible, we established chimeras using bone marrow from 2D2+CD24+/+ or 2D2+CD24−/− mice to reconstitute irradiated wild-type (WT) or CD24-deficient mice. As shown in Fig. 3,A, CD24 expression on thymocytes indicates a nearly complete replacement of the hematopoietic compartment in the chimera mice. In spleens, we detected intermediate to high levels of CD24 expression on CD11c+ cells from 2D2+CD24+/+ > CD24−/− chimeras but not on CD11c+ cells from 2D2+CD24−/− > CD24+/+ chimeras (Fig. 3,B). Thus, the chimeric mice can be used to evaluate the contribution of CD24 in clonal deletion of 2D2 T cells. Mature 2D2 T cells (CD4-single positive (SP)) were generated in 2D2+CD24+/+ > CD24+/+ chimeras but not in 2D2+CD24−/− > CD24−/− chimeras (Fig. 3, C and D, left and right panels). CD24 expression on the radio-resistant stromal cells (Fig. 3, C and D, middle left panel), but not on bone marrow-derived cells (Fig. 3, C and D, middle right panel), rescued transgenic TCRαβ+ cells at the CD4+CD8+ stage; however, because no mature CD4+ 2D2 T cells were generated (Fig. 3 D, middle left), it is likely that CD24 expression on radio-resistant thymic stromal cells alone is not sufficient for rescuing 2D2 T cell deletion. Thus, these bone marrow chimera data suggest that CD24 expression on radio-resistant stromal cells inhibits deletion of transgenic T cells at the CD4+CD8+ stage.

FIGURE 3.

CD24 on radio-resistant thymic stromal cells is necessary but not sufficient for the generation of mature CD4 T cells specific for MOG peptide. CD24−/− and CD24+/+ mice were lethally irradiated (1000 Rad) and reconstituted with 2D2+CD24−/− or 2D2+CD24+/+ donor bone marrow cells. A, CD24 expression on the thymocytes from different bone marrow chimeric mice. B, CD24 expression on splenic CD11c+ cells from different bone marrow chimeras. Splenocytes were digested with collagenase IV, and the resulting mononuclear cells were stained for CD11c and CD24 followed by flow cytometry analysis. Data shown were gated on CD11c-positive cells. C, Generation of 2D2 T cells in the thymi of bone marrow chimeras. Thymocytes were stained for Vα3.2, Vβ11, CD4, and CD8 followed by flow cytometry analysis. D, 2D2 T cell subsets in bone marrow chimeras. Data shown were gated on Vα3.2+Vβ11+ thymocytes. The result shown represents three independent experiments with similar results.

FIGURE 3.

CD24 on radio-resistant thymic stromal cells is necessary but not sufficient for the generation of mature CD4 T cells specific for MOG peptide. CD24−/− and CD24+/+ mice were lethally irradiated (1000 Rad) and reconstituted with 2D2+CD24−/− or 2D2+CD24+/+ donor bone marrow cells. A, CD24 expression on the thymocytes from different bone marrow chimeric mice. B, CD24 expression on splenic CD11c+ cells from different bone marrow chimeras. Splenocytes were digested with collagenase IV, and the resulting mononuclear cells were stained for CD11c and CD24 followed by flow cytometry analysis. Data shown were gated on CD11c-positive cells. C, Generation of 2D2 T cells in the thymi of bone marrow chimeras. Thymocytes were stained for Vα3.2, Vβ11, CD4, and CD8 followed by flow cytometry analysis. D, 2D2 T cell subsets in bone marrow chimeras. Data shown were gated on Vα3.2+Vβ11+ thymocytes. The result shown represents three independent experiments with similar results.

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Because TECs, especially medullary epithelial cells, are the major nonhematopoietic cells that are involved in negative selection (14, 30, 31, 32), we analyzed expression of CD24 on TECs. As shown in Fig. 4,A, about 50% of CD45-negative thymic stromal cells express CD24; almost 100% of medullar epithelial cells (B7+) (33) express CD24. These data are consistent with a role of CD24 on radio-resistant TECs. To determine whether CD24 deficiency affects expression of MOG Ag in the thymus, we compared MOG mRNA expression in the thymus and enriched-thymic stromal cells. As shown in Fig. 4 B, similar levels of MOG-transcripts were detected in WT and CD24-deficient thymi, regardless of whether RNA from whole thymus extract or from enriched-stromal cells were compared. As such, the function of CD24 is unlikely through regulation of peripheral Ag expression in the thymus.

