Grafts of supplementary thymuses injected with allogeneic pancreatic islets protect nonobese diabetic mice against diabetes

In nonobese diabetic (NOD) mice, the autoimmune attack of the beta-cells in pancreatic islets is now believed to result from abnormal thymic selection. Accordingly, grafts of thymic epithelium from NOD donors to athymic recipients promote autoimmune islet inflammation in normal strains, and intrathymic islet grafts decrease the incidence of disease in NOD animals. Two competing hypotheses of abnormal thymic selection in diabetic mice have been proposed: deficient negative selection with poor elimination of aggressive organ-specific T cells vs. deficient positive selection of protective T regulatory cells. We have now addressed these alternatives by grafting, into young NOD mice whose own thymus was left intact, newborn NOD thymuses containing allogeneic pancreatic islets. If the NOD defect represented poor negative selection, these animals would develop disease at control rates, as the generation of autoreactive T cells proceeds undisturbed in the autologous thymus. In contrast, if NOD thymuses are defective in the production of T regulatory cells, lower disease incidence is expected in the chimeras, as more protective cells can be produced in the grafted thymus. The results show a reduced incidence of diabetes in the chimeras (24%) as compared with control (72%) NOD mice, throughout adult life. We conclude that amelioration of NOD mice by intrathymic islet grafts is not caused by enhanced negative selection and suggest that autoimmune diabetes in this system is the result of inefficient generation of T regulatory cells in the thymus.     

Impaired thymic negative selection causes autoimmune graft-versus-host disease

Animal models with impaired thymic negative selection do not always cause autoimmune diseases despite the development of an autoreactive T-cell repertoire. We investigated the requirements for the development of systemic autoimmune disease by using bone marrow chimeras that lacked expression of major histocompatibility complex (MHC) class II on thymic antigen-presenting cells (APCs), leading to impaired negative selection. We found that impaired negative selection mediated by absence of MHC class II, but not MHC class I, permitted the development of systemic autoimmune disease that is indistinguishable from acute graft-versus-host disease (GVHD). Thymectomy prevented disease, confirming the causal association of the thymus with its development. Adoptive transfer of CD4+ T cells caused GVHD in secondary hosts only when they were irradiated, and cotransfer of peripheral CD4+ and CD8+ T cells from naive mice prevented the disease. These results demonstrate that impaired thymic negative selection can cause lethal autoimmune disease indistinguishable from acute GVHD in the context of a proinflammatory milieu when peripheral regulatory mechanisms are absent.     

Induction of antigen-specific tolerance by intrathymic injection of lentiviral vectors

Immune tolerance to self-antigens is established during lymphocyte differentiation in the thymus, but a simple means to induce antigen-specific tolerance in the thymus is still elusive. We show here that intrathymic injection of a lentiviral vector expressing the hemagglutinin antigen (HA) in TCR-HA transgenic mice resulted in negative selection of HA-specific effector T cells and sustained positive selection of HA-specific regulatory T cells (Tregs). This positive selection increased the number of HA-specific Tregs 10-fold and was comparable with the one observed in TCR-HA transgenic mice crossed with transgenic mice expressing HA under the control of the insulin promoter (Ins-HA). HA expression by radioresistant thymic epithelial cells was sufficient to drive Treg generation. Intrathymic injection of the lentiviral vector also resulted in an enrichment of HA-specific Tregs in peripheral lymphoid organs, which prevented diabetes induced in Ins-HA mice by transfer of HA-specific effector T cells. In this model, HA-specific Tregs inhibited effector T-cell division in pancreatic lymph nodes. Finally, we show that intrathymic injection of a lentiviral vector expressing preproinsulin-2 could reduce the occurrence of spontaneous diabetes in nonobese diabetic mice. Intrathymic gene transfer using lentiviral vectors thus offers new means to manipulate antigen-specific tolerance.     

Interferon autoantibodies associated with AIRE deficiency decrease the expression of IFN-stimulated genes

Neutralizing autoantibodies to type I, but not type II, interferons (IFNs) are found at high titers in almost every patient with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), a disease caused by AIRE gene mutations that lead to defects in thymic T-cell selection. Combining genome-wide expression array with real time RT-PCR assays, we here demonstrate that antibodies against IFN-alpha cause highly significant down-regulation of interferon-stimulated gene expression in cells from APECED patients' blood by blocking their highly dilute endogenous IFNs. This down-regulation was lost progressively as these APECED cells matured in cultures without neutralizing autoantibodies. Most interestingly, a rare APECED patient with autoantibodies to IFN-omega but not IFN-alpha showed a marked increase in expression of the same interferon-stimulated genes. We also report unexpected increases in serum CXCL10 levels in APECED. Our results argue that the breakdown of tolerance to IFNs in AIRE deficiency is associated with impaired responses to them in thymus, and highlight APECED as another autoimmune disease with associated dysregulation of IFN activity.     

