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J. Biol. Chem., Vol. 281, Issue 50, 38617-38624, December 15, 2006

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NF-B2 Is Required for the Control of Autoimmunity by Regulating the Development of Medullary Thymic Epithelial Cells^*

Baochun Zhang¹, Zhe Wang¹, Jane Ding, Pärt Peterson, William T. Gunning, and Han-Fei Ding²

From the Department of Biochemistry and Cancer Biology, Medical University of Ohio, Toledo, Ohio 43614 and Molecular Pathology, IGMP, Biomedicum, Ravila 19, University of Tartu, 50414 Tartu, Estonia

Received for publication, July 14, 2006 , and in revised form, August 25, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Medullary thymic epithelial cells function as antigen-presenting cells in negative selection of self-reactive T cell clones, a process essential for the establishment of central self-tolerance. These cells mirror peripheral tissues through promiscuous expression of a diverse set of tissue-restricted self-antigens. The genes and signaling pathways that regulate the development of medullary thymic epithelial cells are not fully understood. Here we show that mice deficient in NF-B2, a member of the NF-B family, display a marked reduction in the number of mature medullary thymic epithelial cells that express CD80 and bind the lectin Ulex europaeus agglutinin-1, leading to a significant decrease in the extent of promiscuous gene expression in the thymus of NF-B2^-/- mice. Moreover, NF-B2^-/- mice manifest autoimmunity characterized by multiorgan infiltration of activated T cells and high levels of autoantibodies to multiple organs. A subpopulation of the mice also develops immune complex glomerulonephritis. These findings identify a physiological function of NF-B2 in the development of medullary thymic epithelial cells and, thus, the control of self-tolerance induction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the thymus, self-reactive T cells are eliminated through negative selection in which the T cell receptor of a thymocyte engages a high affinity peptide-major histocompatibility complex ligand presented by an antigen-presenting cell, leading to the apoptotic death of the thymocyte (1). Although it has been known for many years that medullary thymic epithelial cells (mTECs)³ have a crucial role in negative selection by acting as antigen-presenting cells (2-4), only recently is the underlying mechanism beginning to emerge. mTECs express a broad spectrum of peripheral tissue-restricted self-antigens, termed promiscuous gene expression (5, 6). Evidence for a crucial role of this promiscuous gene expression in self-tolerance induction comes from analysis of mice lacking Aire, a transcription factor that is mutated in the human disease autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) (7, 8). mTECs from mice deficient in Aire have diminished expression of tissue-restricted self-antigens, and these mice develop a multiorgan autoimmune syndrome similar to APECED (9). The development of mTECs is accompanied by an increase in the expression levels of CD80 and a carbohydrate epitope that binds the lectin UEA-1 (10-12). Both UEA-1^- and UEA-1⁺ mTECs display promiscuous gene expression (5). However, a more recent study shows a close correlation between the expression levels of CD80 and the extent of promiscuous gene expression (6). The genes and signaling pathways that regulate the development of mTECs are not fully understood.

NF-B2 is a member of the NF-B family of transcription factors that also include p105/p50 (NF-B1), RelA (p65), RelB, and c-Rel. The full-length NF-B2 protein p100 is preferentially associated with RelB in the cytoplasm (13, 14), which prevents RelB nuclear translocation and represses RelB-dependent transcription. Phosphorylation of the C terminus of p100 by IKK, which itself is activated by NF-B-inducing kinase (NIK), leads to proteolytic processing of p100 into p52 (15, 16). The resulting p52-RelB heterodimers then translocate into the nucleus and activate the transcription of their target genes. This alternative NF-B signaling pathway is activated by engagement of receptors for B cell-activating factor, LT, and CD40 ligand (14, 17-19). Previous studies with NF-B2^-/- mice demonstrate a crucial role of NF-B2 in B cell development and secondary lymphoid organogenesis. These mice present a marked decrease in the B cell population in peripheral lymphoid organs and the absence of discrete perifollicular marginal and mantle zones and of germinal centers in the spleen (20, 21).

