Myasthenia gravis-specific aberrant neuromuscular gene expression by medullary thymic epithelial cells in thymoma

Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG is frequently developed in thymoma patients, the etiologic factors for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-seq, we identified ectopic expression of neuromuscular molecules in MG-associated thymoma (MG-thymoma). These molecules were originated from a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we named neuromuscular mTECs (nmTECs). MG-thymoma also exhibited microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell-cell interaction analysis also predicted the interaction between nmTECs and T/B cells via CXCL12-CXCR4. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma was further confirmed by immunohistochemically and by cellular composition estimation from MG-thymoma transcriptome. Altogether, this study suggests that nmTECs play a significant role in MG pathogenesis via ectopic expression of neuromuscular molecules.

Failures in thymus medulla regeneration during immune recovery cause tolerance loss and prime recipients for auto-GVHD

Bone marrow transplantation (BMT) is a widely used therapy for blood cancers and primary immunodeficiency. Following transplant, the thymus plays a key role in immune reconstitution by generating a naive αβT cell pool from transplant-derived progenitors. While donor-derived thymopoiesis during the early post-transplant period is well studied, the ability of the thymus to synchronize T cell development with essential tolerance mechanisms is poorly understood. Using a syngeneic mouse transplant model, we analyzed T cell recovery alongside the regeneration and function of intrathymic microenvironments. We report a specific and prolonged failure in the post-transplant recovery of medullary thymic epithelial cells (mTECs). This manifests as loss of medulla-dependent tolerance mechanisms, including failures in Foxp3+ regulatory T cell development and formation of the intrathymic dendritic cell pool. In addition, defective negative selection enables escape of self-reactive conventional αβT cells that promote autoimmunity. Collectively, we show that post-transplant T cell recovery involves an uncoupling of thymopoiesis from thymic tolerance, which results in autoimmune reconstitution caused by failures in thymic medulla regeneration.

Integrative analysis of scRNAs-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator

SummaryMedullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by transcriptional regulator AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected by using Fucci technology express a minimal level of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit amplifying Aire+mTEC precursors during Aire+mTEC differentiation process of the postnatal thymus.

A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer

Medullary thymic epithelial cells (mTECs) which produce and present self-antigens are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with specific subset of DCs. Using several relevant Cre-based mouse models controlling the expression of fluorescent proteins, we found that in regards to CAT, each subset of thymic DCs preferentially targets distinct mTEC subset(s) and importantly, XCR1+ activated DCs represented the most potent subset in CAT. Interestingly, one thymic DC can acquire antigen repetitively and of these, monocyte-derived DCs (moDC) were determined to be the most efficient in repetitive CAT. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

Aire regulates chromatin looping by evicting CTCF from domain boundaries and favoring accumulation of cohesin on superenhancers

Aire controls immunological tolerance by driving promiscuous expression of a large swath of the genome in medullary thymic epithelial cells (mTECs). Its molecular mechanism remains enigmatic. High-resolution chromosome-conformation capture (Hi-C) experiments on ex vivo mTECs revealed Aire to have a widespread impact on higher-order chromatin structure, disfavoring architectural loops while favoring transcriptional loops. In the presence of Aire, cohesin complexes concentrated on superenhancers together with mediator complexes, while the CCCTC-binding factor (CTCF) was relatively depleted from structural domain boundaries. In particular, Aire associated with the cohesin loader, NIPBL, strengthening this factor’s affiliation with cohesin’s enzymatic subunits. mTEC transcripts up-regulated in the presence of Aire corresponded closely to those down-regulated in the absence of one of the cohesin subunits, SA-2. A mechanistic model incorporating these findings explains many of the unusual features of Aire’s impact on mTEC transcription, providing molecular insight into tolerance induction.

The AIRE G228W mutation results in a longer-lasting AIRE-SIRT1 interaction

The in silico and in vitro binding of a peptide covering a part of the autoimmune regulator (AIRE) SAND domain with the SIRT1 protein provides a powerful model system for studying the mechanism of the dominant SAND G228W mutation, which is the causative of APS-1 autoimmune syndrome. It is known that the mutant G228W AIRE protein accumulates more within the nucleus of cells than its wild-type counterpart does. This accumulation is not physiological and is associated with loss of AIRE function. However, the precise molecular mechanism that leads to AIRE accumulation is not yet known. AIRE works as a tetramer and interacts with partner proteins to form the “AIRE complex” that pushes RNA Pol II stalling in the chromatin of medullary thymic epithelial cells. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates Lys residues of the SAND domain. Once AIRE is deacetylated, the binding with SIRT1 is undone, allowing the complex to proceed downstream. Here, we integrate molecular modeling, docking, dynamics, and surface plasmon resonance approaches to compare the structure and energetics of binding/release between AIRE G228 (wild-type) or W228 (mutant) peptides to SIRT1. We find that the proximity of G228W mutation to a K aminoacid residue in the SAND domain promotes a longer-lasting AIRE-SIRT1 interaction. The lasting interaction might cause a delay in the AIRE SAND domain to be released from the SIRT1 catalytic site, which might cause accumulation of the defective AIRE mutant protein in the nuclei of cells.SignificanceThis report reveals the mechanism of the pathogenic and dominant G228W mutation in the AIRE SAND domain. The G228W mutation is found in APS-1 syndrome patients, and it is critical to understand the molecular basis of loss of self-representation, a challenging aspect for immunology. Through modeling, molecular dynamics, and protein binding kinetics, we found that the G228W mutation leads to a stronger physical interaction between the AIRE SAND domain and the SIRT1 protein when compared to the equivalent wild-type segment. The short-term consequence of this stronger interaction is that the release of the AIRE-SIRT1 binding is slower. This might explain the reason that cells carrying the G228W mutation accumulate AIRE protein in their nuclei. This finding reveals with precision the AIRE-SIRT1 binding and the molecular mechanism of the human AIRE G228W mutation.