Autoreactive thymic B cells are efficient antigen-presenting cells of cognate self-antigens for T cell negative selection

The thymus contains a population of B cells that colocalize with dendritic cells and medullary thymic epithelial cells in the thymic medulla. The development and functional significance of these cells are largely unknown. Using recombination-activating gene 2 GFP reporter mice along with parabiosis experiments, we demonstrate that the vast majority of thymic B cells develop from progenitors within the thymus. Thymic B cells express unique phenotypic markers compared with peripheral B cells; particularly they express high levels of MHC class II, suggesting that they are poised to present self-antigens efficiently. Using Ig knock-in and T-cell receptor transgenic mice specific for the self-antigen glucose-6-phosphate isomerase, we show that autoreactive thymic B cells serve as efficient antigen-presenting cells for T cell negative selection even when they are present at low frequencies. Furthermore, the endogenous thymic B-cell repertoire also functions in this capacity. These results suggest that developing thymic B cells could efficiently capture a broad array of autoantigens through their B-cell receptors, presenting peptides derived from those autoantigens to developing thymocytes and eliminating cognate T cells.Negative selection purges autoreactive T cells from the immune repertoire and is the major mechanism of central tolerance in the thymus. This process depends on presentation of self-peptides to developing thymocytes by antigen-presenting cells (APCs). The question of which APC presents self-antigen for negative selection has been investigated extensively. Initial studies using bone marrow chimeras found that bone-marrow-derived hematopoietic cells are required for negative selection (reviewed in refs. 1 and 2). Many subsequent studies have demonstrated that cortical and medullary thymic epithelial cells (mTECs) can be quite efficient for negative selection as well (1–3). The role of mTECs in deleting T cells specific for tissue-restricted antigens has been highlighted by autoimmunity in both humans and mice possessing mutations in the AIRE gene, which controls the expression of tissue-specific self-antigens in mTECs (4).Bone-marrow-derived APCs include dendritic cells (DCs), B cells, and macrophages. In vitro assays comparing their relative antigen presentation efficiency showed that DCs were the most efficient, leading to the conclusion that DCs were largely responsible for negative selection in the thymus (5). Although B cells are poor at presenting antigens via nonspecific uptake, they capture and internalize cognate antigens that are bound by their B-cell receptors and present them very efficiently (6, 7). Therefore, antigen-specific B cells could be the most efficient APC on a per cell basis for a particular antigen.The thymus contains a small population of B cells that make up around 0.1–0.5% of thymocytes (8–12), similar to the proportion of DCs and mTECs in the thymus (13–15). The origin of thymic B cells has been debated, and development from intrathymic progenitors and migration from the peripheral circulation have both been suggested (10, 12). Because thymic B cells preferentially reside at the junction of thymic cortex and medulla, an area where negative selection is thought to occur, they have been proposed to play a role in T cell negative selection (8, 9). Although the capacity of thymic B cells to mediate T cell negative selection has been shown in superantigen and self-antigen overexpression models (16, 17), it remains unclear what kinds of antigens thymic B cells present under normal conditions, the role of their antigen specificity, and what their overall influence on the T-cell repertoire is.In these studies, we demonstrate that the thymic B cells develop from Rag-expressing progenitors within the thymus, and that recirculating peripheral B cells play a minor role in sustaining this population. Using Ig knock-in mice and T-cell receptor (TCR) transgenic mice that are specific for the same cognate self-antigen glucose-6-phosphate isomerase (GPI), we show that anti-GPI B cells are efficiently selected into the thymic B-cell compartment and express high levels of MHC class II and activation makers compared with those in periphery. Increasing the frequency of anti-GPI B cells results in more stringent T cell negative selection in the thymus in a B cell-autonomous manner. Furthermore, in B cell-deficient mice, negative selection toward GPI is decreased suggesting that the wild-type thymic B-cell repertoire contributes to negative selection for this physiologically relevant self-antigen. These results suggest that thymic B cells could efficiently capture a broad array of autoantigens through their B-cell receptors (BCRs) and present peptides derived from these autoantigens to developing thymocytes, linking B-cell autoreactivity with a mechanism for removing T cells with a shared specificity in the thymus.     