Recently, several studies provide convincing evidence for a critical role of the LTR signaling pathway in regulation of mTEC development. Mice deficient in LTR, IKK, or carrying a loss-of-function mutant of NIK (NIK^aly/aly) all display disorganized thymic medulla, reduced numbers of mTECs, and overt autoimmunity (22-24). As the LTR signaling pathway is intimately involved in activation of NF-B2 (25, 26), these findings also implicate a role for NF-B2 in the development of mTECs (12). However, defects in LTR signaling not only impair processing of NF-B2 p100 into p52 but also result in accumulation of p100, which may lead to repression of RelB-dependent transcription. In fact, it was recently suggested that it is the increase in the p100 levels, rather than the absence of p52, that might be responsible for the impaired mTEC development observed in IKK-deficient and NIK^aly/aly mice (24).

In this report, we describe an autoimmune phenotype for NF-B2^-/- mice that lack both p100 and p52 and present evidence for a physiological function of NF-B2 in the development of mTECs. Our findings, in conjunction with studies of other mutant mouse strains, delineate an NF-B2 activation-signaling pathway that links thymic organogenesis to the establishment of self-tolerance.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mice—NF-B2^-/- mice (20) were crossed to B6129SF1/J (Jackson Laboratory), and the heterozygous offspring were interbred to obtain NF-B2^-/-, heterozygous, and wild-type littermates. NOD.SCID/NCr mice were purchased from the NCI, National Institutes of Health, at Frederick. All of the animals were maintained under specific pathogen-free conditions at the animal facility of the Medical University of Ohio, and all of the animal procedures were pre-approved by the Institutional Animal Care and Use Committee.

Histology and Immunohistochemistry—Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, sectioned at 5 µm, and stained with hematoxylin and eosin. Histological examination of silver-stained lung sections for possible Pneumocystis infection was conducted by the Research Animal Diagnostic Laboratory at the University of Missouri. For immunohistochemistry, the paraffin was removed and sections were rehydrated according to standard procedures. For retrieval of B220 and CD3 antigen, the sections were subjected to boiling in 10 mM citrate buffer (pH 6.0) or 1 mM EDTA (pH 8.0) for 10 min, respectively. Following quenching of endogenous peroxidase activity with H₂O₂ and blocking with normal serum, the sections were incubated for 1 h with rat mAb against CD45R/B220 (5 µg/ml, RA3-6B2; BD Biosciences) or CD3 (10 µg/ml, CD3-12; Serotec). Isotype-matched rat mAb (10 µg/ml; BD Biosciences) was used as control. After washing, biotinylated rabbit anti-rat antibody (Vector Laboratories) was applied for 30 min. The sections were then incubated for 30 min with ABC reagent (Vector Laboratories), and the immunostaining was visualized with 3,3'-diaminobenzidine (Sigma). The tissue sections were counterstained with hematoxylin and examined under a light microscope.

