Lineage tracing and cell ablation identify a post-Aire-expressing thymic epithelial cell population.

Thymic epithelial cells in the medulla (mTECs) play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs) and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire), a transcriptional activator present in a subset of mTECs characterized by high CD80 and major histocompatibility complex II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire(+) mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire(-) mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates toward the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance.

Extrathymic Aire-expressing cells are a distinct bone marrow-derived population that induce functional inactivation of CD4⁺ T cells.

The autoimmune regulator (Aire) is essential for prevention of autoimmunity; its role is best understood in the thymus, where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)(hi), CD80(lo), CD86(lo), epithelial cell adhesion molecule (EpCAM)(hi), CD45(lo) bone marrow-derived peripheral antigen-presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4⁺ T cells through a mechanism that does not require regulatory T cells (Treg) and is resistant to innate inflammatory stimuli. Together, these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.

Defective autoimmune regulator-dependent central tolerance to myelin protein zero is linked to autoimmune peripheral neuropathy.

Chronic inflammatory demyelinating polyneuropathy is a debilitating autoimmune disease characterized by peripheral nerve demyelination and dysfunction. How the autoimmune response is initiated, identity of provoking Ags, and pathogenic effector mechanisms are not well defined. The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting thymic expression of self-Ags and deletion of self-reactive T cells. In this study, we used mice with hypomorphic Aire function and two patients with Aire mutations to define how Aire deficiency results in spontaneous autoimmune peripheral neuropathy. Autoimmunity against peripheral nerves in both mice and humans targets myelin protein zero, an Ag for which expression is Aire-regulated in the thymus. Consistent with a defect in thymic tolerance, CD4(+) T cells are sufficient to transfer disease in mice and produce IFN-γ in infiltrated peripheral nerves. Our findings suggest that defective Aire-mediated central tolerance to myelin protein zero initiates an autoimmune Th1 effector response toward peripheral nerves.

An autoimmune response to odorant binding protein 1a is associated with dry eye in the Aire-deficient mouse.

Sjögren's Syndrome (SS) is a human autoimmune disease characterized by immune-mediated destruction of the lacrimal and salivary glands. In this study, we show that the Aire-deficient mouse represents a new tool to investigate autoimmune dacryoadenitis and keratoconjunctivitis sicca, features of SS. Previous work in the Aire-deficient mouse suggested a role for alpha-fodrin, a ubiquitous Ag, in the disease process. Using an unbiased biochemical approach, however, we have identified a novel lacrimal gland autoantigen, odorant binding protein 1a, targeted by the autoimmune response. This novel autoantigen is expressed in the thymus in an Aire-dependent manner. The results from our study suggest that defects in central tolerance may contribute to SS and provide a new and clinically relevant model to investigate the pathogenic mechanisms in lacrimal gland autoimmunity and associated ocular surface sequelae.

Effector mechanisms of the autoimmune syndrome in the murine model of autoimmune polyglandular syndrome type 1.

Mutations in the Aire gene result in a clinical phenomenon known as Autoimmune Polyglandular Syndrome (APS) Type I, which classically manifests as a triad of adrenal insufficiency, hypoparathyroidism, and chronic mucocutaneous infections. In addition to this triad, a number of other autoimmune diseases have been observed in APS1 patients including Sjögren's syndrome, vitiligo, alopecia, uveitis, and others. Aire-deficient mice, the animal model for APS1, have highlighted the role of the thymus in the disease process and demonstrated a failure in central tolerance in aire-deficient mice. However, autoantibodies have been observed against multiple organs in both mice and humans, making it unclear what the specific role of B and T cells are in the pathogenesis of disease. Using the aire-deficient mouse as a preclinical model for APS1, we have investigated the relative contribution of specific lymphocyte populations, with the goal of identifying the cell populations which may be targeted for rational therapeutic design. In this study, we show that T cells are indispensable to the breakdown of self-tolerance, in contrast to B cells which play a more limited role in autoimmunity. Th1 polarized CD4(+) T cells, in particular, are major contributors to the autoimmune response. With this knowledge, we go on to use therapies targeted at T cells to investigate their ability to modulate disease in vivo. Depletion of CD4(+) T cells using a neutralizing Ab ameliorated the disease process. Thus, therapies targeted specifically at the CD4(+) T cell subset may help control autoimmune disease in patients with APS1.

Deletional tolerance mediated by extrathymic Aire-expressing cells.

The prevention of autoimmunity requires the elimination of self-reactive T cells during their development and maturation. The expression of diverse self-antigens by stromal cells in the thymus is essential to this process and depends, in part, on the activity of the autoimmune regulator (Aire) gene. Here we report the identification of extrathymic Aire-expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally derived eTACs express a diverse array of distinct self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using two-photon microscopy, we observed stable antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen-expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.

Mechanisms of an autoimmunity syndrome in mice caused by a dominant mutation in Aire.

Homozygous loss-of-function mutations in AIRE cause autoimmune polyglandular syndrome type 1 (APS 1), which manifests in a classic triad of hypoparathyroidism, adrenal insufficiency, and candidiasis. Interestingly, a kindred with a specific G228W AIRE variant presented with an autosomal dominant autoimmune phenotype distinct from APS 1. We utilized a novel G228W-knockin mouse model to show that this variant acted in a dominant-negative manner to cause a unique autoimmunity syndrome. In addition, the expression of a large number of Aire-regulated thymic antigens was partially inhibited in these animals, demonstrating the importance of quantitative changes in thymic antigen expression in determining organ-specific autoimmunity. Furthermore, the dominant-negative effect of the G228W variant was exerted through recruitment of WT Aire away from active sites of transcription in the nucleus of medullary thymic epithelial cells in vivo. Together, these results may demonstrate a mechanism by which autoimmune predisposition to phenotypes distinct from APS 1 can be mediated in a dominant-negative fashion by Aire.