The thymus provides the environment for the development and selection of T cells. Thymic epithelial cells coordinate virtually all stages of T cell development, providing key cytokines, chemokines and ligands for the commitment, survival and proliferation of thymocytes and also for positive and negative selection of T cell precursors. Work from partner 1 showed that the embryonic thymus is colonize by two consecutive waves of thymic seeding progenitors. The first hematopoietic cells that colonize the thymus display a unique capacity to generate lymphoid tissue inducer cells and invariant gd T cells that together are the first thymocytes to interact with thymic epithelial cells. The second wave produces conventional ab and gd expressing T cells. The first wave of thymic progenitors are of exclusive embryonic origin and induce the differentiation of thymic epithelial progenitors into cortical and medullary epithelial cells as well as their subsequent maturation, which is required for the induction of central tolerance. This project aims to understand the molecular and cellular events that regulate the generation of the first thymic lymphoid cells, their impact in the establishment and maintenance of peripheral tolerance and their involvement in thymic involution. In this project we propose to: (1) Identify the cell-intrinsic molecular mechanisms that regulate the developmental potential of thymic seeding progenitors giving rise to the first invariant T cells and thymic lymphoid tissue inducer cells. For this, we will perform a transcriptomic and epigenomic analysis of the progenitors of thymic seeding cells from both waves, (2) Characterize the role of thymic seeding progenitors in the maturation of medullary thymic epithelial cells and in the development of thymic architecture. This will be carried out using fetal thymic organ cultures of alymphoid (Ragc-/-) thymic lobes colonized with thymic seeding progenitors and (3) Investigate the impact of a deficiency of the first wave thymic seeding progenitors in tolerance induction, autoimmunity and thymic involution. This will be done by reconstituting mice with progenitors that can only generate the second wave of thymic seeding progenitors or by depleting the first wave. This project will lead to a better understanding all conditions of immune deficiencies arising from a compromised thymic function, including the effects of chemotherapy and irradiation in pathological situations of cancer and bone marrow transplantation or during aging. Moreover, since gd T cells are important for the integrity of the alveolar epithelia, understanding the origin and developmental pathways leading to the production of these cells will contribute to cell therapy approaches for the treatment of airway viral infections such as influenza or different types of coronavirus. In this project we will investigate the first interactions between thymocytes and epithelial cells, that are likely to modulate all epithelial compartments for life and thus impact on involution that occurs with ageing or in pathologic conditions. Altogether these experiments will provide new insights in thymic epithelia development and involution and could reveal strategies to maintain or restore thymic function in aging and disease.