RESEARCH ACTIVITIES
Our research group is broadly interested in understanding how immunological tolerance to self is established in the thymus and how breakdown of this process results in autoimmunity. In particular, we focus on a very unique population of the thymic stroma, called the medullary thymic epithelial cells (mTECs). Although these cells constitute less than <0.1% of the thymus, they are essential for the establishment of immunological tolerance. Specifically, mTECs are critical for negative selection of self-reactive T cells and/or generation of natural regulatory T (Treg) cells. Their essential role in the induction of self-tolerance is underlined by their amazing and unique capacity to express, and subsequently present, essentially all body antigens, including those whose expression was originally thought to be restricted only to peripheral organs (e.g. insulin, casein, etc.). Such “promiscuous” expression of tissue-restricted-antigen (TRA) genes in the thymus “foreshadows” the self-antigens that T cells would encounter once they reach maturity and are released into the body. Strikingly, many of these ectopic transcripts are regulated by the product of a single gene, the Autoimmune regulator (Aire). The physiological significance of the Aire-driven “promiscuous” gene expression in the thymus is best illustrated by mouse models and/or human patients with a dysfunctional Aire/AIRE gene, which consequently develop a multi-organ autoimmune syndrome characterized by autoantibodies and immune infiltrates directed at multiple peripheral tissues. Therefore, the Aire-/mTEC-mediated induction of central immunological tolerance is critical for effective protection against devastating autoimmune disorders.
Although the mechanisms underlying the Aire/mTEC-mediated induction of self-tolerance have been a topic of considerable interest over the past decade, a number of outstanding questions regarding Aire function and/or mTEC biology still remain unresolved. Therefore, the ultimate goal of our research group is to tackle some of these open questions.
Specifically, our group aims at:
1) Elucidating what cellular and molecular mechanisms control mTEC function and development?
Both medullary (m) and cortical (c) thymic epithelial cells (TECs) develop from a common bi-potent thymic epithelial progenitor (TEP), which however still remains poorly characterized, in particular in the adult thymus. The development of both thymic epithelial lineages depends on various developmental cues such as fibroblast growth factors (FGFs), bone morphogenic protein-4 (BMP4), Wnt, or retinoic acid (RA) signals, provided by other thymic populations, such as fibroblasts and developing thymocytes. While the signals controlling the development of cTEC lineage still remain elusive, the mTEC developmental program was shown to depend on the non-canonical NFkB pathway, induced via several members of the TNF receptor family, including RANK, CD40 and LTβR. It is however very likely that additional molecular pathways are critical for the development of TEC progenitors into functional, terminally differentiated mTECs capable of inducing immunological tolerance. Moreover, the exact transcriptional programs governing the mTEC and/or cTEC developmental programs still remain largely elusive. Therefore, one of the primary goals set by our lab is to further elucidate the molecular mechanisms regulating mTEC commitment and development.
2) Delineating what are the specific transcriptional and epigenetic programs controlling the Aire-mediated promiscuous gene expression in the thymus?
Although we and others have recently identified several key factors that physically interact with Aire and participate in Aire-mediated gene expression, our understanding of how and why Aire “wakes up” only some specific genes, while it represses or does not regulate others, still remains rather elusive. Therefore, our group is also deeply interested in identification of additional physiological partners of Aire, which are critical for its function and which have not yet been identified. Moreover, we are also very interested in deciphering of the specific “histone code” that characterizes and determines Aire-responsive and Aire-resistant genes. Finally, we also seek to provide a more comprehensive understanding of what kind of chromatin changes (e.g. histone modifications) are induced by Aire on its target genes; how these modifications are induced and what are the specific chromatin-remodeling complexes recruited to the Aire-responsive loci in order to facilitate their expression.
3) Delineating what are the specific transcriptional and epigenetic programs controlling Aire-independent promiscuous gene expression in the thymus?
Aire was shown to control expression of a significant fraction of the tissue-restricted antigen (TRA) genes in mTECs, however about half of the TRA repertoire is controlled by an Aire-independent mechanism(s). This suggests that additional transcription/epigenetic regulators, “Aire2 factors”, must exist in differentiated mTECs and facilitate the expression of the remaining TRA repertoire. We aim at identification and characterization of these still unknown factors/mechanisms.
4) Identifying what transcription factors regulate the expression of Aire itself?
Although the gene targets of Aire in mTECs are now well characterized, our understanding of the transcriptional programs regulating the expression of Aire itself remains almost completely elusive. There is a striking contrast between the loose, noisy, promiscuous gene expression that Aire promotes and the very tight control of its own expression, which is largely confined to terminally differentiated mTECs, and a few rare stromal cells in the lymph nodes. Therefore, one of the key questions addressed in the lab is “What turns Aire on?”
5) Validating some of our research findings in human patients suffering from organ specific autoimmunity
Our group also aims at validating our cell- and/or animal-based laboratory findings in human patients suffering from various organ-specific autoimmune disorders. This specific goal is addressed in a very close collaboration with Prof. Eystein Husebye (University of Bergen, Norway), who recently spent a sabbatical year in our laboratory.
The lab’s research activities combine the use of classical molecular biology techniques with cellular immunology and work with mouse models. This involves a broad spectrum of technologies and procedures such as: generation and analysis of gene-targeted mice, use organoid and 3D cell cultures, in-vitro gene targeting (siRNA, CRISPR, lentigenics); gene expression analyses (microarrays; RNAseq, TaqMan, etc); DNA & chromatin analyses (deep sequencing, ChIPseq); flow cytometry, imaging techniques (confocal microscopy, RNA FISH); protein-protein interactions analyses (co-IP, mass spectrometry); histological analyses; and various techniques for immunomodulation (bone marrow transfers, transplantations, use and development of immunomodulators) and many more.