Aire controls the differentiation program of thymic epithelial cells in the medulla for the establishment of self-tolerance

The roles of autoimmune regulator (Aire) in the expression of the diverse arrays of tissue-restricted antigen (TRA) genes from thymic epithelial cells in the medulla (medullary thymic epithelial cells [mTECs]) and in organization of the thymic microenvironment are enigmatic. We approached this issue by creating a mouse strain in which the coding sequence of green fluorescent protein (GFP) was inserted into the Aire locus in a manner allowing concomitant disruption of functional Aire protein expression. We found that Aire⁺ (i.e., GFP⁺) mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes such as insulin 2 and salivary protein 1, whereas Aire-independent TRA genes such as C-reactive protein and glutamate decarboxylase 67 were expressed from both Aire⁺ and Aire⁻ mTECs. Remarkably, absence of Aire from mTECs caused morphological changes together with altered distribution of mTECs committed to Aire expression. Furthermore, we found that the numbers of mTECs that express involucrin, a marker for terminal epidermal differentiation, were reduced in Aire-deficient mouse thymus, which was associated with nearly an absence of Hassall's corpuscle-like structures in the medulla. Our results suggest that Aire controls the differentiation program of mTECs, thereby organizing the global mTEC integrity that enables TRA expression from terminally differentiated mTECs in the thymic microenvironment.     

Spontaneous autoimmunity prevented by thymic expression of a single self-antigen

The expression of self-antigen in the thymus is believed to be responsible for the deletion of autoreactive T lymphocytes, a critical process in the maintenance of unresponsiveness to self. The Autoimmune regulator (Aire) gene, which is defective in the disorder autoimmune polyglandular syndrome type 1, has been shown to promote the thymic expression of self-antigens. A clear link, however, between specific thymic self-antigens and a single autoimmune phenotype in this model has been lacking. We show that autoimmune eye disease in aire-deficient mice develops as a result of loss of thymic expression of a single eye antigen, interphotoreceptor retinoid-binding protein (IRBP). In addition, lack of IRBP expression solely in the thymus, even in the presence of aire expression, is sufficient to trigger spontaneous eye-specific autoimmunity. These results suggest that failure of thymic expression of selective single self-antigens can be sufficient to cause organ-specific autoimmune disease, even in otherwise self-tolerant individuals.     

Identification of a novel cis-regulatory element essential for immune tolerance

Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. Here, we identify a highly conserved noncoding DNA element that is essential for Aire expression. This element shows enrichment of enhancer-associated histone marks in mTECs and also has characteristics of being an NF-κB-responsive element. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance.The establishment and maintenance of immune tolerance requires the successful detection and control of self-reactive T cells in the thymus and the periphery. In the thymus, medullary thymic epithelial cells (mTECs) play a crucial role in this process, presenting a varied repertoire of antigens and thereby eliminating self-reactive T cells or driving them to a regulatory T cell fate (Derbinski et al., 2001; Malchow et al., 2013). Along with ubiquitous self-antigens, mTECs present thousands of tissue-specific antigens (TSAs), and expression of many of these TSAs depends on the Autoimmune regulator (Aire) gene (Anderson et al., 2002). Humans and mice with mutations in Aire develop an organ-specific autoimmune syndrome, underscoring the critical role of Aire in immune tolerance (Aaltonen et al., 1997; Nagamine et al., 1997; Anderson et al., 2002).Interestingly, Aire expression is highly tissue restricted. It is expressed broadly early in embryogenesis but then restricted to a subset of mature mTECs and rare extrathymic Aire-expressing cells (eTACs), a DC-lineage–derived population present in secondary lymphoid tissues (Gardner et al., 2008, 2013). Previous research has demonstrated the importance of a few TNF receptor superfamily members, particularly Tnfrsf11a (RANK), in the development and maintenance of Aire-expressing mTECs (Rossi et al., 2007; Akiyama et al., 2008; Hikosaka et al., 2008; Khan et al., 2014). Likewise, noncanonical NF-κB family members, which can be activated via RANK signaling, are also essential for the development of Aire-expressing mTECs (Heino et al., 2000; Zhu et al., 2006). Despite this knowledge of factors that help promote expression of Aire, the DNA regulatory elements that help coordinate this process have not been clearly delineated. We sought to identify cis-regulatory elements (CREs) important for regulating Aire expression.In vertebrates, gene regulation involves both proximal CREs—the promoter—and distal CREs, including enhancers and silencers. The sequences of such elements are often conserved (Noonan and McCallion, 2010). In addition, several epigenetic markers, including p300 binding and the histone modification acetylated lysine 27 of histone 3 (H3K27ac), often mark active enhancers (Visel et al., 2009; Creyghton et al., 2010). Here, we searched for candidate Aire-regulating elements through the use of both sequence conservation and chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). This focused our investigation on a highly conserved region ∼3 kb upstream of Aire that is an NF-κB-responsive element and is essential for Aire expression and immune tolerance.     

Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen.

The comparison of measurements of fibronectin and lactoferrin in ejaculates from vasectomized men, subjects with functional deficiency or aplasia of the seminal vesicles, and reference subjects provided evidence that both the fibronectin and the lactoferrin in human seminal fluid originate from the seminal vesicles and the ampullae. The fibronectin is incorporated in the framework of the seminal gel formed during the immediate postejaculatory phase, whereas the lactoferrin remains in solution. In the seminal gel fibronectin is linked to its predominant structural protein, a high molecular weight seminal vesicle protein (semenogelin). Both the gel-bound fibronectin and semenogelin are progressively fragmented and solubilized by the abundant prostatic kallikrein-like protease (prostate-specific antigen) during and after seminal gel liquefaction. Lactoferrin remains essentially unaffected by the seminal proteases.     

Gene dosage--limiting role of Aire in thymic expression, clonal deletion, and organ-specific autoimmunity

Inactivation of the autoimmune regulator (Aire) gene causes a rare recessive disorder, autoimmune polyendocrine syndrome 1 (APS1), but it is not known if Aire-dependent tolerance mechanisms are susceptible to the quantitative genetic changes thought to underlie more common autoimmune diseases. In mice with a targeted mutation, complete loss of Aire abolished expression of an insulin promoter transgene in thymic epithelium, but had no effect in pancreatic islets or the testes. Loss of one copy of Aire diminished thymic expression of the endogenous insulin gene and the transgene, resulting in a 300% increase in islet-reactive CD4 T cells escaping thymic deletion in T cell receptor transgenic mice, and dramatically increased progression to diabetes. Thymic deletion induced by antigen under control of the thyroglobulin promoter was abolished in Aire homozygotes and less efficient in heterozygotes, providing an explanation for thyroid autoimmunity in APS1. In contrast, Aire deficiency had no effect on thymic deletion to antigen controlled by a systemic H-2K promoter. The sensitivity of Aire-dependent thymic deletion to small reductions in function makes this pathway a prime candidate for more subtle autoimmune quantitative trait loci, and suggests that methods to increase Aire activity would be a potent strategy to lower the incidence of organ-specific autoimmunity.     

Medullary thymic epithelial cell depletion leads to autoimmune hepatitis

TRAF6, an E3 ubiquitin protein ligase, plays a critical role in T cell tolerance by regulating medullary thymic epithelial cell (mTEC) development. mTECs regulate T cell tolerance by ectopically expressing self-antigens and eliminating autoreactive T cells in the thymus. Here we show that mice with mTEC depletion due to conditional deletion of Traf6 expression in murine thymic epithelial cells (Traf6ΔTEC mice) showed a surprisingly narrow spectrum of autoimmunity affecting the liver. The liver inflammation in Traf6ΔTEC mice exhibited all the histological and immunological characteristics of human autoimmune hepatitis (AIH). The role of T cells in AIH establishment was supported by intrahepatic T cell population changes and AIH development after transfer of liver T cells into immunodeficient mice. Despite a 50% reduction in natural Treg thymic output, peripheral tolerance in Traf6ΔTEC mice was normal, whereas compensatory T regulatory mechanisms were evident in the liver of these animals. These data indicate that mTECs exert a cell-autonomous role in central T cell tolerance and organ-specific autoimmunity, but play a redundant role in peripheral tolerance. These findings also demonstrate that Traf6ΔTEC mice are a relevant model with which to study the pathophysiology of AIH, as well as autoantigen-specific T cell responses and regulatory mechanisms underlying this disease.     

