Jakub Abramson

Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire

Expression of autoimmune regulator (Aire) by thymic medullary epithelial cells (MECs) is critical for central tolerance of self. To explore the mechanism by which such a rare cell population imposes tolerance on the large repertoire of differentiating thymocytes, we examined the proliferation and turnover of Aire+ and Aire− MEC subsets through flow cytometric analysis of 5-bromo-2′deoxyuridine (BrdU) incorporation. The Aire+ MEC subset was almost entirely postmitotic and derived from cycling Aire− precursors. Experiments using reaggregate thymic organ cultures revealed the presence of such precursors among Aire− MECs expressing low levels of major histocompatibility complex class II and CD80. The kinetics of BrdU decay showed the Aire+ population to have a high turnover. Aire did not have a direct impact on the division of MECs in vitro or in vivo but, rather, induced their apoptosis. We argue that these properties strongly favor a “terminal differentiation” model for Aire function in MECs, placing strict temporal limits on the operation of any individual Aire+ MEC in central tolerance induction. We further speculate that the speedy apoptosis of Aire-expressing MECs may be a mechanism to promote cross-presentation of the array of peripheral-tissue antigens they produce.

Aire's partners in the molecular control of immunological tolerance.

Aire induces the expression of a battery of peripheral-tissue self-antigens (PTAs) in thymic stromal cells, promoting the clonal deletion of differentiating T cells that recognize them. Just how Aire targets and induces PTA transcripts remains largely undefined. Screening via Aire-targeted coimmunoprecipitation followed by mass spectrometry, and validating by multiple RNAi-mediated knockdown approaches, we identified a large set of proteins that associate with Aire. They fall into four major functional classes: nuclear transport, chromatin binding/structure, transcription and pre-mRNA processing. One set of Aire interactions centered on DNA protein kinase and a group of proteins it partners with to resolve DNA double-stranded breaks or promote transcriptional elongation. Another set of interactions was focused on the pre-mRNA splicing and maturation machinery, potentially explaining the markedly more effective processing of PTA transcripts in the presence of Aire. These findings suggest a model to explain Aires widespread targeting and induction of weakly transcribed chromatin regions.

Aire employs a histone-binding module to mediate immunological tolerance, linking chromatin regulation with organ-specific autoimmunity

Aire induces ectopic expression of peripheral tissue antigens (PTAs) in thymic medullary epithelial cells, which promotes immunological tolerance. Beginning with a broad screen of histone peptides, we demonstrate that the mechanism by which this single factor controls the transcription of thousands of genes involves recognition of the amino-terminal tail of histone H3, but not of other histones, by one of Aires plant homeodomain (PHD) fingers. Certain posttranslational modifications of H3 tails, notably dimethylation or trimethylation at H3K4, abrogated binding by Aire, whereas others were tolerated. Similar PHD finger–H3 tail-binding properties were recently reported for BRAF-histone deacetylase complex 80 and DNA methyltransferase 3L; sequence alignment, molecular modeling, and biochemical analyses showed these factors and Aire to have structure–function relationships in common. In addition, certain PHD1 mutations underlying the polyendocrine disorder autoimmune polyendocrinopathy–candidiases–ectodermaldystrophy compromised Aire recognition of H3. In vitro binding assays demonstrated direct physical interaction between Aire and nucleosomes, which was in part buttressed by its affinity to DNA. In vivo Aire interactions with chromosomal regions depleted of H3K4me3 were dependent on its H3 tail-binding activity, and this binding was necessary but not sufficient for the up-regulation of genes encoding PTAs. Thus, Aires activity as a histone-binding module mediates the thymic display of PTAs that promotes self-tolerance and prevents organ-specific autoimmunity.

Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells

Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50–100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II–rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

Dominant Mutations in the Autoimmune Regulator AIRE Are Associated with Common Organ-Specific Autoimmune Diseases.

The autoimmune regulator (AIRE) gene is crucial for establishing central immunological tolerance and preventing autoimmunity. Mutations in AIRE cause a rare autosomal-recessive disease, autoimmune polyendocrine syndrome type 1 (APS-1), distinguished by multi-organ autoimmunity. We have identified multiple cases and families with mono-allelic mutations in the first plant homeodomain (PHD1) zinc finger of AIRE that followed dominant inheritance, typically characterized by later onset, milder phenotypes, and reduced penetrance compared to classical APS-1. These missense PHD1 mutations suppressed gene expression driven by wild-type AIRE in a dominant-negative manner, unlike CARD or truncated AIRE mutants that lacked such dominant capacity. Exome array analysis revealed that the PHD1 dominant mutants were found with relatively high frequency (>0.0008) in mixed populations. Our results provide insight into the molecular action of AIRE and demonstrate that disease-causing mutations in the AIRE locus are more common than previously appreciated and cause more variable autoimmune phenotypes.

Regulation of the mast cell response to the type 1 Fcε receptor

Summary:  The type I Fcε receptor (FcεRI) is one of the better understood members of its class and is central to the immunological activation of mast cells and basophils, the key players in immunoglobulin E (IgE)‐dependent immediate hypersensitivity. This review provides background information on several distinct regulatory mechanisms controlling this receptor’s stimulus–response coupling network. First, we review the current understanding of this network’s operation, and then we focus on the inhibitory regulatory mechanisms. In particular, we discuss the different known cytosolic molecules (e.g. kinases, phosphatases, and adapters) as well as cell membrane proteins involved in negatively regulating the FcεRI‐induced secretory responses. Knowledge of this field is developing at a fast rate, as new proteins endowed with regulatory functions are still being discovered. Our understanding of the complex networks by which these proteins exert regulation is limited. Although the scope of this review does not include addressing several important biochemical and biophysical aspects of the regulatory mechanisms, it does provide general insights into a central field in immunology.

