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Delineation of molecular mechanisms underlying the establishment and breakdown of immunological tolerance in the thymus

Periodic Reporting for period 2 - ThymusTolerance (Delineation of molecular mechanisms underlying the establishment and breakdown of immunological tolerance in the thymus)

Reporting period: 2019-03-01 to 2020-08-31

Medullary thymus epithelial cells (mTECs) play a pivotal role in pruning self-reactive thymocytes from the T cell repertoire. Crucial to the key role of mTECs in this process, is their unique capacity to promiscuously express and present almost all self-antigens, including thousands of tissue-specific antigen (TSA) genes. Strikingly, the expression of most of this TSA repertoire in mTECs is regulated by a single transcriptional regulator called Aire. Indeed, Aire deficiency in mice and human patients results to multi-organ autoimmunity. Although there has been dramatic progress in our understanding of how thymic epithelial cells shape and govern the establishment of adaptive immunity and of immunological self-tolerance, there are still several outstanding questions with no comprehensive answers. Therefore, in the research project supported by ERC-CoG grant we sought to provide more comprehensive answers to these still elusive, but very fundamental questions. Specifically, our main goals were:

AIM 1. Delineation of TEC heterogeneity and elucidation of mechanisms underlying thymus development
Our knowledge of how many different TEC subpopulations constitute a functional thymus and what are their hierarchical relationships, is still far from being comprehensive. Therefore to provide a better and more comprehensive understanding of TEC heterogeneity, we sought to perform single cell RNA seq-based characterisation of the TEC compartment of a mouse thymus at different stages of development. Successful completion of this aim should provide us not only with a more comprehensive understanding of how many different TEC subsets constitute a thymus, but they should also provide us with critical novel molecular insights into our understanding of thymus organogenesis and development.

AIM 2. Delineation of molecular mechanisms underlying promiscuous gene expression in the thymus
Although several recent studies have considerably advanced our understanding of Aire-mediated promiscuous gene expression in the thymus, several seminal questions regarding the molecular regulation of this process remain unanswered. Specifically, the transcriptional machineries that “awaken” Aire-dependent and, in particular, Aire-independent fractions of TSA transcripts have yet to be uncovered. Correspondingly, it is still unclear how the expression of Aire-dependent and –independent TSA genes (in bona fide mTECs) is regulated on chromatin and epigenetic levels. Therefore, in this specific aim we sought to further delineate the molecular mechanisms that underlie Aire-mediated gene expression in the thymus.

AIM 3. Identification and characterization of mono-allelic mutations responsible for the breakdown of thymus-dependent self-tolerance
The Autoimmune Regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Therefore, in this goal we sought to further characterise putative dominant-negative AIRE mutations and elucidate the mechanisms by which they cause breakdown of thymus dependent self-tolerance.
AIM 1.
To provide a better and more comprehensive understanding of TEC heterogeneity in a mouse thymus at different stages of development, we combined single-cell RNA-sequencing, chromatin profiling and gene targeting. Our analysis highlighted four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constituted a new and highly divergent TEC subset with molecular characteristics of tuft cells (Fig 1). Indeed, mice deficient in Pou2f3, a master regulator of tuft cells, showed complete and specific depletion of mTEC IV cells, which resulted in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, this study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus (Bornstein et al, Nature).

AIM 2.
In this specific aim we sought to further delineate the molecular mechanisms that underlie Aire-mediated gene expression in the thymus. In frame of this research project we were able to demonstrate that Aire-dependent promiscuous gene expression in mTECs is facilitated by a rather unconventional mechanism involving Aire-dependent activation of the DNA damage response (DDR) machinery. Specifically, our data demonstrate that in mTECs, Aire induces phosphorylation of several components involved in the DDR pathway, including DNA-PK, Trim28 or H2AX, which then co-localize with Aire in DNA damage foci. Furthermore, our data show that induction of targeted DNA breaks at the vicinity of transcription start sites of Aire-dependent genes results not only in their chromatin relaxation, but also in their transcriptional activation. Finally and most importantly, our data demonstrate that the transcriptional regulator Trim28 operates as the key factor linking Aire-induced DDR with transcriptional activation of its target genes and induction of self-tolerance (Chuprin et al, submitted).

AIM 3.
Using mouse models of various different patient mutations we validated that some monoallic mutants, including a novel variant C446G, indeed abrogate the induction of central tolerance in the thymus. Moreover, we successfully dissected the mechanisms that underlie their dominant-negative capacities, by showing that recessive mutations result in a lack of AIRE protein expression, whereas the dominant mutations augment the expression of both AIRE protein and transcripts. Interestingly, the enhanced AIRE expression was partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, completion of this specific goal not only helped elucidating why some AIRE mutations are recessive while others dominant, but also identified aa novel auto-regulatory mechanism by which AIRE negatively modulates its own expression (Goldfarb et al, submitted).
Our work on AIM1 resulted in identification of several previously uncharacterised TEC populations, including thymic tuft cells. These interesting but unexpected findings prompted us to further follow these novel and highly intriguing TEC subsets. Therefore, our ongoing research aims at their better functional characterisation in the thymus. We plan to complete these studies in frame of the current ERC-CoG funding

Work on AIM2 prompted us to develop a novel methodology, that would allow us to study protein-protein interactions and protein post-transcriptional modifications (PTMs) in rare primary cell populations, such as TECs. Indeed, we were successful in developing a PLIC (proximity ligation imaging cytometry) protocol, which allows studying protein-protein interactions and PTMs at a single cell resolution (Avin et al, Nat Comm).

Our work on AIM3 provided us with critical insights into why some mutations in the AIRE gene follow a recessive while others follow a dominant negative pattern of inheritance (Goldfarb, et all, submitted). Unexpectedly, these studies also revealed that AIRE is critical for its own gene regulation. We are currently further following these intriguing findings.