Sylvie Lesage

Aire regulates negative selection of organ-specific T cells

Autoimmune polyendocrinopathy syndrome type 1 is a recessive Mendelian disorder resulting from mutations in a novel gene, AIRE, and is characterized by a spectrum of organ-specific autoimmune diseases. It is not known what tolerance mechanisms are defective as a result of AIRE mutation. By tracing the fate of autoreactive CD4+ T cells with high affinity for a pancreatic antigen in transgenic mice with an Aire mutation, we show here that Aire deficiency causes almost complete failure to delete the organ-specific cells in the thymus. These results indicate that autoimmune polyendocrinopathy syndrome 1 is caused by failure of a specialized mechanism for deleting forbidden T cell clones, establishing a central role for this tolerance mechanism.

Novel dimeric Nur77 signaling mechanism in endocrine and lymphoid cells.

Within the nuclear receptor family, Nur77 (also known as NGFI-B) distinguishes itself by its ability to bind a target sequence (the NBRE) as a monomer and by its role in T-cell receptor (TCR)-induced apoptosis in T cells. We now report on a novel mechanism of Nur77 action that is mediated by homodimers. These dimers bind a Nur77 response element (NurRE), which has been identified as a target of CRH-induced Nur77 in the pro-opiomelanocortin (POMC) gene promoter. Both halves of the palindromic NurRE are required for responsiveness to physiological signals, like CRH in pituitary-derived AtT-20 cells. Similarly, in T-cell hybridomas, TCR activation induced NurRE but not NBRE reporters. The in vivo signaling function of Nur77 thus appears to be mediated by dimers acting on a palindromic response element of unusual spacing between its half-sites. This mechanism may represent the biologically relevant paradigm of action for this subfamily of orphan nuclear receptors.

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.

Identifying the MAGUK Protein Carma-1 as a Central Regulator of Humoral Immune Responses and Atopy by Genome-Wide Mouse Mutagenesis

In a genome-wide ENU mouse mutagenesis screen a recessive mouse mutation, unmodulated, was isolated with profound defects in humoral immune responses, selective deficits in B cell activation by antigen receptors and T cell costimulation by CD28, and gradual development of atopic dermatitis with hyper-IgE. Mutant B cells are specifically defective in forming connections between antigen receptors and two key signaling pathways for immunogenic responses, NF-kappaB and JNK, but signal normally to calcium, NFAT, and ERK. The mutation alters a conserved leucine in the coiled-coil domain of CARMA-1/CARD11, a member of the MAGUK protein family implicated in organizing multimolecular signaling complexes. These results define Carma-1 as a key regulator of the plasticity in antigen receptor signaling that underpins opposing mechanisms of immunity and tolerance.

Antagonism between Nur77 and Glucocorticoid Receptor for Control of Transcription

Two important functions of glucocorticoids (Gc), namely, suppression of immune system function and feedback repression of the hypothalamo-pituitary-adrenal (HPA) axis, are mediated through repression of gene transcription. Previous studies have indicated that this repression is exerted in part through antagonism between the glucocorticoid receptors (GR) and the AP-1 family of transcription factors. However, this mechanism could not account for repression of the pro-opiomelanocortin (POMC) gene, an important regulator of the HPA axis. Our recent identification of the orphan nuclear receptor Nur77 as a mediator of CRH induction of POMC transcription led us, in the present work, to show that Gc antagonize this positive signal at two levels. First, Gc partly blunt the CRH induction of Nur77 mRNA, and second, they antagonize Nur77-dependent transcription. GR repression is exerted by antagonism of Nur77 action on the NurRE element of the POMC gene. Gc antagonism of NurRE activity was observed in response to physiological stimuli in both endocrine (CRH induction of POMC) and lymphoid (T-cell receptor activation) cells. In transfection experiments, transcriptional activation by Nur77 and the repressor activity of liganded GR titrated each other on their cognate DNA target. In vitro binding experiments as well as mutation analysis of GR suggest that the mechanism of GR antagonism of Nur77 is very similar to that of the antagonism between GR and AP-1. The convergence of positive signals mediated by Nur77 (and also probably by related family members) and negative signals exerted by GR appears to be a general mechanism for control of transcription, since it is active in both endocrine and lymphoid cells.