Detection of an autoreactive T-cell population within the polyclonal repertoire that undergoes distinct autoimmune regulator (Aire)-mediated selection

The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting the display of tissue-specific antigens in the thymus. To study the influence of Aire on thymic selection in a physiological setting, we used tetramer reagents to detect autoreactive T cells specific for the Aire-dependent tissue-specific antigen interphotoreceptor retinoid-binding protein (IRBP), in the polyclonal repertoire. Two class II tetramer reagents were designed to identify T cells specific for two different peptide epitopes of IRBP. Analyses of the polyclonal T-cell repertoire showed a high frequency of activated T cells specific for both IRBP tetramers in Aire(-/-) mice, but not in Aire(+/+) mice. Surprisingly, although one tetramer-binding T-cell population was efficiently deleted in the thymus in an Aire-dependent manner, the second tetramer-binding population was not deleted and could be detected in both the Aire(-/-) and Aire(+/+) T-cell repertoires. We found that Aire-dependent thymic deletion of IRBP-specific T cells relies on intercellular transfer of IRBP between thymic stroma and bone marrow-derived antigen-presenting cells. Furthermore, our data suggest that Aire-mediated deletion relies not only on thymic expression of IRBP, but also on proper antigen processing and presentation of IRBP by thymic antigen-presenting cells.     

Cytotoxic T lymphocytes and autoimmunity

PURPOSE OF REVIEW: The possibility of the recognition by cytotoxic T lymphocytes of tissue autoantigens has been largely ignored in explaining organ-specific autoimmune diseases. Recent advances in the understanding of human leukocyte antigen class I-binding peptides motifs have led to the detection and the characterization of those autoreactive CD8(+) cytotoxic T lymphocytes involved in organ-specific autoimmune diseases. The purpose of this review is to discuss recent studies that shed light on the implication of cytotoxic T lymphocytes in several autoimmune disorders as well as the mechanisms underlying their stimulation. RECENT FINDINGS: Significant progress has been made in the characterization of autoantigens targeted by cytotoxic T lymphocytes in several class I-restricted autoimmune diseases, including Beh?et's disease and ankylosing spondylitis, and their implication in systemic autoimmune disease such as systemic lupus erythematosus. Moreover, the signals involved in the activation of autoreactive cytotoxic T lymphocytes have been better characterized, particularly the molecular requirements of the antigen presentation at the surface of the dendritic cell system, mainly because of a better understanding of the Toll-like receptor-induced signals or the discovery of a defect in regulatory T cells. SUMMARY: New findings in the pathophysiology of cytotoxic T lymphocytes in autoimmunity and especially a better comprehension of their activation may give a new impetus for the development of targeted immunologic therapies in various autoimmune disorders.     

Dissociation of thymic positive and negative selection in transgenic mice expressing major histocompatibility complex class I molecules exclusively on thymic cortical epithelial cells

Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8(+) T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8(+) cells were observed in the periphery. Upon specific activation, transgenic CD8(+) T cells efficiently lysed syngeneic MHC class I(+) targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.     

The etiopathogenesis of autoimmunity

Acquisition by mammals of an adaptive immune system was an evolutionary leap, but occurred at the cost of autoimmunity. The necessity for self-recognition was appreciated in 1900, but autoimmune disease did not become a clinical reality until the 1950s-still the perimeters are indistinct. Autoimmune responses recapitulate the complex events of normal immune responses but cannot shut down. Immune tolerance is established during repertoire development centrally in thymus or bone marrow by deletion of self-reactive immunocytes, and is supplemented peripherally by regulatory T cells (Tregs). A startling discovery is the autoimmune regulator AIRE gene that enables expression of organ-specific autoantigens for intrathymic deletional tolerance of T cells. The origins, activities, and markers of Tregs are under intensive investigation. Of genes implicated in autoimmune, only few are characterized; contributions of environment are similarly uncertain. Time-latency considerations implicate stochastic factors or chance in the etiopathogenesis of autoimmunity, whether they are somatic mutations or successive random gene-environment interactions. Solutions to etiopathogenesis require novel experimental models and finely designed gene-environment studies on human populations. Perhaps immunomodulatory therapies will effect cures before causes are fully understood.     