FIGURE 4.

Expression of CD24 and MOG in thymic stromal cells. A, Thymi from CD24−/− and CD24+/+ C57BL6 mice were digested with collagenase, 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 CD45 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.

FIGURE 4.

Expression of CD24 and MOG in thymic stromal cells. A, Thymi from CD24−/− and CD24+/+ C57BL6 mice were digested with collagenase, 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 CD45 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.

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As a comparison to the development of autoreactive transgenic T cells, we also studied the development of transgenic T cells specific for OVA in WT and CD24-deficient hosts. As shown in Fig. 5, the TCR distribution (Fig. 5,A), thymic cellularity (Fig. 5,B), and subset distribution (Fig. 5,C) are similar in OTII+CD24+/+ and OTII+CD24−/− transgenic mice. Moreover, the OTII T cells in the periphery are fully functional (Fig. 5 D). These results demonstrate that CD24 does not have a general effect on the development of all transgenic T cells. Because the development of the OTII T cells requires positive selection, our data emphasize that the CD24 gene does not regulate positive selection.

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 CD24−/− mice for two generations. The resulting OTII+CD24−/− 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 numbers in mice of different genotypes. C, Flow cytometry analysis of the thymi of OTII TCR transgenic mice with or without CD24. OTII+CD24+/+ and OTII+CD24−/− mice revealed similar generation of OTII T cells. D, Splenocytes from OTII+CD24+/+ and OTII+CD24−/− mice show similar proliferation in response to OVA peptide. Data presented in A, C, and D represent five independent experiments with similar results.

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 CD24−/− mice for two generations. The resulting OTII+CD24−/− 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 numbers in mice of different genotypes. C, Flow cytometry analysis of the thymi of OTII TCR transgenic mice with or without CD24. OTII+CD24+/+ and OTII+CD24−/− mice revealed similar generation of OTII T cells. D, Splenocytes from OTII+CD24+/+ and OTII+CD24−/− mice show similar proliferation in response to OVA peptide. Data presented in A, C, and D represent five independent experiments with similar results.

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The VSAgs react with the variable regions of the TCR β-chain (Vβ). Because T cells expressing any given Vβ can be monitored by flow cytometry, quantification for the frequencies of these T cells is a useful assay available to evaluate clonal deletion in mice without a transgenic TCR. BALB/c mice have integrations of mouse mammary tumor provirus types 6, 8, and 9. As a result, the majority of T cells expressing Vβ3, Vβ5, Vβ11, and Vβ12 have been deleted (34). We, therefore, generated CD24−/− BALB/c mice by five generations of marker-assisted backcross to achieve nearly 100% of the BALB/c genome. The CD24+/− BALB/c mice were intercrossed, and the CD24+/+ and CD24−/− littermates were compared for the Vβ3, 5, 8, 11, and 12 to determine whether CD24 deficiency increased the efficacy of clonal deletion. As shown in Fig. 6,A, VSAgs-reactive T cells among CD4 or CD8 SP thymocytes from CD24+/+ or CD24−/− BALB/c mice were quantitated. We observed significantly reduced frequencies of Vβ3-, 5-, 11-, and 12-positive CD4 SP thymocytes (Fig. 6,B); Vβ3- and 12-positive CD8 SP thymocytes were also significantly reduced (Fig. 6 B). These data demonstrate that CD24 deficiency increased the efficiency of clonal deletion of VSAg-reactive T cells. Therefore, the function of CD24 is not limited to MOG-specific autoreactive T cells.

FIGURE 6.

Frequencies of VSAg-reactive T cells are reduced in CD24−/− BALB/c mice. CD24+/− BALB/c mice were bred with CD24+/− BALB/c mice, and CD24+/+ BALB/c and CD24−/− BALB/c mice were generated. Thymocytes from mice (4–5 wk old) were stained for CD4, CD8, and a single Vβ. Frequencies of each type of Vβ+ cells (Vβ3, 5, 8, 11, 12) were quantitated. A total of 200,000 cells were harvested for each sample. A, A representative flow cytometry profile of CD4+CD8 and CD4CD8+ thymocytes from one pair of mice is shown. B, Summary of frequencies of VSAg-reactive CD4+CD8 and CD4CD8+ thymocytes. Each triangle represents the number from a single mouse. A closed triangle represents the number from a CD24+/+ BALB/c mouse; an open triangle represents the number from a CD24−/− BALB/c mouse. Student’s t test was used for the comparison.