Acute and chronic prostatitis

In summary, prostatitis is a complex syndrome that spans a spectrum from acute prostatitis with a straightforward presentation to CP-CPPS with a complex array of symptoms. The identification of prostatic or pelvic pain becomes a requirement for the diagnosis of CP-CPPS. The NIH system of prostatitis categorization is a refinement of the traditional classification of prostatitis by Drach et al, which was based on the localization test of Meares and Stamey. The NIH categorization system allows for a framework to define the disease process, and the NIH-CPSI was created to quantify the symptoms of chronic prostatitis. Integral to the classification of prostatitis is the presence or absence of inflammation, determined by looking for leukocytes in the EPS, seminal fluid, and VB3 specimens. In addition, the role of bacteria as a cause in category III prostatitis continues to be debated. Future research into using inflammatory markers (eg, tumor necrosis factor-alpha, interleukin-2) and using PCR to identify the presence of bacteria may further refine the pathophysiology of prostatitis. The mainstream treatment of chronic prostatitis involves antimicrobials, non-steroidal anti-inflammatory medications, and alpha-blockers. The potential role of asymptomatic category IV chronic prostatitis in the etiology of prostate cancer may be delineated further with future research.     

Failure to censor forbidden clones of CD4 T cells in autoimmune diabetes

Type 1 diabetes and other organ-specific autoimmune diseases often cluster together in human families and in congenic strains of NOD (nonobese diabetic) mice, but the inherited immunoregulatory defects responsible for these diseases are unknown. Here we track the fate of high avidity CD4 T cells recognizing a self-antigen expressed in pancreatic islet beta cells using a transgenic mouse model. T cells of identical specificity, recognizing a dominant peptide from the same islet antigen and major histocompatibility complex (MHC)-presenting molecule, were followed on autoimmune susceptible and resistant genetic backgrounds. We show that non-MHC genes from the NOD strain cause a failure to delete these high avidity autoreactive T cells during their development in the thymus, with subsequent spontaneous breakdown of CD4 cell tolerance to the islet antigen, formation of intra-islet germinal centers, and high titre immunoglobulin G1 autoantibody production. In mixed bone marrow chimeric animals, defective thymic deletion was intrinsic to T cells carrying diabetes susceptibility genes. These results demonstrate a primary failure to censor forbidden clones of self-reactive T cells in inherited susceptibility to organ-specific autoimmune disease, and highlight the importance of thymic mechanisms of tolerance in organ-specific tolerance.     

超声引导联合药物注射治疗难治性前列腺炎

目的探讨超声引导联合药物注射治疗难治性前列腺炎的可行性.方法采用经直肠超声引导对34例难治性前列腺炎患者行前列腺两侧外腺(或回声减低区)及精囊腺联合药物注射治疗,每周2次,4～5周为一疗程,观察疗效及副作用.结果28例治愈,6例有效.术后小腹胀痛15例,大小便带血或血性精液10例,均自行消失,无感染或大出血发生.结论经直肠超声引导前列腺两侧外腺和精囊腺内联合药物注射是治疗难治性前列腺炎的一种安全有效的方法.     

