Thymic epithelial cells : post-natal maintenance, regeneration and autoimmune-associated abnormalities

Normal thymocyte development, and therefore good health, depends on an intact, functional thymic stromal microenvironment. The thymic niche consists of a 

series of heterogenous, spatially distinct epithelial compartments through which thymocytes at particular stages of development must progress in order to continue 

maturation. Of the stroma, thymic epithelial cells (TEC) interact most closely with thymocytes, and play a primary role in editing the T cell repertoire and establishing self-tolerance. Accordingly, TEC defects and abnormalities are often associated with autoimmune disease, and yet the study of TEC and other stromal cells is still very limited, largely due to technical difficulties in working with this 

relatively rare, heterogenous, sensitive population of cells.

This thesis aimed, firstly, to develop a more stringent method of thymic stromal cell analysis. We show that previous methods, relying on sequential enrichment of 

stromal cells from pooled thymi, were selectively isolating medullary cell types.  Cortex was also lost in early digestion fractions, leading to incorrect assumptions 

about the relative composition of the thymus. A new method is described, using single thymi and a non-enriching digestion protocol, which permits analysis with 

extremely low variability between mice. It is possible to identify small variations between groups, with full statistical analysis as a routine part of each experiment.  This method permits routine and rigorous thymic stromal analysis by flow cytometry, which was previously not possible.

Using these methods, we identified a population of medullary TEC (mTEC) which upregulate MHC class II during thymic regeneration in vivo to restore the 

Aire-expressing mTEC-hi subset; cells intrinsically involved in negative selection of autoreactive thymocytes. These cells expressed an antigen identified by the antibody MTS24, previously found to enrich for high-efficiency thymic epithelial stem cells in the embryo, and this antigen was lost upon differentiation. Further work is required to definitively identify thymic epithelial stem cells (TESC) 

believed to reside within this population, including BrdU pulse-chase experiments to find and fully phenotype the label retaining population, followed by molecular 

analysis for genes important to maintenance of known epithelial stem cells from other tissues. Postnatal TEC sensitivity to isolation is such that these cells lose the 

ability to later reaggregate, for unknown reasons, and therefore direct differentiation studies in vivo are difficult to perform, but would be required for ultimate demonstration of stem cell ability. We also defined the regeneration 

kinetics, in vivo, of Aire-expressing mTEC, important for restoration of tolerance processes, and identified the lymphotoxin signalling pathway as an important 

regulator of chemokine and tissue-restricted antigen expression (TRA) - also vital to tolerance induction - in mTEC.

Dysregulation of the thymic epithelial network is a hallmark in many models of spontaneous autoimmunity. Despite being the subject of decades of study, the aetiology and functional significance of these thymic defects have proven difficult to unravel. Using flow cytometry and q-PCR of sorted thymic subsets, we have shown that the New Zealand Black mouse strain, which develops spontaneous, 

systemic autoimmunity, possesses thymic abnormalities more complex than previously appreciated. Their TEC - particularly mTEC - did not expand normally, and they exhibited a strong deficiency in Aire. They also showed 

evidence for sluggish NF- kB2 signalling in the mature, tolerance-inducing mTEC subset, and chemokine abnormalities, which may contribute to thymocyte arrest in 

the medulla. Taken together, we show evidence for several defects that are strongly linked to autoimmune susceptibility. Whether these defects develop 

independently or downstream of a central defect remains to be determined, but experiments such as bone marrow chimeras and stromal transplants may allow further characterisation of any separate origins. We examined thymi from the non-obese diabetic (NOD) Iddll congenic strain, which is resistant to development of type I diabetes, and compared them to wildtype NOD mice. We found that the Iddll allele did not contribute to development of the thymic defect, and that the defect was indistinguishable from NOD despite reduced disease.  As a secondary focus of this thesis, we examined the effect of sex steroid ablation (SSA) on immune regeneration in states of immunosenescence and autoimmunity.  We firstly showed that SSA confers the same immune benefits to female mice as it does to males, which suggests women undergoing severe immunodepletion may also benefit from reversible SSA therapy.  We also examined the effect of SSA on the thymi of aged NOD mice, identifying several severe, unreported defects in early T cell development.  Although SSA increased thymus size compared to sham-ovariectomised controls, it was unable to reverse these striking abnormalities.  Transfer of ETP and TN cells from young NOD mice, as well as differentiation assays using the aged bone marrow progenitor cells will be required to determine if the defect lies in T lineage commitment, or entry to the thymus, or whether the stroma is perhaps incapable of supporting early T developmental events.

Lastly, we were interested in testing the function of recovering peripheral T cells, since significant dysfunction has been reported in T cells created by ex-thymic 

expansion, rather than thymic-dependent, naive T cell generation. Although SSA demonstrably increases naive T cell output in aged or immunodeficient animals, their function in disease models has not been tested. We show that castration improves the immune response against influenza, by increasing numbers of cytotoxic T lymphocytes (CTL), and that the age-related defect is due to a numerical, rather than functional deficiency.

While many questions remain to be answered, this work opens several new avenues of investigation and deepens our understanding of the development and 

maintenance of thymic epithelial cells, as well as identifying new targets, both stromal and haemopoietic, for further study and possible therapeutic intervention 

in states of autoimmunity. The development of new methods for thymic stromal analysis provides an invaluable tool for the further study of this important cell 

type, whose function is so strongly tied to human health.

This thesis was scanned from the print manuscript for digital preservation and is copyright the author.

Author requested conversion to open access 27 Oct 2022