FIGURE 6.

Frequencies of VSAg-reactive T cells are reduced in CD24−/− BALB/c mice. CD24+/− BALB/c mice were bred with CD24+/− BALB/c mice, and CD24+/+ BALB/c and CD24−/− BALB/c mice were generated. Thymocytes from mice (4–5 wk old) were stained for CD4, CD8, and a single Vβ. Frequencies of each type of Vβ+ cells (Vβ3, 5, 8, 11, 12) were quantitated. A total of 200,000 cells were harvested for each sample. A, A representative flow cytometry profile of CD4+CD8 and CD4CD8+ thymocytes from one pair of mice is shown. B, Summary of frequencies of VSAg-reactive CD4+CD8 and CD4CD8+ thymocytes. Each triangle represents the number from a single mouse. A closed triangle represents the number from a CD24+/+ BALB/c mouse; an open triangle represents the number from a CD24−/− BALB/c mouse. Student’s t test was used for the comparison.

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To understand the immunological consequence of CD24-mediated clonal deletion for autoimmune diseases, we compared the function and pathogenicity of 2D2 T cells developed in the presence or absence of the CD24 gene. In spleens of 2D2+CD24+/+ mice, the Vα3.2+Vβ11+ population of cells are mainly CD4+, while the Vα3.2+Vβ11+ cells from 2D2+CD24−/− mice do not express CD4 (Fig. 7,A); thus, no mature 2D2 T cells (CD4+Vα3.2+Vβ11+) are present in the spleens of 2D2+CD24−/− mice (Fig. 7,B). Immunostaining of the Vα3.2+Vβ11+ T cells from 2D2+CD24−/− mice suggests that the majority of the transgenic T cells are of a naive phenotype, as is reflected by high expression of CD62L and low or no expression of CD25 and CD69 (Fig. 7,C). TCR expression levels on 2D2 T cells from 2D2+CD24−/− mice were down-regulated compared with 2D2 T cells from 2D2+CD24+/+ mice (Fig. 7,C). Thus, it is likely that TCR-positive T cells in the peripheral lymphoid organs of 2D2+CD24−/− mice were anergic. Functional analysis suggests that splenocytes from 2D2+CD24−/− mice failed to respond to MOG-peptide stimulation, whereas splenocytes from 2D2+CD24+/+ mice had vigorous proliferative responses to MOG peptide even at low concentrations (Fig. 8,A). This difference is not attributable to the costimulatory activity of CD24 on APC as purified T cells from WT and CD24−/− 2D2 transgenic mice exhibit the same difference when WT APC were used (Fig. 8 B).

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 Vα3.2+Vβ11+ cells in the peripheral lymphoid organs of 2D2+CD24−/− mice are mainly CD4CD8. B, The peripheral lymphoid organs (spleen) of 2D2+CD24−/− mice contain no mature 2D2 (CD4+Vα3.2+Vβ11+) T cells. Significant, but reduced, numbers of Vα3.2+Vβ11+ cells were detected in the spleens of 2D2+CD24−/− 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, Vα3.2+Vβ11+ cells in the peripheral lymphoid organs of 2D2+CD24−/− mice are of a naive phenotype with down-regulated TCR expression. Cell suspensions of spleen were stained for Vα3.2, Vβ11, and one of the other cell surface markers. Data shown were gated on Vα3.2+Vβ11+ cells. Three independent experiments were done with similar results.

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 Vα3.2+Vβ11+ cells in the peripheral lymphoid organs of 2D2+CD24−/− mice are mainly CD4CD8. B, The peripheral lymphoid organs (spleen) of 2D2+CD24−/− mice contain no mature 2D2 (CD4+Vα3.2+Vβ11+) T cells. Significant, but reduced, numbers of Vα3.2+Vβ11+ cells were detected in the spleens of 2D2+CD24−/− 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, Vα3.2+Vβ11+ cells in the peripheral lymphoid organs of 2D2+CD24−/− mice are of a naive phenotype with down-regulated TCR expression. Cell suspensions of spleen were stained for Vα3.2, Vβ11, and one of the other cell surface markers. Data shown were gated on Vα3.2+Vβ11+ cells. Three independent experiments were done with similar results.

