The studies conducted above were used to develop a suitable method by which to characterise Treg both functionally and phenotypically. Although there is still much debate surrounding a truly reliable human Treg phenotype it was decided that CD4+CD25+FOXP3+ cells were to be used in all experiments on the basis that these were commonly used markers within the literature. Due to the high running costs of the project, Treg were isolated via Dynal magnetic beads. This was compared to MACS but this was found to be too expensive to use on a regular basis. Critical to the success of the project was the expansion of these cells to larger populations than those obtained on isolation from whole blood. After comparing the cost/purity benefits of different stimuli used in expansion it was decided that an CDβκ superagonist would be used alongside high concentrations of IL-2. This allowed breakage of the anergic phenotype of Treg and thus provided suitably pure and large populations of cells with which downstream experiments could be conducted. The expansion of Treg is limited to 42 days as continued stimulation led to a down-regulation of key Treg markers as well as a loss of suppressive activity.
Treg demonstrated dose dependent suppression of both CD4+CD25- and CD8+ T cells. This provides the basis for a functional tool with which to alter the suppressive effects of Treg pharmacologically. Interestingly, the cytokine milieu generated by Treg suppression can be substantially altered, depending on the stimuli added. Mitogenic stimulation leads to the production of IFN- and IL-10 from Treg whereas TCR engagement with CDγ/ CDβκ does not. This could be attributed to the altered proliferative pathways that exist within Treg. It also
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indicates that induction of certain molecular pathways, in particular the NF- B pathway may be of interest in altering Treg function.
After the establishment of Treg suppression, it became apparent that the less Treg present within the environment, the greater the proliferation of the effector population. This observation, together with those found in literature suggests that strategies to either manipulate Treg function or decrease the number of Treg within the tumour microenvironment will be of clinical benefit (82). The following chapters of this thesis will seek to alter the suppressive and migratory capacity of Treg as well as enhance the generation of a tumour antigen-specific immune response.
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