The thymus generates from immature precursors, named thymocytes, functional T cells, key players of the adaptive immunity. The thymus is also in charge of preventing autoimmune diseases via negative selection of developing thymocytes, operated principally by medullary thymic epithelial cells (mTECs). mTECs express tissue restricted antigens (TRAs) to preview the peripheral self to maturing thymocytes. Thymocytes that strongly bind for those antigens will undergo clonal deletion or diversion into the regulatory T cell lineage, thereby enforcing central tolerance. Although Aire gene has been implicated as a major regulator of TRAs presentation, recent work has refocused attention on the presence of medullary "mimetic cells" in mouse. Single-cell RNA-sequencing analysis uncovered transcriptomic equivalents in medullary subpopulations, including ciliated, myoid, and neuroendocrine mTECs, suggesting the possibility of compartmentalised and non-random TRAs presentation. These "diverse cells" all depend on the expression of lineage-defining transcription factors specific for each of their subtypes e.g., FoxI for ionocytes, Insm1 for neuroendocrine cells and MyoG for skeletal muscle medullary cells. While all this work has been performed in mice, the coordinated provision of TRAs by 'mimetic cells' has important consequences for our understanding of autoimmune syndromes in humans. In the clinic most autoimmune diseases are still treated with immune suppressing drugs rather than targeting specific molecular defects. 'Mimetic cells' may play a key role in altering the tolerogenic microenvironment that leads to loss of tolerance towards a coherent set of antigens, resulting in specific patterns of autoimmunity. We recently reported that thymus contains epithelial stem cells with pleiotropic multilineage potency that give rise to several cell types that were not previously considered to have shared origin such as cornified Hassall's-Bodies, ionocytes, myoid and neuroendocrine cells. The latter derive from a common ASCL1-expressing medullary progenitor. ASCL1 function in transcription and regulation of the chromatin landscape might be critical for differentiation and function of neuroendocrine cells in the thymus. In this project, we will (i) dissect the molecular mechanisms of ASCL1 and related TFs in driving neuroendocrine fate by determining its chromatin binding profiles and interactor partners; (ii) perform loss and gain of function studies in human primary TEC cultures and generate Ascl1 knockout and overexpressing mouse models to gain insight on the function of thymic neuroendocrine cells. Finally, (iii) we aim at understanding their role in shaping the medullary tolerogenic microenvironment. In collaboration with clinical specialists, we will characterise medullary compartments in the context of autoimmune diseases such Myastenia Gravis, and syndromic inborn errors of immunity such as deletion syndrome (22q11.2DS) that are frequently associated with autoimmune manifestations. Of note, altered differentiation of thymic medullary epithelial progenitors may be responsible of autoimmune phenotypes observed in most of thymic epithelial tumours (i.e., thymoma and thymic carcinoma). Thymic tumours are highly heterogeneous and their molecular profiles are poorly characterised. In conclusion, the acquired knowledge on human thymic epithelial cells and the molecular mechanisms regulating medullary cell subtypes will be essential to improve diagnosis and develop targeted therapies for some of the severe autoimmune and neoplastic conditions related to thymic dysfunction.