Immunofluorescence—Thymi from 4- to 6-week-old NF-B2^-/- and wild-type mice were embedded in OCT compound (Sakura) and snap frozen. Sections at 5 µm were cut from the frozen blocks, fixed in cold acetone, rehydrated in PBS plus 0.1% saponin (Sigma), and blocked for 1 h at room temperature with 5% goat serum in PBS. The sections were incubated with rabbit anti-mouse Aire polyclonal antibody (27) (1:1000), hamster anti-mouse CD11c mAb (HL3, 1:200), rat anti-mouse Ep-CAM mAb (G8.8, 1:100), biotinylated hamster anti-mouse CD80 mAb (16-10A1, 1:200), and biotinylated UEA-1 (2 µg/ml, Sigma) for 1 h at room temperature. After washing with PBS, the sections were incubated with FITC goat anti-rabbit IgG (1:500; Molecular Probes), FITC mouse anti-hamster IgG (1:200), and biotin mouse anti-rat IgG2a (1:100) for 1 h at room temperature. The biotin-conjugated antibodies were detected with PE-streptavidin (Southern Biotechnology Associates). For direct immunofluorescence staining, sections were incubated with FITC rat anti-mouse B220 mAb (RA3-6B2, 1:1000) or PE rat anti-mouse IgM mAb (R6-60.2, 1:200). Unless indicated, all antibodies were obtained from BD Biosciences. 4',6-Diamidino-2-phenylindole (300 nM; Molecular Probes) was used for counterstaining of nuclei. For detection of immune complexes in renal glomeruli, cryostat sections of kidney were fixed in cold acetone for 15 min, rehydrated in PBS, and blocked with 10% goat serum/3% bovine serum albumin in PBS for 2 h at room temperature. The sections were then incubated with FITC goat anti-mouse IgG (1:500; Molecular Probes) for 1 h at room temperature. To detect autoantibodies, cryostat sections of various organs from 8-week-old NOD.SCID/NCr mice were fixed in cold methanol for 5 min, blocked with 10% goat serum in PBS for 1 h at room temperature, and incubated with 1:40 dilutions of the serum from individual 1-year-old NF-B2^-/- and wild-type littermates. The sections were then incubated with FITC goat anti-mouse IgM (1:200; Southern Biotechnology Associates) for 1 h. 4',6-Diamidino-2-phenylindole was used for counterstaining of nuclei as described above. Fluorescent images were taken on a Nikon Eclipse E800 microscope.

Real-time PCR—Real-time PCR quantification was conducted with cDNA prepared from total RNA extracted from individual thymi of 4-week-old NF-B2^-/- and wild-type mice. The primers and probes for Aire, Spt1, fatty acid-binding protein, glutamic acid decarboxylase 67, and glyceraldehyde phosphodehydrogenase were as previously described (28, 29). PCR reactions in triplicate were performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) and run on an Applied Biosystems 7500 real-time PCR system according to the manufacturer's instruction.

In Vivo Anti-CD3 Antibody-induced Apoptosis—NF-B2^-/- and wild-type control mice (4-6 weeks) were injected intraperitoneally with 20 µg of hamster anti-mouse CD3 mAb (145-2C11; BD Biosciences) or, as control, with PBS. Mice were sacrificed 40 h later. Thymocytes were collected and counted, and cell subset distribution was determined by flow cytometry (Epics Elite; Beckman-Coulter) after staining with FITC rat anti-mouse CD4 (GK1.5) and PE rat anti-mouse CD8 (53-6.7, both from BD Biosciences).

Analysis of Lung-infiltrating Cells—The infiltrating cells were isolated as described (28). Briefly, lung tissues were finely minced and digested for 30 min in 15 ml of RPMI 1640 containing 1 mg/ml collagenase VIII (Sigma) and 2% fetal bovine serum at 37 °C. Cell suspensions were passed through a 100-µm Nitex filter, and red blood cells were depleted with ACK lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.3). The cells were stained with PE rat anti-mouse CD4 (RM4-5), PE rat anti-mouse CD8, FITC rat anti-mouse CD44 (IM7), FITC hamster anti-mouse CD69 (H1.2F3, all from BD Biosciences) and then analyzed by flow cytometry.

Analysis of Thymic Stromal Cells—Thymic stromal cells were prepared as described (30, 31). Briefly, thymi of 4- to 6-week-old mice were minced and slowly stirred in the medium (RPMI 1640 plus 2% fetal bovine serum) to release the majority of thymocytes. The tissue fragments were sequentially digested with 0.5 mg/ml collagenase D (Roche Applied Science) and collagenase D/dispase I (0.2 mg/ml each; Roche Applied Science) in the presence of 25 µg/ml DNase I (Worthington) in the same medium to release epithelial cells. The digests were pooled, disrupted by 5 mM EDTA, and separated on a discontinuous Percoll gradient (Pharmacia) with densities of = 1.115, 1.06, and 1.0 g/ml (from bottom to top) by centrifugation at 1,350 x g for 30 min at 4 °C. The thymic epithelial cell fraction was collected from the interphase between = 1.06 and 1.0 and subjected to three-step staining with Mouse BD Fc Block (2.4G2), with rat anti-mouse Ep-CAM (G8.8) and Biotin-UEA-1 or Biotin-CD80 (16-10A1), and then with PE Cy5-conjugated CD45 (30-F11), PE-conjugated Ly51 (BP-1), FITC-conjugated mouse anti-rat IgG2a (RG7/1.30), and PE Texas Red-conjugated streptavidin (all from BD Biosciences). Cells were sorted on Epics Elite (Beckman-Coulter) with appropriate forward and side scatter setting to exclude thymocytes, and the data were analyzed with WinMDI 2.8 software.