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FIGURE 8.

Vα3.2+Vβ11+ cells in the peripheral lymphoid organs of 2D2+CD24−/− mice failed to proliferate in response to their cognate peptide Ag. Splenocytes from mice with different genotypes were cultured in the presence of titrated MOG peptide. [3H]Tritium assay was used to quantify DNA synthesis in response to MOG peptide stimulation. A, Splenocytes from 2D2+CD24+/+ mice proliferated vigorously to MOG peptide stimulation, while splenocytes from 2D2+CD24−/− mice failed to respond to MOG peptide. B, Purified 2D2 T cells from 2D2+CD24−/− mice failed to respond to MOG Ag. 2D2 T cells from 2D2+CD24−/− and 2D2+CD24+/+ mice were purified from spleens by negative selection using Dynabeads. Equal numbers of purified 2D2 T cells were then used as responders, while irradiated splenocytes were used as APCs. Because the two lines representing purified T cells only are overlapping, only three lines can be seen in this figure. Data presented in A and B represent three independent experiments with similar results.

FIGURE 8.

Vα3.2+Vβ11+ cells in the peripheral lymphoid organs of 2D2+CD24−/− mice failed to proliferate in response to their cognate peptide Ag. Splenocytes from mice with different genotypes were cultured in the presence of titrated MOG peptide. [3H]Tritium assay was used to quantify DNA synthesis in response to MOG peptide stimulation. A, Splenocytes from 2D2+CD24+/+ mice proliferated vigorously to MOG peptide stimulation, while splenocytes from 2D2+CD24−/− mice failed to respond to MOG peptide. B, Purified 2D2 T cells from 2D2+CD24−/− mice failed to respond to MOG Ag. 2D2 T cells from 2D2+CD24−/− and 2D2+CD24+/+ mice were purified from spleens by negative selection using Dynabeads. Equal numbers of purified 2D2 T cells were then used as responders, while irradiated splenocytes were used as APCs. Because the two lines representing purified T cells only are overlapping, only three lines can be seen in this figure. Data presented in A and B represent three independent experiments with similar results.

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2D2 T cells are encephalitogenic, as the injection of pertussis toxin induces EAE in 2D2 TCR transgenic mice (24). We, therefore, used this model to determine the immunological consequence of CD24-mediated escape of autoreactive T cells. As shown in Fig. 9,A, two doses of pertussis toxin at days 0 and 2 induced severe EAE symptoms in 7 of 10 2D2+CD24+/+ mice, with disease onset at about day 10, peaked at about day 18, and persisted over the observation period. In contrast, only 1 of 10 2D2+CD24−/− mice that received the same dose of pertussis toxin developed EAE. Histologic analysis further revealed that inflammatory cells infiltrated the cerebellum and spinal cords of 2D2+CD24+/+ mice and caused demyelination, while the cerebellum and spinal cords of 2D2+CD24−/− mice were largely devoid of inflammation and demyelination (Fig. 9, B and C). We also detected severe inflammation in the optic nerves of 2D2+CD24+/+ mice but not in optic nerves of 2D2+CD24−/− mice (Fig. 9 D). Therefore, CD24-mediated escape of clonal deletion preserved autopathogenic T cells that can potentially cause autoimmune diseases under the condition of inflammatory insults.

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, 2D2+CD24+/+ mice developed severe EAE while 2D2+CD24−/− mice show no signs of EAE, with the sole exception of one 2D2+CD24−/− mouse which became severely disabled later. B, H&E and fast blue stainings of cerebellum and spinal cords revealed severe inflammation and demyelination in 2D2+CD24+/+ but not in 2D2+CD24−/− mice. Blown up sections are shown to the left. C, Summary of histology scores in each group. The median score for 2D2+CD24+/+ mice was 3.0 and only one 2D2+CD24−/− mouse reached a score of 3.0. D, H&E staining of the optic nerves revealed 2D2+CD24+/+ but not 2D2+CD24−/− mice have inflammation in response to pertussis toxin (bottom row shows the blown up images).