SH2 Domain-Containing Inositol Polyphosphate 5′-Phosphatase Is the Main Mediator of the Inhibitory Action of the Mast Cell Function-Associated Antigen

The mast cell function-associated Ag (MAFA) is a type II membrane glycoprotein originally found on the plasma membrane of rat mucosal-type mast cells (RBL-2H3 line). A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering has previously been shown to suppress the secretory response of these cells to the FcεRI stimulus. Here we show that the tyrosine of the ITIM undergoes phosphorylation, on MAFA clustering, that is markedly enhanced on pervanadate treatment of the cells. Furthermore, the Src homology 3 domain of the protein tyrosine kinase Lyn binds directly to a peptide containing nonphosphorylated MAFA ITIM and PAAP motif. Results of both in vitro and in vivo experiments suggest that Lyn is probably responsible for this ITIM phosphorylation, which increases the Src homology domain 2 (SH2) affinity of Lyn for the peptide. In vitro measurements established that tyrosine-phosphorylated MAFA ITIM peptides also bind the SH2 domains of inositol 5′-phosphatase (SHIP) as well as protein tyrosine phosphatase-2. However, the former single domain is bound 8-fold stronger than both of the latter. Further support for the role of SHIP in the action of MAFA stems from in vivo experiments in which tyrosine-phosphorylated MAFA was found to bind primarily SHIP. In RBL-2H3 cells overexpressing wild-type SHIP, MAFA clustering causes markedly stronger inhibition of the secretory response than in control cells expressing normal SHIP levels or cells overexpressing either wild-type protein tyrosine phosphatase-2 or its dominant negative form. In contrast, on overexpression of the SH2 domain of SHIP, the inhibitory action of MAFA is essentially abolished. Taken together, these results suggest that SHIP is the primary enzyme responsible for mediating the inhibition by MAFA of RBL-2H3 cell response to the FcεRI stimulus.

An unusual inhibitory receptor--the mast cell function-associated antigen (MAFA).

The mast cell function-associated antigen (MAFA) is a type II membranal glycoprotein that was first identified on the surface of rat mucosal-type mast cells of the RBL-2H3 line. A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. Human and mouse homologues of MAFA have been discovered recently. However, they are expressed also or only by NK and T-cells, where they most probably play different roles. MAFA clustering by its specific antibody mAb G63 has been previously shown to cause a dose-dependent inhibition of the secretory response of these cells to the FcepsilonRI stimulus. More recent results established that MAFAs inhibitory action involves at least two different enzymes: Following the tyrosyl-phosphorylation of MAFA ITIM by the PTK Lyn, two phosphatases SHIP and SHP2 are recruited to it at the plasma membrane where they propagate the inhibitory signals. The following is a brief report on this unusual inhibitory receptor and its functional activities.

Dok protein family members are involved in signaling mediated by the type 1 Fcε receptor

Aggregation of type 1 Fcϵ receptors (FcϵRI) on mast cells activates a biochemical cascade that culminates in secretion of inflammatory mediators, as well as in changes of cell morphology and adhesion properties. Some of the intracellular components involved in the early coupling events are still unidentified. Here we show that two adaptor proteins, downstream of tyrosine kinases (Dok)‐1 and Dok‐2, are involved in the FcϵRI coupling cascade in the rat mucosal‐type mast cells of the RBL‐2H3 line. Dok‐1 is found to be constitutively associated with the FcϵRI, even in untreated cells, and this interaction is not affected by this receptors aggregation. Both Dok forms undergo a fast and relatively long‐term tyrosyl‐phosphorylation. This modification of Dok‐1 increases its association with RasGAP, suggesting that it is modulating Ras activity. Indeed, we further found that FcϵRI‐mediated Ras/Raf1/Erk signaling as well as the de novo synthesis of TNF‐α are markedly reduced in cells overexpressing Dok‐1. Moreover, FcϵRI clustering causes both Dok‐1 and Dok‐2 to become docking sites for other signaling molecules including Nck, CrkL and Cas. The latter proteins have been implicated particularly in regulation of the actin‐cytoskeletal reorganization. Hence Dok‐1/Dok‐2 may also be involved in the FcϵRI‐stimulated processes of cytoskeleton rearrangement required for cell adhesion, membrane ruffling and exocytosis.

Thymic Epithelial Cells

Intrathymic T cell development is a complex process that depends upon continuous guidance from thymus stromal cell microenvironments. The thymic epithelium within the thymic stroma comprises highly specialized cells with a high degree of anatomic, phenotypic, and functional heterogeneity. These properties are collectively required to bias thymocyte development toward production of self-tolerant and functionally competent T cells. The importance of thymic epithelial cells (TECs) is evidenced by clear links between their dysfunction and multiple diseases where autoimmunity and immunodeficiency are major components. Consequently, TECs are an attractive target for cell therapies to restore effective immune system function. The pathways and molecular regulators that control TEC development are becoming clearer, as are their influences on particular stages of T cell development. Here, we review both historical and the most recent advances in our understanding of the cellular and molecular mechanisms controlling TEC development, function, dysfunction, and regeneration.