The Role of Resident Stem Cells, Extracellular Matrix and Transcriptional Networks in Thymic Biology

The thymus is the main site of T cell production and attends to the establishment of central tolerance. Despite its critical role in the immune system, it undergoes progressive atrophy with age that results in chronically decreased T-cell output. This poses a great risk to immunocompromised patients who may be subjected to chemotherapy regimes, infected with immunotropic viruses or challenged with a new vaccine. Additionally, the reduced immune plasticity found in the elderly may contribute to graft rejection and to an increased risk of developing autoimmune diseases. Proposed clinical approaches to solving this entail cytokine- or growth factor- induced stimulation, androgen suppression, or the administration of hormones. More focussed methodologies aiming at reactivating resident stem cells or creating de novo thymic tissue are currently lacking. Cellular and molecular events pertaining thymus organogenesis and the onset of age-induced involution are yet to be fully elucidated and remain crucial to the advancement of these protocols. Determining the identity of endogenous adult thymic stem cells and the role played by native extracellular matrix represent the next challenges towards achieving efficient in vivo transplantation, thymus rejuvenation, and controlled in vitro culture of thymic epithelial cells. Foxn1, a thymic master regulator, has been extensively studied due to its critical role in thymus development. Although numerous transgenic cell lines and mouse models have been created for this gene, none could combine faithful replication of its physiological expression with the ability of selecting Foxn1-expressing live cells. Here we developed a Foxn1eGFP knock-in mouse that achieves both these aims. We have then utilised this to identify a putative adult progenitor within the cortical compartment of thymic epithelium, characterized by a low level of MHCII and high expression of a6 integrin and Sca-1. Additionally, we employed these cells to establish a robust 3D culture method for ex vivo purified TECs whilst demonstrating their dependence on Bmp4 supplementation to sustain their Foxn1 expression and self-renewal. In order to reinstate their lost differentiation capacity, Bmp4-supplemented cultured were further optimized in a serum-free medium with the introduction of decellularized thymic matrix comprising of native protein components. This enabled TECs to give rise to fully differentiated MHCIIhi cells and, when used as a scaffold for in vivo grafting, this facilitated the formation of a complete thymic microenvironment able to foster the production of naïve T cells that reached the periphery in nude mice. By sorting cell subsets from the developing pharynx and thymus, we investigated the transcriptional networks involved in thymus organogenesis. Novel differentially expressed genes and miRNAs were identified as putative markers, such as Krt15, that may aid in defining the adult stem cell phenotype whilst elucidating additional factors involved in thymic senescence. Taken together, these data further advance our understanding of basic processes involved in thymic biology, from the role of secreted extracellular components to key factors crucial for the maintenance of thymic epithelial identity. These were found intertwined in pathways that drive thymus organogenesis and subsequent decline from its origin in the pharyngeal endoderm to its involution with age.