EGFR和PDGFR在慢性前列腺炎中的表达及其意义

目的检测Wistar大鼠慢性前列腺炎动物模型的表皮生长因子受体（EGFR）和血小板衍生的生长因子受体（PDGFRl的表达,为慢性前列腺芡和前列腺癌的的关联性提供理论依据。方法实验组和对照组各采用50只Wistar大鼠慢性细菌性前列腺炎组织和正常前列腺组织．应用免疫组化方法分别检测两组大鼠前列腺组织的EGFR和PDGFR的表达情况。结果实验组大鼠慢性前列腺炎组织EGFR和PDGFR表达均较对照组明显增加,差异均有统计学意义（u分别=3．35、9．05,P均〈0．05）。结论慢性细菌性前列腺炎组织中EGFR和PDGFR的表达高于正常组织,可能与前列腺癌存在一定关联。     

慢性前列腺炎患者血清PSA的检测及其意义

摘 要 目的：探讨血清前列腺特异性抗原（PSA）浓度变化在慢性前列腺炎发生、发展及疗效评价中的意义。方法：用ELISA法测定60例慢性前列腺炎患者直肠指检前的血清PSA浓度，与正常组30例对照，并对比慢性前列腺炎治疗前后患者血清PSA浓度的变化。结果：慢性前列腺炎组和对照组的血清PSA浓度分别为（1.6±0.3）和（0.5±0.2）μg.L-1， 两组间有极显著性差异（t =11.68，P<0.01）；45例慢性前列腺炎治疗有效者，治疗后血清PSA浓度较治疗前明显下降（P<0.01），而15例治疗无效者，血清PSA浓度较治疗前下降不明显（P>0.05）。 结论：慢性前列腺炎时血清PSA浓度明显升高，经治疗随着炎症的消失，PSA浓度可逐渐恢复正常，PSA浓度可作为慢性前列腺炎疗效评价的指标之一。关键词 慢性前列腺炎 前列腺特异性抗原中图分类号：R697.44 文献标识码：A     

Thymic Gene Transfer of Myelin Oligodendrocyte Glycoprotein Ameliorates the Onset but Not the Progression of Autoimmune Demyelination

Tolerance induction, and thus prevention of autoimmunity, is linked with the amount of self-antigen presented on thymic stroma. We describe that intrathymic (i.t.) delivery of the autoantigen, myelin oligodendrocyte glycoprotein (MOG), via a lentiviral vector (LV), led to tolerance induction and prevented mice from developing fulminant experimental autoimmune encephalomyelitis (EAE). This protective effect was associated with the long-term expression of antigen in transduced stromal cells, which resulted in the negative selection of MOG-specific T cells and the generation of regulatory T cells (Tregs). These selection events were effective at decreasing T-cell proliferative responses and reduced Th1 and Th17 cytokines. In vivo, this translated to a reduction in inflammation and demyelination with minimal, or no axonal loss in the spinal cords of treated animals. Significantly intrathymic delivery of MOG to mice during the priming phase of the disease failed to suppress clinical symptoms despite mice being previously treated with a clearing anti-CD4 antibody. These results indicate that targeting autoantigens to the thymic stroma might offer an alternative means to induce the de novo production of tolerant, antigen-specific T cells; however, methods that control the number and or the activation of residual autoreactive cells in the periphery are required to successfully treat autoimmune neuroinflammation.     

Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance

Thymic central tolerance is a critical process that prevents autoimmunity but also presents a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of tumor-specific effector T cells. Recent work has implicated the TNF family members RANK and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the thymus to create a window of defective negative selection. Furthermore, we demonstrate that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that persistence of these tumor-specific effector T cells promoted increased host survival in response to tumor challenge. These results indicate that modulating central tolerance through RANKL can alter thymic output and potentially provide therapeutic benefit by enhancing anti-tumor immunity.Medullary thymic epithelial cells (mTECs) contribute to self-tolerance through the ectopic expression of tissue-specific antigens (TSAs) in the thymus (Derbinski et al., 2001; Anderson et al., 2002; Metzger and Anderson, 2011). This TSA expression in mTECs is largely dependent on autoimmune regulator (Aire), which is expressed in mature mTECs (Gäbler et al., 2007; Gray et al., 2007; Metzger and Anderson, 2011). Through the recognition of TSAs, developing autoreactive T cells are either negatively selected from the pool of developing thymocytes or recruited into the regulatory T (T reg) cell lineage (Liston et al., 2003; Anderson et al., 2005; DeVoss et al., 2006; Shum et al., 2009; Taniguchi et al., 2012; Malchow et al., 2013). The overall importance of this process is underscored by the development of a multi-organ autoimmune syndrome in patients or mice with defective AIRE expression (Consortium, 1997; Nagamine et al., 1997; Anderson et al., 2002).Although central tolerance provides protection against autoimmunity, this process also represents a challenge for anti-tumor immunity (Kyewski and Klein, 2006; Malchow et al., 2013). Because many of the TSAs expressed in the thymus are also expressed in tumors, high-affinity effector T cells capable of recognizing tumor self-antigens may normally be deleted in the thymus (Bos et al., 2005; Cloosen et al., 2007; Träger et al., 2012; Zhu et al., 2013). Transiently suppressing central tolerance by depleting mTECs or modulating Aire expression may provide a therapeutic window for the generation of T cells capable of recognizing tumor self-antigens. Many current cancer immune therapies rely on activating relatively weak tumor-specific T cell responses through modulating peripheral tolerance (Swann and Smyth, 2007; Chen and Mellman, 2013). In contrast, manipulation of central tolerance has the potential to increase the pool and affinity of effector T cells that can recognize and contribute to effective anti-tumor responses. Furthermore, such high-affinity, self-reactive T cells may be more resistant to peripheral tolerance mechanisms that typically restrain an anti-tumor response (Swann and Smyth, 2007). Thus, the development of methods that selectively and transiently deplete Aire-expressing mTECs may be an attractive method to enhance tumor-specific immune responses.Previous work has identified agents that can inhibit the growth and development of TECs such as corticosteroids, cyclosporine, and some inflammatory cytokines (Anz et al., 2009; Fletcher et al., 2009). Despite their clear inhibitory effects on TECs, however, these agents do not appear to have selectivity for blocking mTEC development. Interestingly, recent studies have demonstrated a role for TNF family member pairs RANK–RANKL and CD40-CD40L in the embryological development of Aire⁺ mTECs (Rossi et al., 2007; Akiyama et al., 2008; Hikosaka et al., 2008; Roberts et al., 2012). Recent work has also demonstrated that mTECs in particular have a relatively fast turnover in adult mice with an estimated half-life of ∼2 wk (Gäbler et al., 2007; Gray et al., 2007). Given these findings, we speculated that in vivo blockade of RANKL in adult hosts could both selectively and transiently inhibit the development and turnover of mTECs with potential to alter central T cell tolerance. To this end, we performed in vivo RANKL blockade in adult mice and investigated its effects on both TECs and developing thymocytes. We show that anti-RANKL treatment not only depleted mTECs but could also be used therapeutically to break central tolerance and, as a result, increase the generation of tumor-specific T cells.     

Enforced Bcl-2 Expression Inhibits Antigen-mediated Clonal Elimination of Peripheral B Cells in an Antigen Dose–dependent Manner and Promotes Receptor Editing in Autoreactive, Immature B Cells

The mechanisms that establish immune tolerance in immature and mature B cells appear to be distinct. Membrane-bound autoantigen is thought to induce developmental arrest and receptor editing in immature B cells, whereas mature B cells have shortened lifespans when exposed to the same stimulus. In this study, we used Eμ–bcl-2-22 transgenic (Tg) mice to test the prediction that enforced expression of the Bcl-2 apoptotic inhibitor in B cells would rescue mature, but not immature, B cells from tolerance induction. To monitor tolerance to the natural membrane autoantigen H-2K^b, we bred 3–83μδ (anti-K^k,b) Ig Tg mice to H-2^b mice or to mice expressing transgene-driven K^b in the periphery. In 3–83μδ/bcl-2 Tg mice, deletion of autoreactive B cells induced by peripheral K^b antigen expression in the liver (MT-K^b Tg) or epithelia (KerIV-K^b Tg), was partly or completely inhibited, respectively. Furthermore, Bcl-2 protected peritoneal B-2 B cells from deletion mediated by acute antigen exposure, but this protection could be overcome by higher antigen dose. In contrast to its ability to block peripheral self-tolerance, Bcl-2 overexpression failed to inhibit central tolerance induced by bone marrow antigen expression, but instead, enhanced the receptor editing process. These studies indicate that apoptosis plays distinct roles in central and peripheral B cell tolerance.     

