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.

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.

A novel myelin P0-specific T cell receptor transgenic mouse develops a fulminant autoimmune peripheral neuropathy.

Autoimmune-prone nonobese diabetic mice deficient for B7-2 spontaneously develop an autoimmune peripheral neuropathy mediated by inflammatory CD4(+) T cells that is reminiscent of Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy. To determine the etiology of this disease, CD4(+) T cell hybridomas were generated from inflamed tissue-derived CD4(+) T cells. A majority of T cell hybridomas were specific for myelin protein 0 (P0), which was the principal target of autoantibody responses targeting nerve proteins. To determine whether P0-specific T cell responses were sufficient to mediate disease, we generated a novel myelin P0-specific T cell receptor transgenic (POT) mouse. POT T cells were not tolerized or deleted during thymic development and proliferated in response to P0 in vitro. Importantly, when bred onto a recombination activating gene knockout background, POT mice developed a fulminant form of peripheral neuropathy that affected all mice by weaning age and led to their premature death by 3-5 wk of age. This abrupt disease was associated with the production of interferon gamma by P0-specific T cells and a lack of CD4(+) Foxp3(+) regulatory T cells. Collectively, our data suggest that myelin P0 is a major autoantigen in autoimmune peripheral neuropathy.

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.

Lessons on immune tolerance from the monogenic disease APS1.

Autoimmunity is a complex disease process that results from a breakdown in the ability of the immune system to discriminate self from non-self. One approach to unraveling how autoimmunity occurs is to study monogenic diseases, for which a single gene defect is responsible. Recent work on the monogenic autoimmune disease 'autoimmune polyglandular syndrome type 1' (APS1) and on the causal gene of this disorder--autoimmune regulator (AIRE)--is providing new lessons on how immune tolerance is maintained.

A suppressive oligodeoxynucleotide enhances the efficacy of myelin cocktail/IL-4-tolerizing DNA vaccination and treats autoimmune disease.

Targeting pathogenic T cells with Ag-specific tolerizing DNA vaccines encoding autoantigens is a powerful and feasible therapeutic strategy for Th1-mediated autoimmune diseases. However, plasmid DNA contains abundant unmethylated CpG motifs, which induce a strong Th1 immune response. We describe here a novel approach to counteract this undesired side effect of plasmid DNA used for vaccination in Th1-mediated autoimmune diseases. In chronic relapsing experimental autoimmune encephalomyelitis (EAE), combining a myelin cocktail plus IL-4-tolerizing DNA vaccine with a suppressive GpG oligodeoxynucleotide (GpG-ODN) induced a shift of the autoreactive T cell response toward a protective Th2 cytokine pattern. Myelin microarrays demonstrate that tolerizing DNA vaccination plus GpG-ODN further decreased anti-myelin autoantibody epitope spreading and shifted the autoreactive B cell response to a protective IgG1 isotype. Moreover, the addition of GpG-ODN to tolerizing DNA vaccination therapy effectively reduced overall mean disease severity in both the chronic relapsing EAE and chronic progressive EAE mouse models. In conclusion, suppressive GpG-ODN effectively counteracted the undesired CpG-induced inflammatory effect of a tolerizing DNA vaccine in a Th1-mediated autoimmune disease by skewing both the autoaggressive T cell and B cell responses toward a protective Th2 phenotype. These results demonstrate that suppressive GpG-ODN is a simple and highly effective novel therapeutic adjuvant that will boost the efficacy of Ag-specific tolerizing DNA vaccines used for treating Th1-mediated autoimmune diseases.

Multiple elements of the allergic arm of the immune response modulate autoimmune demyelination.

Analysis of mRNA from multiple sclerosis lesions revealed increased amounts of transcripts for several genes encoding molecules traditionally associated with allergic responses, including prostaglandin D synthase, histamine receptor type 1 (H1R), platelet activating factor receptor, Ig Fc epsilon receptor 1 (Fc epsilon RI), and tryptase. We now demonstrate that, in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), mediated by T helper 1 (Th1) T cells, histamine receptor 1 and 2 (H1R and H2R) are present on inflammatory cells in brain lesions. Th1 cells reactive to myelin proteolipid protein expressed more H1R and less H2R than Th2 cells. Pyrilamine, an H1R antagonist, blocked EAE, and the platelet activating factor receptor antagonist CV6209 reduced the severity of EAE. EAE severity was also decreased in mice with disruption of the genes encoding Ig Fc gamma RIII or both Fc gamma RIII and Fc epsilon RI. Prostaglandin D synthase and tryptase transcripts were elevated in EAE brain. Taken together, these data reveal extensive involvement of elements of the immune response associated with allergy in autoimmune demyelination. The pathogenesis of demyelination must now be viewed as encompassing elements of both Th1 responses and "allergic" responses.

Analysis of neuronal gene expression with laser capture microdissection.

The brain is a heterogeneous tissue in which the numbers of neurons, glia, and other cell types vary among anatomic regions. Gene expression studies performed on brain homogenates yield results reflecting mRNA abundance in a mixture of cell types. Therefore, a method for quantifying gene expression in individual cell populations would be useful. Laser capture microdissection (LCM) is a new technique for obtaining pure populations of cells from heterogeneous tissues. Most studies thus far have used LCM to detect DNA sequences. We developed a method to quantify gene expression in hippocampal neurons from mouse brain using LCM and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). This method was optimized to permit histochemical or immunocytochemical visualization of nerve cells during LCM while minimizing RNA degradation. As an example, gene expression was quantified in hippocampal neurons from the Tg2576 mouse model for Alzheimer's disease.