PDGFRalpha-expressing mesenchyme regulates thymus growth and the availability of intrathymic niches

The thymus provides a specialized site for the production of T cells capable of recognizing foreign antigens in the context of self-major histocompatibility complex (MHC) molecules. During development, the thymus arises from an epithelial rudiment containing bipotent progenitors that differentiate into distinct cortical and medullary epithelial cells to regulate the maturation and selection of self-tolerant CD4+ and CD8+ T cells. In addition to their differentiation, thymic epithelial cells undergo cellular expansion to ensure that sufficient intrathymic cellular niches are available to support the large number of immature thymocytes required to form a self-tolerant T-cell pool. Thus, intrathymic T-cell production is intimately linked to the formation and availability of niches within thymic microenvironments. Here, we show the increase in intrathymic niches caused by the proliferation of the epithelium in the developing thymus is temporally regulated, and correlates with the presence of a population of fetal thymic mesenchyme defined by platelet-derived growth factor receptor alpha (PDGFRalpha) expression. Depletion of PDGFRalpha+ mesenchyme from embryonic thymi prior to their transplantation to ectopic sites results in the formation of functional yet hypoplastic thymic tissue. In summary, we highlight a specialized role for PDGFRalpha+ fetal mesenchyme in the thymus by determining availability of thymic niches through the regulation of thymic epithelial proliferation.     

Thymic emigrants isolated by a new method possess unique phenotypic and functional properties

T cells that emigrate from the thymus have primarily been studied in vivo using fluorescent dye injection of the thymus. This study examined the properties of thymocytes that emigrate from cultured thymic lobes in organ culture. Under these conditions, thymic emigrants displayed the expected phenotype, that of mature thymocytes expressing high levels of T-cell receptor (TCR-alphabeta) and either CD4 or CD8, and were observed to emigrate within 24 hours of positive selection. Emigration was inhibited by cytochalasin D, pertussis toxin, or Clostridium difficile toxin B, implicating an active motility process. Most of the surface markers on alphabeta-thymic emigrants (Thy1, CD44, CD69, CD25, leukocyte functional antigen-1, intercellular adhesion molecule-1, alpha(4)-integrin, alpha(5)-integrin, CD45, and CD28) were expressed at a surface density similar to that on mature intrathymic cells and peripheral splenic T cells. Heterogeneous expression of L-selectin and heat-stable antigen (HSA) suggested that subsets emerge from the thymus with a commitment to different migration patterns. The only marker on emigrants not found on either intrathymic cells or mature spleen T cells was CTLA-4, which could dampen the response of emigrants to peripheral antigens. Antigen responsivenes measured in vitro against allogeneic dendritic cells showed a proliferative response comparable to that of splenic T cells. In vivo, however, thymic emigrants failed to induce an acute graft-versus-host reaction in allogeneic severe combined immunodeficiency recipients. This suggests that a mechanism operating in vivo, perhaps tolerance or migration pattern, attenuates the response of emigrants against antigens that did not induce their deletion in the thymus.     

Immune tolerance and autoimmune uveoretinitis: the role of the ocular microenvironment

Two major self-antigens, S-antigen and interphotoreceptor retinoid-binding protein, which can induce uveoretinitis, exist in the eye. However, immunologic tolerance to these self-antigens is generated and maintained. Two major mechanisms have been demonstrated by which tolerance to tissue-specific self-antigens is maintained. One is central tolerance in the thymus where autoreactive T cells are deleted by medullary thymic epithelial cells expressing the autoimmune regulator gene (Aire) and the other is peripheral tolerance mediated by regulatory T cells such as Foxp3(+)CD25(+)CD4(+) T cells. In addition, the eye is an immune privileged site where indigenous immunomodulatory mechanisms allow immune protection of the eye in a manner that is largely devoid of immunogenic inflammation.     