View larger version (81K): [in this window] [in a new window]   FIGURE 1. Multiorgan infiltration of activated T cells in NF-B2-/- mice. A, survival curves of NF-B2-/-, NF-B2+/-, and wild-type mice. Numbers of mice for each genotype are indicated. B, infiltration of peripheral organs revealed by hematoxylin and eosin staining of formalin-fixed sections of the liver, lung, and salivary gland from a 10-month-old NF-B2-/- mouse, with an age-matched wild-type mouse as control. Scale bars, 100 µm. C, immunohistochemical staining of formalin-fixed lung sections from a 1-year-old NF-B2-/- mouse. Most of the infiltrating cells were stained strongly for CD3, a T cell marker, and a significant number of the infiltrating cells stained positively for B220, a B cell marker. Data are representatives of four mice for each genotype. Scale bars, 100 µm. D, CD4+ T cell infiltrates in the lung of a 1-year-old NF-B2-/- mouse show increased expression of the memory marker CD44 and the activation marker CD69 in comparison with an age-matched wild-type mouse. Numbers indicate percentages of the gated populations. Data are representatives of four mice for each genotype.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Multiorgan Lymphocytic Infiltration in NF-B2^-/- Mice—During a study of apoptosis regulation in thymocytes and T cells by NF-B2 p100 and its processed product p52, we noticed that a significant number of NF-B2^-/- mice died prematurely when compared with their heterozygous and wild-type littermates (Fig. 1A). Histological examination of various tissue samples revealed that, of the 20 deceased NF-B2^-/- mice, 5 had leukemia or lymphoma, indicated by complete effacement of normal bone marrow and spleen architecture by massive infiltration of medium-sized lymphocytes with pleomorphic nuclei and abundant cytoplasm (data not shown). However, the remaining 15 deceased NF-B2^-/- mice showed no obvious sign of leukemia in their bone marrow and spleen samples. Instead, these mice had extensive perivascular infiltration of morphologically normal lymphocytes in multiple organs (data not shown, see also Fig. 1B). The cellular infiltrates might result in organ dysfunction, leading to premature death.

We next examined 20 NF-B2^-/- mice at 10-12 months of age to determine whether the multiorgan lymphocytic infiltration observed in the deceased mice was a consistent feature in NF-B2^-/- mice. All of the NF-B2^-/- mice showed marked lymphocytic infiltration in the lung, liver, and salivary gland (Fig. 1B). Of note, many of the lung lesions appeared to have lymphocytic invasion of artery walls, consistent with vasculitis (Fig. 1B). Immunohistochemical staining of lung sections revealed that most of the infiltrating cells were CD3⁺ T cells (Fig. 1C). No significant lymphocytic infiltration was found in major organs of the 15 age-matched wild-type littermates examined (Fig. 1B). This phenotype of multiorgan lymphocytic infiltration in NF-B2^-/- mice is very similar to that described for various mouse strains with autoimmunity, such as those lacking Aire, LTR, IKK, or with NIK mutation (9, 22-24, 28), suggesting that NF-B2^-/- mice may also develop autoimmunity.

Given the profound defect in B cell-mediated responses in NF-B2^-/- mice (20, 21), we examined the possibility that the multiorgan infiltration might represent responses to chronic infection by opportunistic pathogens such as Pneumocystis carinii. Histological examination of silver-stained sections of the lungs from 1-year-old wild-type (n = 2) and NF-B2^-/- (n = 3) mice revealed no indication of Pneumocystis infection (data not shown).