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 β 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.

Expression of the self‐marker CD47 on dendritic cells governs their trafficking to secondary lymphoid organs

Dendritic cells (DCs) capture and process Ag in the periphery. Thus, traffic through lymphatic vessels is mandatory before DCs relocate to lymph nodes where they are dedicated to T‐cell priming. Here, we show that the ubiquitous self‐marker CD47 selectively regulates DC, but not T and B cell trafficking across lymphatic vessels and endothelial barriers in vivo. We find an altered skin DC migration and impaired T‐cell priming in CD47‐deficient mice at steady state and under inflammatory conditions. Competitive DC migration assays and active immunization with myeloid DCs demonstrate that CD47 expression is required on DCs but not on the endothelium for efficient DC trafficking and T‐cell responses. This migratory defect correlates with the quasi‐disappearance of splenic marginal zone DCs in nonmanipulated CD47‐deficient mice. Nonetheless, CCR7 expression and CCL19‐driven chemotaxis remain intact. Our data reveal that CD47 on DCs is a critical factor in controlling migration and efficient initiation of the immune response.

Genetic lesions in T‐cell tolerance and thresholds for autoimmunity

Summary:  The cause of common organ‐specific autoimmune diseases is poorly understood because of genetic and cellular complexity in humans and animals. Recent advances in the understanding of the mechanisms of the defects underlying autoimmune disease in autoimmune polyendocrinopathy syndrome type 1 and non‐obese diabetic mice suggest that failures in central tolerance play a key role in predisposition towards organ‐specific autoimmunity. The lessons from such rare monogenic autoimmune disorders and well‐characterized polygenic traits demonstrate how subtle quantitative trait loci can result in large changes in the susceptibility to autoimmunity. These data allow us to propose a model relating efficiency of thymic deletion to T‐cell tolerance and susceptibility to autoimmunity.

T Cell Tolerance to a Neo-Self Antigen Expressed by Thymic Epithelial Cells: The Soluble Form Is More Effective Than the Membrane-Bound Form

We have previously shown that transgenic (Tg) mice expressing either soluble or membrane-bound hen egg lysozyme (sHEL or mHEL, respectively) under control of the αA-crystallin promoter develop tolerance due to thymic expression of minuscule amounts of HEL. To further address the mechanisms by which this tolerance develops, we mated these two lines of Tg mice with the 3A9 line of HEL-specific TCR Tg mice, to produce double-Tg mice. Both lines of double-Tg mice showed deletion of HEL-specific T cells, demonstrated by reduction in numbers of these cells in the thymus and periphery, as well as by reduced proliferative response to HEL in vitro. In addition, the actual deletional process in thymi of the double-Tg mice was visualized in situ by the TUNEL assay and measured by binding of Annexin V. Notably, the apoptosis localized mainly in the thymic medulla, in line with the finding that the populations showing deletion and increased Annexin V binding consisted mainly of single- and double-positive thymocytes. Interestingly, the thymic deletional effect of sHEL was superior to that of mHEL in contrast to the opposite differential tolerogenic effects of these HEL forms on B cells specific to this Ag. Analysis of bone marrow chimeras indicates that both forms of HEL are produced by irradiation-resistant thymic stromal cells and the data suggest that sHEL is more effective in deleting 3A9 T cells due mainly to its higher accessibility to cross-presentation by dendritic APC.

A comprehensive review of the phenotype and function of antigen-specific immunoregulatory double negative T cells

Double negative T cells that lack the expression of both CD4 and CD8 T cell co-receptors exhibit a most unique antigen-specific immunoregulatory potential first described over a decade ago. Due to their immunoregulatory function, this rare T cell population has been studied in both mice and humans for their contribution to peripheral tolerance and disease prevention. Consequently, double negative cells are gaining interest as a potential cellular therapeutic. Herein, we review the phenotype and function of double negative T cells with emphasis on their capacity to induce antigen-specific immune tolerance. While the phenotypic and functional similarities between double negative T cells identified in mouse and humans are highlighted, we also call attention to the need for a specific marker of double negative T cells, which will facilitate future studies in humans. Altogether, due to their unique properties, double negative T cells present a promising therapeutic potential in the context of various disease settings.