Periodic Reporting for period 4 - ThymusTolerance (Delineation of molecular mechanisms underlying the establishment and breakdown of immunological tolerance in the thymus)
Reporting period: 2022-03-01 to 2023-02-28
Medullary thymus epithelial cells (mTECs) play a pivotal role in pruning self-reactive thymocytes from the T cell repertoire, thanks to their 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, whose deficiency results to multi-organ autoimmunity. Although there has been dramatic progress in our understanding of how mTEC govern the establishment of adaptive immunity and of immunological self-tolerance, there are still several outstanding questions with no comprehensive answers, which served as a basis for the following research aims:
AIM 1. Delineation of mTEC heterogeneity and elucidation of mechanisms underlying thymus development
AIM 2. Delineation of molecular mechanisms underlying promiscuous gene expression in the thymus
AIM 3. Identification and characterization of mono-allelic mutations responsible for the breakdown of thymus-dependent self-tolerance
AIM 1. Delineation of mTEC heterogeneity and elucidation of mechanisms underlying thymus development
AIM 2. Delineation of molecular mechanisms underlying promiscuous gene expression in the thymus
AIM 3. Identification and characterization of mono-allelic mutations responsible for the breakdown of thymus-dependent self-tolerance
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.
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.
Work on AIM1 resulted in identification of several previously uncharacterised TEC populations, including thymic tuft cells, endocrine cells or microfold cells. These interesting but unexpected findings prompted us to further follow these novel and highly intriguing TEC subsets, which consequently affected and somewhat amended the key focus of the original proposal. All the studies were completed (and are either published or accepted for publication) 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).
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, JEM 2021). Unexpectedly, these studies also revealed that AIRE is critical for its own gene regulation.
In addition, work on we aim 3 resulted in initiation of another project, in which we successfully isolated and cloned several prostate-and ovary-specific auto antibodies from Arie-deficient mice and exploited them for their potential use for cancer immunotherapy. This project will be completed after the ERC 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).
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, JEM 2021). Unexpectedly, these studies also revealed that AIRE is critical for its own gene regulation.
In addition, work on we aim 3 resulted in initiation of another project, in which we successfully isolated and cloned several prostate-and ovary-specific auto antibodies from Arie-deficient mice and exploited them for their potential use for cancer immunotherapy. This project will be completed after the ERC funding.