Search

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance. [ABSTRACT FROM AUTHOR]

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina. [ABSTRACT FROM AUTHOR]

Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomain-containing protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire:Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire:Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers.

Roughly one-half of mice with partial defects in two immune tolerance pathways (Aire GW/+ Lyn -/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4 + T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP -/- mice and greatly defective in Aire GW/+ mice. Most P2.U2 +/- mice and all P2.U.2 +/- Aire GW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina., Competing Interests: Competing interests statement:The authors declare no competing interest.

Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomaincontaining protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire:Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire:Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers. [ABSTRACT FROM AUTHOR]

Medullary thymic epithelial cells (mTECs) are essential for the establishment of T cell central tolerance. The transcription factor Aire plays a key role in this process, but other factors remain understudied. We found that a small population of mTECs expressed the coinhibitory receptor cytotoxic T lymphocyte-associated protein 4 (CTLA-4). These CTLA-4 + cells were detectable in perinates, peaked around young adulthood and expanded sixfold in the absence of Aire. Single-cell transcriptomics revealed CTLA-4 + mTECs to express a distinct gene signature encoding molecules associated with antigen presentation and interferon-gamma signaling. Mice conditionally lacking CTLA-4 in thymic epithelial cells had no major immunological deficiencies but displayed a mildly increased inflammatory tone and a partial defect in the generation of Foxp3 + CD4 + regulatory T cells. Consequently, these mice developed modest levels of autoantibodies and lymphocytic infiltration of peripheral tissues. Thus, CTLA-4 expression in mTECs complements Aire to establish T cell central tolerance.

Peripheral tolerance to developmentally regulated antigens is necessary to sustain tissue homeostasis. We have now devised an inducible and reversible system that allows interrogation of T-cell tolerance induction in endogenous naïve and memory CD8 T cells. Our data show that peripheral CD8 T-cell tolerance can be preserved through two distinct mechanisms, antigen addiction leading to anergy for naïve T cells and ignorance for memory T cells. Induction of antigen in dendritic cells resulted in substantial expansion and maintenance of endogenous antigen-specific CD8 T cells. The self-reactive cells initially exhibited effector activity but eventually became unresponsive. Upon antigen removal, the antigen-specific population waned, resulting in development of a self-specific memory subset that recalled to subsequent challenge. In striking contrast to naïve CD8 T cells, preexisting antigen-specific memory CD8 T cells failed to expand after antigen induction and essentially ignored the antigen despite widespread expression by dendritic cells. The inclusion of inflammatory signals partially overcame memory CD8 T-cell ignorance of self-antigen. Thus, peripheral CD8 T-cell tolerance for naïve CD8 T cells depended on the continuous presence of antigen, whereas memory CD8 T cells were prohibited from autoreactivity in the absence of inflammation. [ABSTRACT FROM AUTHOR]

Thymic medullary epithelial cells (MECs) express a broad repertoire of peripheral-tissue antigens (PTAs), many of which depend on the transcriptional regulatory factor Aire. Although Aire is known to be critically important for shaping a self-tolerant T cell repertoire. its role in MEC maturation and function remains poorly understood. Using a highly sensitive and reproducible single-cell PCR assay, we demonstrate that individual Aire-expressing MECs transcribe a subset of PTA genes in a probabilistic fashion, with no signs of preferential coexpression of genes characteristic of particular extrathymic epithelial cell lineages. In addition, Aire-dependent PTA genes in MECs are transcribed monoallelically or biallelically in a stochastic pattern, in contrast to the usually biallelic transcription of these same genes in the relevant peripheral cells or of Aire-independent genes in MECs. Expression of PTA genes in MECs depends on transcriptional regulators and uses transcriptional start sites different from those used in peripheral cells. These findings support the "terminal differentiation" model of Aire function: as MECs mature, they transcribe more and more PTA genes, culminating in a cell population that is both capable of presenting antigens (MHCII[suphi], CD80[suphi]) and can draw on a large repertoire of antigens to present. [ABSTRACT FROM AUTHOR]

Both central (thymic) and peripheral (nonthymic) mechanisms are important for the induction and maintenance of T cell tolerance. Mice with a defect in Foxp3, required for the generation and activity of CD4+CD25+ regulatory T cells, exhibit massive lympho-proliferation and severe inflammatory infiltration of multiple organs, in particular the lungs, liver, and skin. We have explored how this phenotype is influenced by an additional defect in central tolerance induction, generated by either crossing in a null mutation of the Air-gene or substituting the nonobese diabetic (NOD) genetic background. The double-deficient mice had fulminant autoimmunity in very early life and a gravely shortened lifespan vis-â-vis single-deficient littermates. They showed massive lymphoproliferation and exacerbated inflammatory damage, particularly in the lungs and liver. Yet, the range of affected sites was not noticeably extended, and, surprisingly, many organs, or regions of organs, remained untouched, suggesting additional important mechanisms to enforce immunological self-tolerance. [ABSTRACT FROM AUTHOR]