AIM 1.
To provide a better and more comprehensive understanding of mTEC heterogeneity, we combined single-cell RNA-sequencing, chromatin profiling and gene targeting. Indeed, our analysis highlighted several novel mTEC populations with distinct molecular functions, epigenetic landscapes and lineage regulators, which shared striking similarities with functionally well-defined parenchymal populations, including tuft cells (Bornstein et al, Nature 2018), endocrine cells, microfold cells or myocytes (Givony et al, Nature 2023). By further focusing on endocrine-TEC, microfold-TEC and tuft-TEC populations, we found that while the endocrine-TEC required Insm1 for their development, and were critical in maintaining thymus cellularity in ghrelin dependent manner, the microfold-TEC required Spib for their development, and were essential for the generation of thymic IgA+ plasma cells (Givony et al, Nature 2023). Similarly, tuft-TEC required Pou2f3 for their development (Bornstein et al, Nature 2018) and were required for effective regeneration of the thymus after dexamethasone-induced damage, via activation of innate lymphoid cells type-2 (ILC2) (Nevo, et al resubmitted). Collectively, our study reveals that mTEC have the potential to differentiate into various types of molecularly functional defined cells, which not only contribute to the induction of central tolerance, but also regulate homeostasis of other thymus-resident populations.
In addition, completion of this aim provided important insights into molecular mechanisms which control the expression of the master regulator of TEC development - the transcription factor Foxn1 (Kadouri et al, Sci Immunol 2022).
The results stemming from this specific aim were not only disseminated in the corresponding scientific publications, but also in review articles (Kadouri et al, Nat Rev Immunol 2020), as well as in various international conferences and popular lectures.
AIM 2.
In this specific aim we sought to further delineate the molecular mechanisms underlying promiscuous gene expression in the thymus. This specific aim yielded 2 published and 3 unpublished manuscripts. In the first study, we identified and validated several key interaction of AIRE with other proteins (e.g. Sirt1, histones) using a novel methodology we developed and called PLIC (proximity ligation imaging cytometry) (Avin et al, Nat Comm).
In a separate project, we identified 3 distinct types of promiscuous gene expression in the thymus for the purpose of tolerance induction. While the first two (1a and 1b) are dependent on Aire (Gome et al, in preparation), the type-2 mechanism is largely AIRE independent and is instead orchestrated by cell-specific transcription factors, which induce a specific gene signature (Givony et al, Nature 2023). In addition, we also identified TRIM28 as one of the key factors essential for Arie-dependent regulation of promiscuous gene expression (Chuprin et al in preparation) and SMARCA5/Baz1b complex as another regulator of this process (Elenekave et al in preparation).
AIM 3.
We have identified several novel mono-allelic AIRE mutations in humans suffering from various organ-specific autoimmune disorders and generated the corresponding mouse models. Indeed, using these mouse models 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 (Goldfarb et al, J. Exp. Med, 2021).
In addition, in frame of this specific aim, we were also successful in elucidating how loss of function mutations in the AIRE gene result in chronic candida infection and enamel dystrophy in AIRE-deficient patients.
Specifically, we found that extrathymic (rather then thymic) expression of Aire in Roryt+ APCs in lymph nodes is essential for induction of effective Th17 response to candida infection, as specific deletion of AIRE in these Roryt+ APCs results in failure to generate candida specific Th17 cells and failure to clear candida (Dobes et al Nat Immunol 2022).
Moreover, a study spearheaded by a PhD student Yael Gruper delineating the mechanisms by which Aire deficiency results in enamel dystrophy identified a new form of autoimmune disorder - Autoimmune Amelogenesis Imperfecta (AAI), which is characterized by development of IgA autoantibodies to various enamel matrix proteins (Gruper et al manuscript resubmitted to Nature).
The results stemming from this specific aim were not only disseminated in the corresponding scientific publications, but also in review articles (Abramson et al, Nat Rev Immunol 2023), as well as in various international conferences and popular lectures.