View larger version (87K): [in this window] [in a new window]   FIGURE 2. Autoimmune diseases in NF-B2-/- mice. A, hematoxylin and eosin staining of formalin-fixed renal sections of deceased NF-B2-/- and wild-type mice. The renal cortex of the NF-B2-/- mouse shows mesangial proliferation, crescent formation, and diffuse interstitial lymphocytic infiltrates. B, the presence of IgG-containing immune complexes in glomeruli of the same NF-B2-/- mouse, revealed by staining cryostat renal sections with FITC-labeled anti-mouse IgG. C, NF-B2-/- mice have autoantibodies to multiple organs, revealed by FITC-labeled anti-mouse IgM staining of NOD.SCID mouse tissue sections of liver, lung, salivary gland, and pancreas preincubated with serum from individual 1-year-old NF-B2-/- or wild-type mice. Nuclei were stained with 4',6-diamidino-2-phenylindole. Shown is representative staining of the tissue sections with sera from six mice for each genotype. Scale bars, 100 µm.

Autoimmune Diseases in NF-B2^-/- Mice—Given the apparent autoimmune phenotype of NF-B2^-/- mice, we further examined these mice for signs of autoimmune disease. Of the deceased NF-B2^-/- mice (n = 20), 55% showed pathological evidence of glomerulopathy, including mesangial proliferation, crescent formation, and diffuse interstitial lymphocytic infiltrates (Fig. 2A). Immunofluorescence staining of cryostat-sectioned renal tissues using anti-IgG revealed deposits of immune complexes with a granular pattern in both the mesangial matrix and capillary loops (Fig. 2B), suggesting that these mice had immune complex glomerulonephritis. We also examined 20 NF-B2^-/- mice at 10-12 months of age and found 30% of them had immune complex glomerulonephritis. None of the 15 age-matched or the two deceased wild-type littermates showed any pathological features of autoimmune renal disease.

It has been shown previously that, despite a marked reduction in the peripheral B cell population and impaired B cell-mediated immune response, NF-B2^-/- mice had a significant increase (4.5-fold) in the levels of serum IgM in comparison with control wild-type littermates (20). Immunostaining of NOD. SCID mouse tissues with sera from 1-year-old NF-B2^-/- mice (n = 6) revealed the presence of high levels of IgM autoantibodies against multiple organs, including the liver, lung, salivary gland, and pancreas (Fig. 2C). We also detected high levels of antibodies against double-stranded DNA in two of the six serum samples from NF-B2^-/- mice but not in any of the six serum samples from age-matched wild-type littermates (data not shown). Together with the results of kidney histopathological analyses, these findings indicate that NF-B2^-/- mice developed systemic autoimmune disease, a phenotype consistent with a broad defect in self-tolerance induction.

NF-B2 Deficiency Has No Significant Effect on Activation-induced Thymocyte Apoptosis and the Number of Aire-expressing Cells in the Thymus—Self-tolerance is maintained by multiple mechanisms such as expression and presentation of self-antigens in the thymus, induction of apoptosis during negative selection, and production of regulatory T cells (32). Defects in any one of these mechanisms could lead to autoimmunity. As NF-B2 regulates death receptor-mediated apoptosis (33), which has been shown to play a role in negative selection (34), we examined NF-B2^-/- mice for the ability of anti-CD3 antibody to induce apoptosis in CD4⁺CD8⁺ thymocytes, a widely used model of negative selection (35). No significant difference was observed in the levels of thymocyte death between NF-B2^-/- mice and their age-matched wild-type littermates after injection of anti-CD3 antibody (Fig. 3A). We also performed in vitro assays of anti-CD3-induced apoptosis of thymocytes and obtained similar results (data not shown). Thus, NF-B2 deficiency has no apparent effect on the ability of thymocytes to undergo apoptosis induced by T cell receptor ligation, at least in the model systems examined.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Effects of NF-B2 deficiency on activation-induced thymocyte apoptosis and Aire expression. A, in vivo anti-CD3-induced apoptosis in CD4+CD8+ immature thymocytes in 4- to 6-week-old NF-B2-/- and wild-type mice. Thymocytes were collected 40 h after intraperitoneal injection of 20 µg of anti-CD3 (145-2C11) and analyzed by flow cytometry. Data represent means ± S.D. in percentages of total thymocytes from three mice for each genotype. B, immunofluorescence staining for Aire expression of cryostat thymic sections from 4-week-old NF-B2-/- and wild-type mice. Shown is representative staining of thymic sections from three mice for each genotype. Scale bars, 100 µm. C, real-time PCR analysis of relative abundance of thymic Aire mRNA in 4- to 6-week-old NF-B2-/- and wild-type mice. Data represent means ± S.D. from four mice for each genotype.

