Phenotypic and functional characterisation of adult thymic epithelial progenitors in the mouse

Our immune system is an effective line of defence against foreign, infectious material and also functions to eradicate transformed cells. T lymphocytes (T cells) are a vital component of the adaptive arm of the immune system that develop and mature in the thymus. The thymus is broadly compartmentalised into an outer cortex and inner medulla and is populated by developing T cells (thymocytes) together with thymic stromal cells (TSC) that include cells of mesenchymal, neural, myeloid and epithelial lineage; with the latter being the predominant cell type within the stromal compartment. Thymic epithelial cells (TEC) provide molecular cues that promote thymocyte maturation and in turn, receive essential signals from thymocytes that encourage their differentiation. It is in this specialised thymic microenvironment that stringent selection processes occur, to ensure that a functional yet self-tolerant repertoire of mature T cells exit into the periphery. Immune senescence occurs as we age and one of the most evident signs of this is the gradual contraction of the thymus organ, known as age-associated (chronic) thymic involution. The loss of functional thymus tissue in particular, the epithelial compartment, is a main feature and leads to an abnormal microenvironment unable to support proper thymocyte development. Other extrinsic factors may also exacerbate thymic involution, such as conditioning regimes (cytoreductive treatments) prior to hematopoietic stem cell transplants, chemotherapy and malnutrition. Ultimately these factors contribute to the overall demise of immune function, resulting in a diminished response to invading pathogens, increased incidence of autoimmunity and inadequate surveillance of malignant cells. Several strategies to regenerate peripheral T cells show promising results, however there is only a transient immune recovery with no current therapy available that rescues sustained TEC function. One approach would be to activate endogenous repair programmes to rejuvenate the involuted or damaged thymus. Recent studies have elucidated the presence of thymic epithelial progenitor cells (TEPC) mainly in the fetal thymus however the identity of adult TEPC remains enigmatic. In this project, we sought to identify and characterise potential adult TEPC candidates. We revealed that phenotypically immature cortical TEC (cTEC) contain hallmark features of epithelial SC. We initially optimised an enzymatic digestion protocol to enable complete dissociation of adult thymus tissue which released the maximum number of TEC for downstream phenotypic and functional analyses. TEC subsets were then extensively assessed for the expression of epithelial SC-associated markers. Interestingly, a subset of cTEC expressing low (lo) levels of major histocompatibility complex II (MHC II) (cTEClo) was found to be enriched with SC-associated cell surface antigens and gene transcripts. Label retention studies revealed that cTEClo were relatively quiescent and that label retaining cells were largely localised to the cortico-medullary junction; an anatomical location in which bipotent TEPC are presumed to reside. More importantly, cTEClo showed bipotent and multilineage potential when purified and reaggregated with supporting fetal thymic stroma for subsequent ectopic engraftment. Adult cTEClo could form epithelial networks and differentiate into medullary TEC (mTEC); including the mature mTEC lineage that expresses the autoimmune regulator (Aire) transcription factor. Using a novel 3-dimensional (3D) epithelial culture assay, we further demonstrate exclusive and enriched colony forming potential within adult cTEClo. Colonies could be serially passaged whilst retaining thymic identity in vitro and when returned back to in vivo reaggregate grafts. Collectively, these data identify, for the first time, adult TEPC function within cTEClo and a possible differentiation series, in which cortical progenitors generate the medullary lineages in a stepwise manner. The findings and assays developed here may help streamline future TEPC research, in order to harness their potential as therapeutic targets for thymus regeneration.