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

The thymus is a primary lymphatic organ where T-cell progenitors travel to from the bone marrow and undergo commitment, differentiation and maturation into fully functional T-lymphocytes. Cells responsible for this process are highly specialised cells called cortical thymic epithelial cells (cTEC) and medullary thymic epithelial cells (mTEC). Positive selection (selecting T-cells that can interact with MHC) and T-cell receptor gene rearrangement is primarily mediated by epithelial cells of the thymic cortex (cTECs). Whereas negative selection, a process whereby auto-reactive T-cells are removed from the repertoire produced, is primarily mediated by epithelial cells of the thymic medulla (mTECs). Interestingly so, the thymus involutes with age and is replaced with fat tissue which contributes to making our immune systems weaker with age.

Many primary immunodeficiencies (PID) occur from problems in development or function of the thymus. One such PID is DiGeorge syndrome (DGS) in which patients partially or wholly lack thymus function. It is widely known that such diseases can be treated with transplantation of neonatal human thymus tissue, however, the availability of this is limited and generates a need for an alternative source of thymic tissue.

It has recently been shown that induced expression of the forkhead transcription factor FOXN1 in mouse embryonic fibroblasts (MEFs) can convert these cells into thymic epithelial cells (TEC) referred to from here out as induced Thymic Epithelial Cells (iTEC). These iTECs can support in vitro T-cell development and when transplanted into mice create a functional thymus. By creating iTECs on large scale it could potentially be very important for treating patients with DGS as well as boosting the immune system in many immunocompromised individuals such as people suffering from AIDS, bone-marrow transplant recipients and elderly patients.
The aim of the project overall will be to examine iTEC as a tool for repairing adaptive immunity. Specifically, transplantation of iTEC-derived thymi will be examined to see if they can repair the immune defect in athymic nude mice (animal model for DGS/ spontaneous deletion in FOXN1 gene), by investigating the quality and functionality of the T-cell repertoires produced, and the stability and longevity of iTEC-derived thymic transplants. A series of grafts will be performed under the kidney capsule in syngeneic mice and thymocyte development, thymic architecture and phenotype will be inspected at different timepoints (2 and 3 weeks post transplantation) with FACS and immunohistochemistry. Following this, the longevity of the grafts in syngeneic mice will also be established by recovering the grafts at different timepoints over the course of several months. The phenotype and functionality of the T-cell repertoire produced by the iTEC-derived thymic transplants will also be examined. The above experiment will then be repeated but grafting will take place in nude athymic mice and their capacity to reject skin grafts and respond to vaccines will be tested and autoimmunity monitored. T-cell receptor (TCR) sequencing may also be performed to examine the diversity of the T-cell repertoire produced. Grafting under the skin will also be investigated to reduce invasiveness of the procedure and establish a vastly improved recovery period and decreased chance of post-surgical infections.

In addition to this, genome editing will be used in conjunction with iTECs to test whether gene corrected mouse and/or human DGS iPS cells (TBX1 mutants- major candidate for DGS) can generate functional iTEC upon transplantation.