Induction of factor VIII‐specific unresponsiveness by intrathymic factor VIII injection in murine hemophilia A

Summary. Background: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen‐specific immune tolerance by interferon‐γ (IFN‐γ)‐dependent T‐cell anergy in hemophilia A mice. Objective: The thymus plays crucial roles in self‐tolerance, with negative selection of self‐reactive effector T cells and positive selection of self‐reactive regulatory T cells. We investigated the possibility of the induction of antigen‐specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. Methods: Hemophilia A mice were injected with recombinant FVIII into the thymus under real‐time high‐resolution image guidance. Results: Anti‐FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 ± 2.3 vs. 122.5 ± 27.6 BU mL^?1, respectively, P = 0.00078). The CD4⁺ T cells from thymic‐injected mice could not proliferate or produce interleukin (IL)‐2, IL‐12 and IFN‐γ in response to FVIII. The CD4⁺CD25⁺ T cells generated from thymic‐treated mice but not from naïve mice efficiently suppressed the in vitro proliferative response of CD4⁺ T cells and blocked the in vivo development of anti‐FVIII antibodies in the adoptive transfer. Conclusion: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII‐specific regulatory T cells.     

Induction of tolerance to autoimmune diabetes with islet antigens

The development of T cell tolerance to self-antigens is imparted principally through negative selection events during thymic ontogeny. However, this tolerance may be limited to antigens that are expressed in the thymus, and additional mechanisms are probably required to regulate autoimmune responses to tissue-specific antigens. Autoimmune diabetes can be induced experimentally by treating susceptible stains of mice with multiple low doses of streptozotocin (STZ). In this report we show that transplantation of isolated islets of Langerhans into the thymuses of adult C57BL/KsJ mice will induce tolerance to the subsequent induction of autoimmune diabetes. This tolerance is tissue specific and thymus dependent. It was not induced by thymic transfer of adrenal tissue or by kidney transfer of islets. Furthermore, depletion of mature T cells was required and the tolerant state was abrogated by the adoptive transfer of normal splenocytes. It is interesting that pretreatment of the islets with STZ enhanced their ability to induce tolerance, and suggests that antigen shedding induced by tissue damage may facilitate transfer of islet antigens to tolerizing cells in the thymus. These findings indicate that thymic tolerance specific for tissue can be stimulated to occur in the presence of atopic tissue-specific intrathymic antigens. Elimination of disease-related T cells in the absence of global immunosuppression represents a novel approach for the prevention of autoimmune disease.     

Massive thymic deletion results in systemic autoimmunity through elimination of CD4+ CD25+ T regulatory cells

Incomplete deletion of KRN T cells that recognize the ubiquitously expressed self-antigen glucose-6-phosphate-isomerase (GPI) initiates an anti-GPI autoimmune cascade in K/BxN mice resulting in a humorally mediated arthritis. Transgenic (Tg) expression of a KRN T cell receptor (TCR) agonist under the major histocompatibility complex class II promoter resulted in thymic deletion with loss of anti-GPI T and B cell responses and attenuated arthritis course. However, double Tg mice succumbed to systemic autoimmunity with multiorgan inflammation and autoantibody production. Extensive thymic deletion resulted in lymphopenia and elimination of CD4+ CD25+ regulatory T cells (Tregs), but spared some CD4+ T cells expressing endogenous TCR, which oligoclonally expanded in the periphery. Disease was transferred by these T cells and prevented by cotransfer of CD4+ CD25+ Tregs. Moreover, we extended our findings to another TCR system (anti-hen egg lysozyme [HEL] TCR/HEL mice) where similarly extensive thymic deletion also resulted in disease. Thus, our studies demonstrated that central tolerance can paradoxically result in systemic autoimmunity through differential susceptibility of Tregs and autoreactive T cells to thymic deletion. Therefore, too little or too much negative selection to a self-antigen can result in systemic autoimmunity and disease.