On the role of thymic epithelium vs. bone marrow-derived cells in repertoire selection of T cells

T lymphocytes mature in the thymus to become functional T cells. Studies with chimeric mice and T cell receptor (TCR) transgenic (tg) mice have indicated that the major histocompatibility gene complex (MHC) of thymic radio-resistant (presumed to be epithelial) cells positively select the MHC-restricted T cell repertoire. Surprisingly, mice without a thymus reconstituted with an MHC-incompatible thymus generate effector T cells which are, in general, specific for the host and not for the thymic MHC. The present study reanalyzed this longstanding paradox in nude mice that were reconstituted with an MHC-incompatible thymus plus or minus immunologically defective bone marrow-derived cells or in nude mice expressing a transgenic T cell receptor. A pathway of thymus-dependent but thymic MHC-independent T cell maturation is revealed where expansion of the antiviral T cell repertoire depends on the MHC of bone marrow-derived cells. These results indicate an alternative, if not a general, pathway of T cell maturation and selection: the thymus may function essentially as an organ promoting T cell receptor expression; T cell specificity, however, reflects repertoire expansion plus cell survival and effector T cell induction driven by the MHC of bone marrow-derived cells. Therefore pure thymus defects can be efficiently reconstituted by allo- and xenogeneic thymic grafts.     

Early fetal gene delivery utilizes both central and peripheral mechanisms of tolerance induction

OBJECTIVE: We previously reported the induction of stable immune tolerance following direct injection of retroviral vectors into preimmune fetal sheep. In the present studies, we conduct detailed analysis of the thymus of recipients of in utero gene transfer (IUGT) to delineate the mechanism of the observed immune tolerance and assess the impact of recipient age on this process. MATERIALS AND METHODS: Fetal sheep at varying gestational ages received the MSCV-NeoR-RFP retroviral vector. The thymus was then collected from these animals at 27 to 30 days postinjection and analyzed for evidence of transduction of key immunoregulatory thymic cells. RESULTS: Our results reveal that both thymic epithelial cells (TEC), crucial for presentation of self-antigen during T-cell thymic selection, and the cells comprising the Hassall's corpuscles, which can present antigen directly and also instruct dendritic cells to induce the formation of CD4(+)CD25(+) T-regulatory cells in the thymus, were only efficiently transduced if IUGT was performed early in gestation. CONCLUSIONS: Our findings thus demonstrate, for the first time, that early IUGT can potentially take advantage of multiple tolerogenic avenues in the fetus, transducing both TEC, which promote central tolerance, and Hassall's corpuscles, which induce formation of T regulatory cells that could act to maintain peripheral tolerance to the transgene products.     

Aire regulates the transfer of antigen from mTECs to dendritic cells for induction of thymic tolerance

To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4(+) or CD8(+) T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells.     

Antigen presentation by keratinocytes directs autoimmune skin disease

The antigen-presenting cells that initiate and maintain MHC class II-associated organ-specific autoimmune diseases are poorly defined. We now describe a new T cell antigen receptor (TCR) transgenic (Tg) model of inflammatory skin disease in which keratinocytes activate and are the primary target of autoreactive CD4(+) T cells. We previously generated keratin 14 (K14)-A(beta)b mice expressing MHC class II only on thymic cortical epithelium. CD4(+) T cells from K14-A(beta)b mice fail to undergo negative selection and thus have significant autoreactivity. The TCR genes from an autoreactive K14-A(beta)b CD4 hybridoma were cloned to produce a TCR Tg mouse, 2-2-3. 2-2-3 TCR Tg cells are negatively selected in WT C57BL6 mice but not in 2-2-3K14-A(beta)b mice. Interestingly, a significant number of mice that express both the K14-A(beta)b transgene and the autoreactive 2-2-3 TCR spontaneously develop inflammatory skin disease with mononuclear infiltrates, induction of MHC class II expression on keratinocytes, and T helper 1 cytokines. Disease can be induced by skin inflammation but not solely by activation of T cells. Thus, cutaneous immunopathology can be directed through antigen presentation by tissue-resident keratinocytes to autoreactive TCR Tg CD4(+) cells.     

Quorum sensing allows T cells to discriminate between self and nonself

T cells orchestrate pathogen-specific adaptive immune responses by identifying peptides derived from pathogenic proteins that are displayed on the surface of infected cells. Host cells also display peptide fragments from the host’s own proteins. Incorrectly identifying peptides derived from the body’s own proteome as pathogenic can result in autoimmune disease. To minimize autoreactivity, immature T cells that respond to self-peptides are deleted in the thymus by a process called negative selection. However, negative selection is imperfect, and autoreactive T cells exist in healthy individuals. To understand how autoimmunity is yet avoided, without loss of responsiveness to pathogens, we have developed a model of T-cell training and response. Our model shows that T cells reliably respond to infection and avoid autoimmunity because collective decisions made by the T-cell population, rather than the responses of individual T cells, determine biological outcomes. The theory is qualitatively consistent with experimental data and yields a criterion for thymic selection to be adequate for suppressing autoimmunity.