Extrathymic Aire-expressing cells support maternal-fetal tolerance

Healthy pregnancy requires tolerance to fetal alloantigens as well as syngeneic embryonic and placental antigens. Given the importance of the autoimmune regulator (Aire) gene in self-tolerance, we investigated the role of Aire-expressing cells in maternal-fetal tolerance. We report that maternal ablation of Aire-expressing (Aire+) cells during early mouse pregnancy caused intrauterine growth restriction (IUGR) in both allogeneic and syngeneic pregnancies. This phenotype is immune mediated, as IUGR was rescued in Rag1-deficient mice, and involved a memory response, demonstrated by recurrence of severe IUGR in second pregnancies. Single-cell RNA sequencing demonstrated that Aire+ cell depletion in pregnancy results in expansion of activated T cells, particularly T follicular helper cells. Unexpectedly, selective ablation of either Aire-expressing medullary thymic epithelial cells or extrathymic Aire-expressing cells (eTACs) mapped the IUGR phenotype exclusively to eTACs. Thus, we report a previously undescribed mechanism for the maintenance of maternal-fetal immune homeostasis and demonstrate that eTACs protect the conceptus from immune-mediated IUGR.

Autoimmune regulator act in synergism with thymocyte adhesion in the control of lncRNAs in medullary thymic epithelial cells

The autoimmune regulator (Aire) gene in medullary thymic epithelial cells (mTECs) encodes the AIRE protein, which interacts with its partners within the nucleus. This Aire complex induces stalled RNA Pol II on chromatin to proceed with transcription elongation of a large set of messenger RNAs and microRNAs. Considering that RNA Pol II also transcribes long noncoding RNAs (lncRNAs), we hypothesized that Aire might be implicated in the upstream control of this RNA species. To test this, we employed a loss-of-function approach in which Aire knockout mTECs were compared to Aire wild-type mTECs for lncRNA transcriptional profiling both in vitro and in vivo model systems. RNA sequencing enables the differential expression profiling of lncRNAs when these cells adhere in vitro to thymocytes or do not adhere to them as a way to test the effect of cell adhesion. Sets of lncRNAs that are unique and that are shared in vitro and in vivo were identified. Among these, we found the Aire-dependent lncRNAs as for example, Platr28, Ifi30, Morrbid, Malat1, and Xist. This finding represents the first evidence that Aire mediates the transcription of lncRNAs in mTECs. Microarray hybridizations enabled us to observe that temporal thymocyte adhesion modulates the expression levels of such lncRNAs as Morrbid, Xist, and Fbxl12o after 36h of adhesion. This finding shows the existence of a synergistic mechanism involving a link between thymocyte adhesion, Aire, and lncRNAs in mTECs that might be important for immune self-representation.

miR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells

Background: The autoimmune regulator (Aire) gene is critical for the appropriate establishment of central immune tolerance. As one of the main controllers of promiscuous gene expression in the thymus, Aire promotes the expression of thousands of downstream tissue-restricted antigen (TRA) genes, cell adhesion genes and transcription factor genes in medullary thymic epithelial cells (mTECs). Despite the increasing knowledge about the role of Aire as an upstream transcriptional controller, little is known about the mechanisms by which this gene could be regulated. Results: Here, we assessed the posttranscriptional control of Aire by miRNAs. The in silico miRNA-mRNA interaction analysis predicted thermodynamically stable hybridization between the 3UTR of Aire mRNA and miR-155, which was confirmed to occur within the cellular milieu through a luciferase reporter assay. This finding enabled us to hypothesize that miR-155 might play a role as an intracellular posttranscriptional regulator of Aire mRNA. To test this hypothesis, we transfected a murine mTEC cell line with a miR-155 mimic in vitro, which reduced the mRNA and protein levels of Aire. Moreover, large-scale transcriptome analysis showed the modulation of 311 downstream mRNAs, which included 58 TRA mRNAs. Moreover, miR-155 mimic-transfected cells exhibited a decrease in their chemotaxis property compared with control thymocytes. Conclusion: Overall, the results indicate that miR-155 may posttranscriptionally control Aire mRNA as well as a crucial process by which mTECs allow migration of thymocytes through chemotaxis.