Impaired mTEC Development in NF-B2^-/- Mice—To search further for the mechanism underlying the autoimmune phenotype of NF-B2^-/- mice, we investigated a possible role of NF-B2 in the development of the thymic medulla and its cellular constituents. Histological examination of the thymi from 4- to 6-week-old NF-B2^-/- mice revealed no gross alterations in the thymic architecture (Fig. 4A), as previously reported (20). We also visualized thymic dendritic cells (DCs) with an antibody against the DC marker CD11c and epithelial cells with the antibody G8.8 that recognizes the marker epithelial cell adhesion molecule (Ep-CAM) (5). Immunofluorescence staining showed no differences in the numbers and distribution patterns of these cells between age-matched NF-B2^-/- and wild-type mice (Fig. 4B). In contrast, NF-B2^-/- mice have a marked reduction in the number of mTECs that bind the lectin UEA-1 (Fig. 4B).

To confirm these findings, we performed flow cytometry analysis of thymic epithelial cell populations (CD45^-G8.8⁺) in 4- to 6-week-old NF-B2^-/- and wild-type mice (Fig. 4C). No significant difference in the numbers of cortical TECs (Ly51⁺) was observed between the knock-out and wild-type mice (Fig. 4D). However, the number of CD80⁺ cells was reduced by 88% in the thymus of NF-B2^-/- mice in comparison with their wild-type littermates (Fig. 4D). CD80 is a marker for mature mTECs (12) but also expressed on activated B cells (37, 38). Dual immunofluorescence staining of thymic sections from both wild-type and NF-B2^-/- mice revealed that all of the CD80⁺ cells were negative for the B cell markers B220 and surface IgM (data not shown). Taken together, these data indicate that NF-B2^-/- mice have a marked reduction in the number of CD80⁺ mTECs, suggesting that NF-B2 has a specific and crucial role in the differentiation of mTECs and/or the survival of mature mTECs.

NF-B2^-/- Mice Show Defects in Promiscuous Gene Expression in the Thymus—It was recently shown that CD80⁺ mTECs display the highest degree of promiscuous gene expression (6). As NF-B2^-/- mice show a marked reduction in the number of CD80⁺ mTECs, they may have defects in promiscuous expression of tissue-restricted self-antigens in the thymus. We investigated this possibility by examining the expression levels of three representative tissue-restricted self-antigens, Spt1, fatty acid-binding protein, and glutamic acid decarboxylase 67 (9, 22-24). Real-time PCR analysis revealed that the expression levels of Spt1 and fatty acid-binding protein in the thymus of NF-B2^-/- mice were decreased by 65 and 85%, respectively, in comparison with their age-matched, wild-type littermates (Fig. 5A). However, NF-B2 deficiency had no apparent effect on the expression of glutamic acid decarboxylase 67 in the thymus (Fig. 5A). These data suggest that NF-B2^-/- mice are defective in promiscuous expression of some, but not all, tissue-restricted self-antigens, which may impair the process of self-tolerance induction, leading to the development of autoimmunity.

