Regulatory T cells (Tregs) have been implicated in the pathogenesis of many human diseases from classical autoimmune diseases to cancer, and their role in inducing tolerance in transplantation and at the foetal-maternal interface is increasingly recognised. Tregs can express a huge variety of surface markers giving rise to phenotypically diverse subsets. This thesis therefore aimed to carry out a phenotypic and functional analysis of subsets of Tregs in healthy donors and in the context of pregnancy. In the first experimental chapter, our key hypothesis was that Treg subsets with differing surface phenotypes would display differing functional properties. Flow cytometric analysis was carried out in 10 healthy donors using both manual gating and unbiased clustering methods. Using manual gating, we identified several previously reported Tregs subsets, while unbiased analyses enabled us to identify novel populations. We then simultaneously compared the function of several Treg subsets in healthy donors using an
in vitro Treg suppression assay optimised for small cell numbers. Results from this assay showed that naïve Tregs expressing CD31 were significantly more suppressive than bulk Tregs and memory Treg subsets. We also found preliminary evidence suggesting an enhanced ability of these cells to suppress IL-2 production. The second experimental chapter then further investigated the ex vivo expansion properties of this subset. We found that naïve CD31+ Tregs had an equal expansion potential to bulk, memory, or naïve Tregs. As analysed by flow cytometry, this subset also had stable FoxP3 expression throughout expansion, and expressed significantly higher Helios than all the other expanded Tregs at 28 days of expansion. A large proportion of naïve Tregs co-expressed CD45RA and CD95 at the end of 6 weeks of expansion involving strong TCR stimulation. Therefore, in the following chapter we hypothesised that Tregs with stem cell-like memory potential could exist in the Treg compartment. As analysed by a 16-marker flow cytometry panel, we observed FoxP3+Helios+ Tregs with a stem cell-like memory phenotype. However, we saw no conclusive evidence of stem cell-like memory function in these Tregs after expanding the cells in culture. In the final experimental chapter, we investigated the phenotype and prevalence of Treg subsets in healthy pregnancy compared to pregnant women with pre-eclampsia and gestational diabetes mellitus (GDM) stratified for 3 different treatment interventions. Through manual gating and unbiased clustering analyses we found that Tregs from GDM and pre-eclampsia mothers were skewed towards a memory phenotype, expressing surface markers such as CD45R0, CCR4, and CD95. Additionally, all GDM groups had a significantly reduced frequency of CXCR5+ Tregs, and unbiased Treg clusters which were significantly more abundant in healthy pregnant women compared to GDM groups and pre-eclampsia were predominantly of a naïve CD73+ phenotype. Finally, we found that the
in vitro suppressive capacity of bulk Tregs from GDM and pre-eclampsia mothers was significantly reduced compared to healthy pregnant controls and an inverse correlation between CD45R0 expression and suppression was found. Collectively data from this thesis show that the surface phenotype of Tregs dictates the functional properties of Treg subsets and that the balance of frequencies of Treg subsets may differ in immune pathologies.
A functional investigation of human regulatory T cell subsets in health and pregnancy related diseases
Harris, F. (Author). 1 Sept 2023
Student thesis: Doctoral Thesis › Doctor of Philosophy