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, 2D2+CD24+/+ mice developed severe EAE while 2D2+CD24−/− mice show no signs of EAE, with the sole exception of one 2D2+CD24−/− mouse which became severely disabled later. B, H&E and fast blue stainings of cerebellum and spinal cords revealed severe inflammation and demyelination in 2D2+CD24+/+ but not in 2D2+CD24−/− mice. Blown up sections are shown to the left. C, Summary of histology scores in each group. The median score for 2D2+CD24+/+ mice was 3.0 and only one 2D2+CD24−/− mouse reached a score of 3.0. D, H&E staining of the optic nerves revealed 2D2+CD24+/+ but not 2D2+CD24−/− mice have inflammation in response to pertussis toxin (bottom row shows the blown up images).

Close modal

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 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 enhancement can be observed with transgenic T cells specific for autoantigen MOG and polyclonal T cells specific for VSAgs. Importantly, CD24 deficiency enhanced clonal deletion without affecting self-Ag expression (Fig. 4). These data have established that an active suppressive mechanism exists to enable escape of autoreactive T cells from negative selection. The biological benefits of the CD24-mediated prevention of thymic negative selection of autoreactive clones are unclear; considering the majority of tumor Ags are self Ags (40, 41), it is conceivable to speculate that CD24-mediated protection of self-reactive T cells may be required for the immune system to preserve antitumor immunity.

Another interesting issue is the cellular mechanisms by which CD24 suppresses clonal deletion. Recent studies (30, 31) have revealed that both CD4+CD8+ double positive (DP) as well as semimature SP thymocytes are targets of negative selection. Medullary TECs can synthesize peripheral Ags and, thereby, are predicted to play a central role in negative selection (4, 14). However, there is also argument that other TECs can also mediate negative selection (31). In addition, considerable evidence exists that peripheral Ags can come from the blood, captured and presented by DC to immature thymocytes (42, 43). Although our results have demonstrated the importance of radio-resistant TEC in rescuing DP thymocytes, CD4 SP thymocytes are not rescued in bone marrow chimeric mice with CD24 expression only on TEC (Fig. 3). Thus, it is conceivable to speculate that CD24 on DC can rescue immature SP thymocytes. Additional experiments are required to prove this point. Taken together, we propose that CD24 on TEC and perhaps on DC transmit an inhibitory signal in immature thymocytes during negative selection, which inhibits deletion of autoreactive T cells.

The molecular mechanisms of how CD24 inhibits negative selection remains to be determined. Increased understanding of the signaling pathways that regulate negative selection has recently been achieved (44). c-Jun NH2-terminal kinase (JNK) is activated in DP thymocytes in vivo in response to signals that initiate negative selection (45). Studies have revealed that the JNK pathway is required for the deletion of DP thymocytes by apoptosis in response to TCR-derived signals (45, 46, 47). JNK is a signaling molecule downstream of kinase MINK and upstream of proapoptotic molecule Bim (44, 48). All three molecules have been shown to be critically involved in promoting negative selection (45, 49, 50), thus, it is interesting to determine whether CD24 directly affects activation/induction of the JNK/Bim signaling pathway during negative selection.

CD24 is a critical checkpoint for the pathogenesis of autoimmune diseases (51). Mice with targeted mutation of CD24 are resistant to EAE (20, 52). Moreover, CD24 polymorphisms affect the risk of both organ-specific and systemic autoimmune disease in humans (21, 22). Our previous studies have demonstrated several mechanisms by which CD24 facilitates autoimmune diseases, including expansion of autoreactive T cells in the target organ (25) and regulation of homeostatic proliferation (53, 54). Our study presented here suggests a novel mechanism by which CD24 mediates pathogenesis of autoimmune diseases, namely the escape of autoreactive T cells from clonal deletion.

We thank Dr. V. K. Kuchroo (Harvard Medical School, Boston, MA) for providing us 2D2 TCR-transgenic mice.

The authors have no financial conflict of interest.

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 (RG 3638 to X.-F.B.) and Ohio Department of Development.

3

Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; WT, wild type; TEC, thymic epithelial cells; DP, double positive; SP, single positive; DC, dendritic cell; VSAg, viral superantigen.

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