We next examined the possibility that the reduced number of mTECs and the resulting defect in promiscuous gene expression in NF-B2^-/- mice may impair the production of CD4⁺CD25⁺ regulatory T cells (39, 40), a population of T cells important for suppression of CD4⁺ T cell-mediated organ-specific autoimmune diseases (41, 42). Flow cytometry analysis revealed that spleens from NF-B2^-/- mice actually have a 2.2-fold increase in the percentage of CD4⁺CD25⁺ T cells compared with their wild-type littermates (Fig. 5B). We wanted to point out that NF-B2^-/- mice also have more CD4⁺ T cells in their spleens (Fig. 5B), and therefore the ratio of CD4⁺CD25⁺ to CD4⁺ T cells is about same between NF-B2^-/- (10.5%) and wild-type (10.6%) mice. Given the activated phenotype of peripheral CD4⁺ T cells in NF-B2^-/- mice (Fig. 1D), which may co-express CD25, we further analyzed the CD4⁺CD25⁺ T cells for the expression of Foxp3 (Fig. 5C), a recently identified marker for regulatory T cells (43-45). The analysis revealed that NF-B2^-/- mice had a modest (27%) increase in the number of regulatory T cells in the spleen compared with their wild-type littermates (Fig. 5D). We also examined the frequency of regulatory T cells in the thymus and found no significant differences between NF-B2^-/- and wild-type mice (data not shown). Together, these data indicate that NF-B2 deficiency does not significantly affect the production of regulatory T cells. Thus, the autoimmune phenotype of NF-B2^-/- mice probably results from impaired elimination of auto-reactive T cells.

View larger version (79K): [in this window] [in a new window]   FIGURE 4. Impaired mTEC development in NF-B2-/- mice. A, hematoxylin and eosin staining of formalin-fixed thymic sections of 4- to 6-week-old NF-B2-/- and wild-type mice. Shown is representative staining of thymic sections from five mice for each genotype. Scale bars, 500 µm. M, medulla; C, cortex. B, immunofluorescence staining of thymic sections of 4- to 6-week-old NF-B2-/- and wild-type mice for G8.8 (an epithelial cell marker), CD11c (a DC marker), or UEA-1 (a marker for mature mTECs). Shown is representative staining of thymic sections from five mice for each genotype. Scale bars, 100 µm. C and D, flow cytometry analysis of thymic epithelial cell populations. Pooled thymic stromal cells from three NF-B2-/- or wild-type mice 4 to 6 weeks of age were stained with antibodies to CD45, G8.8, CD80, and Ly51. Mature mTECs are CD45-G8.8+Ly51-CD80+, and cortical TECs are CD45-G8.8+Ly51+. Data in panel D represent means ± S.D. from three independent experiments with a total of nine mice for each genotype. **, Student's t test, p < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (34K): [in this window] [in a new window]   FIGURE 5. NF-B2-/- mice show defects in promiscuous gene expression in the thymus. A, real-time PCR analysis of the relative expression levels of three representative tissue-restricted self-antigens, Spt1, fatty acid-binding protein, and glutamic acid decarboxylase 67. The highest expression levels of these antigens in wild-type mice are defined as 1.0. Data represent means ± S.D. from four mice for each genotype. **, Student's t test, p < 0.01. B, flow cytometry analysis of CD4+CD25+ regulatory T cell populations in spleens of 4- to 6-week-old NF-B2-/- and wild-type mice. Numbers indicate percentages of the CD4+ and CD4+CD25+ T cell populations. Data are representative of six mice for each genotype. C and D, flow cytometry analysis of CD4+CD25+Foxp3+ regulatory T cells in spleens of 4- to 6-week-old NF-B2-/- and wild-type mice. The plots in panel C contain only the CD4+ cells in which percentages of the CD25+Foxp3+ T cells are shown. Data in panel D represent means ± S.D. from four mice for each genotype.

