Induced Thymic Epithelial Cells (iTEC) as a tool for repairing adaptive immunity

Abstract

The thymus is the primary lymphoid organ required for T cell development. Here, haematopoietic progenitor cells undergo T cell lineage commitment, and the subsequent differentiation, selection and maturation processes required to generate a fully functional diverse and self-tolerant T cell repertoire. Cortical and medullary thymic epithelial cells (cTECs and mTECs) are the key elements of the thymus required to mediate this process, termed thymopoiesis. Several primary immunodeficiencies (PID) are associated with aberrant thymus development or function and some of these can be treated by neonatal or fetal thymus transplantation. Donor demand however outweighs tissue availability, generating a need for an alternative source of thymic epithelial cells (TECs), that can be generated or expanded in vitro. In 2014, the Blackburn lab developed a direct reprogramming protocol, in which induced expression of the transcription factor FOXN1 converts mouse embryonic fibroblasts (MEFs) into induced TECs (iTEC). When transplantated with immature thymocytes and fetal thymic mesenchyme (FTM) in reaggregate thymic organ cultures (RTOC), iTEC were able to form an organized and functional thymus. However, the initial study characterised only some aspects of the iTEC system; additionally, for unknown reasons, the system exhibited variability of outcome. This thesis aimed to test whether iTEC, used as the basis of thymic organoids, can instate an adaptive immune system in thymus-dependent PID patients. Specifically, it aimed to: (i) improve the iTEC reprogramming protocol for generating functional TEC for transplantation, (ii) determine the minimal cellular requirements of the grafted iTEC-based RTOC system and (iii) test the ability of iTEC grafts to repopulate athymic recipient mice with a diverse T cell repertoire. The work described herein provides an optimised reprogramming and reaggregation protocol resulting in improved iTEC function. Using this protocol, the minimal cellular requirements required for the production of iTEC-RTOC grafts are established. It is shown that FTM is beneficial but not essential for patterning of iTEC-RTOC grafts into cortical and medullary compartments and for support of thymopoiesis, providing insight into the molecular basis of FTM function in iTEC- RTOC. It further shows that iTEC-RTOC grafts can recruit host lymphoid progenitors and support their development into all major T cell subsets, while the presence of donor thymocytes within iTEC-RTOC results in improved graft histology. Lastly, through in-depth characterisation of T cells generated from iTEC grafts it is demonstrated that this improved iTEC system can repopulate the immune system of athymic mice with a diverse TCR repertoire. These data substantially extend previous findings and provide further support for development of iTEC as a tool for repairing the adaptive immune system in PID patients.