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 singlepositive 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 Tcell 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. (Blood. 2011;118(9): 2462-2472) IntroductionFor many years investigators noted the ectopic expression of tissue-specific antigens (TSAs) in the thymus of normal and transgenic mice 1,2 and other species. 3,4 Although this observation was a fascinating one, the true impact of thymic expression of TSAs was not fully appreciated until the generation and analysis of Aire-deficient mice. [5][6][7] These studies were prompted by the discovery of a recessive autoimmune disease in humans referred to as autoimmune polyglandular syndrome type I or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. 8,9 This disease is associated with circulating tissue-specific autoantibodies that contribute to the destruction of target organs, mainly endocrine glands. [10][11][12] The first symptoms that typically appear during early childhood include chronic mucocutaneous candidiasis, hypoparathyroidism, and primary adrenocortical failure. In adulthood, patients develop endocrine autoimmune diseases, such as gonadal atrophy, type 1 diabetes, hypothyroidism, and hepatitis. In addition, several ectodermic diseases may arise. 10 This disease was mapped to the AIRE locus, where several mutations have now been reported. 13 The generation of Airedeficient mice revealed 2 important observations: first, that many TSAs shown to have ectopic thymic expression were regulated by Aire and, second, that mice deficient in Aire were prone to autoimmune disease. 14 The initial report by Anderson et al 5 suggested that Aire controlled self-tolerance by enabling ectopic expression of TSAs in the medullary thymic epithelial cells (mTECs), although not all TSA genes appeared to be controlled by Aire. The ability of Aire to control thymic tolerance by regulating expression of TSAs was formally demonstrated by use of the RIP-HEL transgenic system, where the model antigen hen egg lysozyme (HEL) was expressed...

Autoimmune regulator (AIRE) is an important transcription regulator that mediates a role in central tolerance via promoting the “promiscuous” expression of tissue-specific Ags in the thymus. Although several mouse models of Aire deficiency have been described, none has analyzed the phenotype induced by a mutation that emulates the common 13-bp deletion in human APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) by disrupting the first plant homeodomain in exon 8. Aire-deficient mice with a corresponding mutation showed some disturbance of the medullary epithelial compartment, but at the phenotypic level their T cell compartment appeared relatively normal in the thymus and periphery. An increase in the number of activated T cells was evident, and autoantibodies against several organs were detected. At the histological level, lymphocytic infiltration of several organs indicated the development of autoimmunity, although symptoms were mild and the quality of life for Aire-deficient mice appeared equivalent to wild-type littermates, with the exception of male infertility. Vβ and CDR3 length analysis suggested that each Aire-deficient mouse developed its own polyclonal autoimmune repertoire. Finally, given the prevalence of candidiasis in APECED patients, we examined the control of infection with Candida albicans in Aire-deficient mice. No increase in disease susceptibility was found for either oral or systemic infection. These observations support the view that additional genetic and/or environmental factors contribute substantially to the overt nature of autoimmunity associated with Aire mutations, even for mutations identical to those found in humans with APECED.

NALP5 appears to be a tissue-specific autoantigen involved in hypoparathyroidism in patients with APS-1. Autoantibodies against NALP5 appear to be highly specific and may be diagnostic for this prominent component of APS-1.

Key Points Ezh2 represses Ifng, Gata3, and Il10 loci in naïve CD4+T cells, and its deficiency leads to Th1 skewing and IL-10 overproduction in Th2 cells. Ezh2 deficiency activates multiple death pathways in differentiated effector Th cells.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is an autoimmune disorder caused by mutations in the autoimmune regulator gene AIRE. We examined the expression of Aire in different organs (thymus, spleen, and lymph nodes) in C57BL/6 mice, using a novel rat mAb, specific for murine Aire. Using flow cytometry, directly fluorochrome-labeled mAb revealed Aire expression in a rare thymic cellular subset that was CD45−, expressed low levels of Ly51, and was high for MHC-II and EpCam. This subset also expressed a specific pattern of costimulatory molecules, including CD40, CD80, and PD-L1. Immunohistochemical analysis revealed that Aire+ cells were specifically localized to the thymus or, more precisely, to the cortico-medulla junction and medulla, correlating with the site of negative selection. Although in agreement with previous studies, low levels of Aire mRNA was detected in all dendritic cell subtypes however lacZ staining, immunohistochemistry and flow cytometry failed to detect Aire protein. At a cellular level, Aire was expressed in perinuclear speckles within the nucleus. This report provides the first detailed analysis of Aire protein expression, highlighting the precise location at both the tissue and cellular level.

Background The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored.ResultsHere we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements.ConclusionsHere we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0064-6) contains supplementary material, which is available to authorized users.

Key Points Inactivation of Suz12 results in a rapid and marked exhaustion of the HSC pool. Lymphoid development is completely dependent on PRC2, but numerous myeloid lineages develop in the absence of PRC2.

BH3-only proteins, such as Bim and Bad, contribute to tissue homeostasis by initiating apoptosis in a cell type- and stimulus-specific manner. Loss of Bim provokes lymphocyte accumulation in vivo and renders lymphocytes more resistant to diverse apoptotic stimuli and Bad has been implicated in the apoptosis of hematopoietic cells upon cytokine deprivation. To investigate whether their biological roles in apoptosis overlap, we generated mice lacking both Bim and Bad and compared their hematopoietic phenotype with that of the single-knockout and wild-type (wt) animals. Unexpectedly, bad−/− mice had excess platelets due to prolonged platelet life-span. The bim−/−bad−/− mice were anatomically normal and fertile. Their hematopoietic phenotype resembled that of bim−/− mice but lymphocytes were slightly more elevated in their lymph nodes. Although resting B and T lymphocytes from bim−/−bad−/− and bim−/− animals displayed similar resistance to diverse apoptotic stimuli, mitogen-activated bim−/−bad−/− B cells were more refractory to cytokine deprivation. Moreover, combined loss of Bim and Bad enhanced survival of thymocytes after DNA damage and accelerated development of γ-irradiation-induced thymic lymphoma. Unexpectedly, their cooperation in the thymus depended upon thymocyte-stromal interaction. Collectively, these results demonstrate that Bim and Bad can cooperate in the apoptosis of thymocytes and activated B lymphocytes and in the suppression of thymic lymphoma development.