We noticed that NIK^aly/aly and IKK^-/- mice display more severe structural and cellular defects than do NF-B2-deficient mice. Unlike NF-B2^-/- mice, NIK^aly/aly and IKK^-/- mice show disorganization of thymic medulla and a marked reduction in the number of CD4⁺CD25⁺ regulatory T cells (23, 24). Although the underlying mechanism remains to be defined, we speculate that it may be related to the control of RelB expression and activation. NF-B2 p100 is an inhibitor of RelB transcriptional activity by forming a complex with RelB in the cytoplasm, which prevents RelB from associating with other NF-B molecules and entering into the nucleus (13, 14). Therefore, the absence of p100 in NF-B2^-/- mice is expected to result in activation of NF-B complexes containing RelB. On the other hand, the expression of RelB is down-regulated in NIK^aly/aly and IKK^-/- mice (23, 24). Thus, RelB may play a more general role in thymic organogenesis and regulatory T cell production. Consistent with the model, mice lacking tumor necrosis factor receptor-associated factor 6 (TRAF6), in which RelB expression is also down-regulated, show a dramatic reduction in the size of the thymic medulla and in the number of CD4⁺CD25⁺ regulatory T cells (47). Also, the phenotype of RelB^-/- mice more closely resembles that of NIK^aly/aly, IKK^-/-, and TRAF6^-/- mice, with complete disruption of the thymic medulla (48, 49). A further examination of RelB-deficient mice will reveal whether they also have defects in the production of regulatory T cells.

The distinct phenotypes of knock-out mice lacking individual NF-B members suggest that they have non-redundant physiological functions. Because the NF-B family members exert their biological functions as homo- or heterodimers, the functional specificity must be encoded in the particular NF-B dimers. Thus, identification of particular NF-B dimers that drive distinct biological processes is essential for a molecular understanding of NF-B biology. Our study, coupled to the phenotypic analysis of RelB^-/- mice (48, 49), suggests a cell type-dependent specificity in RelB binding partners. The p52/RelB heterodimer appears to have an essential role in the control of mTEC differentiation and maturation, as both RelB^-/- and NF-B2^-/- mice display a significant reduction in the number of UEA-1⁺ mTECs. However, in contrast to NF-B2^-/- mice, RelB^-/- mice also show an absence of thymic DCs (48). Because the predominant B binding activity in thymic extracts is composed of p52/RelB and p50/RelB heterodimers (36), it is most likely that the p50/RelB dimer plays a critical role in the development of thymic DCs. This model is consistent with the reported defect in thymic DC function and development in NF-B1 and NF-B2 double knock-out mice (50).

It was recently demonstrated that mTECs and cortical TECs share a common epithelial precursor (51-53). However, NF-B2 deficiency has no apparent effect on the development of cortical TECs, despite its essential role in the generation and/or maintenance of mTECs. A molecular understanding of this cell type-dependent activation of NF-B2 will likely provide valuable insights into the process of thymic epithelial cell differentiation. Finally, the specific deficiency of mature mTECs in NF-B2^-/- mice provides an experimental system for identifying NF-B2 target genes that regulate the development of mTECs.

	FOOTNOTES

 
^* This work was supported by American Cancer Society Grant RSG-03-173-01-CCG) and NCI, National Institutes of Health Grant R01 CA106550 (to H.-F. D.). 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 Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biochemistry and Cancer Biology, Medical University of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Tel.: 419-383-6653; Fax: 419-383-6228; E-mail: hding{at}meduohio.edu.

³ The abbreviations used are: mTEC, medullary thymic epithelial cell; Aire, autoimmune regulator; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; DC, dendritic cell; FITC, fluorescein isothiocyanate; IKK, IB kinase ; LT, lymphotoxin-; LTR, LT receptor; NIK, NF-B-inducing kinase; PE, phycoerythrin; Spt1, salivary protein 1; UEA-1, Ulex europaeus agglutinin-1; mAb, monoclonal antibody.

⁴ B. Zhang and H.-F. Ding, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Tom Sawyer and Karen Domenico for help with flow cytometry analysis, Judy Meredith, Linda Bauman, and Connie Nowak for assistance in histological studies, and Bristol-Myers Squibb for providing NF-B2 knock-out mice.

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