| Popis: |
Allergic and asthmatic disease is highly prevalent in the UK, however current treatments such as steroids, although effective in many individuals, only relieve disease symptoms, and do not provide long-lasting relief. Allergen immunotherapy can provide long-term alleviation of disease symptoms however is only effective in a proportion of patients, carries significant risk of adverse side effects and needs to be given over a prolonged period of time, often several years, for maximal efficacy. Allergic disease is associated not only with a type 2 adaptive immune response, but also impairment of regulatory T cell function. Allergen immunotherapy is associated with skewing of allergen-specific Th2 responses towards an IL-10 phenotype, suggesting that plasticity of this T cell lineage occurs in vivo. Work from our lab has shown that steroids such as dexamethasone, although non-specific in action, augment IL-10 synthesis by CD4+ T cells, a response that is enhanced by vitamin D. The major focus of this thesis was to assess whether established human CD4+ Th2 cell lines could be deviated towards an IL-10+ profile by dexamethasone and 1α25-dihydroxyvitamin D3, and whether these agents might therefore represent appropriate adjuvants to boost the antigen-specific effects of immunotherapy. Combined drug treatment of established Th2 cell lines increased Foxp3+ expression although these cells were not inhibitory in an in vitro assay of suppression. In contrast Th2 cells co-cultured with dexamethasone, 1α25-dihydroxyvitamin D3 and IL-10 over a 2-week period deviated towards an IL-10+ phenotype as assessed by qPCR and flow cytometry. Analysis of TCR-Vβ receptor usage suggested this did not represent clonal outgrowth. These cells exhibited strong suppression of Th2 cells in vitro, although this was unexpectedly not reversed by addition of neutralizing antibodies to IL-10 or TGFβ to the cell culture. Analysis of several genes previously identified to be upregulated in freshly isolated CD4+ T cells cultured with dexamethasone and 1a25-dihydroxyvitamin D3 did not reveal comparable expression by Th2-deviated IL-10+ T cells. A transcriptional gene expression array was therefore performed in order to search for biomarkers of these deviated cells and clues as to suppressive mechanisms by which they inhibit Th2 cells proliferation. Genes identified to be of interest included PDCD1LG2, BTLA and several granzymes. Granzyme expression was subsequently validated by qPCR. Severe asthma is associated not only with Th2, but also Th17 cells, therefore the capacity of 1α25-dihydroxyvitamin D3 and dexamethasone to deviate Th17-associated cytokine production was also assessed. Dexamethasone failed, and indeed could enhance IL-17A synthesis in cultures of CD4+ T cells, which may contribute to severe steroid refractory asthma. 1α25-dihydroxyvitamin D3 inhibited IL-17A synthesis in a glucocorticoid-independent manner. This work demonstrates that calcitriol and dexamethasone can be used in vitro to manipulate T cell plasticity to skew effector phenotypes towards more regulatory phenotypes. This has the potential to tailor and further develop therapeutic allergen specific regimens in vivo. |
