Characterisation of SHIP-1 as a regulator of T lymphocyte function

PI3Ks have been shown to have many important functions in T cells, including in cell growth and survival, chemotaxis and differentiation; however, the role of SHIP-1 as a negative regulator of PI3K signalling has not been so thoroughly investigated. The use of knockout mouse models has given an insight into the role of SHIP-1 in murine T cells, but these are compromised by loss of function during development which impinges upon mature T cell function. In addition, some work has been undertaken to investigate the role of SHIP-1 in human leukaemic T cell lines, but it is not clear how these findings might translate to SHIP1’ s function in normal cells. Therefore the work in this thesis was undertaken to investigate the role of SHIP-1 in primary human T lymphocytes. Initial experiments demonstrated that SHIP-1 was active both in unstimulated cells and in response to a variety of stimuli. Using a lentiviral method to deliver shRNA against SHIP-1 and a constitutively active form of SHIP-1 it was found that whilst constitutive activation of SHIP-1 led to cell death, silencing of SHIP-1 expression resulted in viable cells which failed to proliferate. Furthermore, silencing of SHIP-1 revealed its crucial role in the regulation of basal PI3K signalling, actin polymerisation, cell morphology and basal motility. Interestingly, there was no additional defect in chemotaxis, in agreement with findings that SHIP-1 and TAPP PH domain probes did not localise to specific regions of the cell membrane during chemotaxis. In addition, silencing of SHIP-1 caused an abnormal profile of cytokine production and in particular, CD4+ T cells were skewed towards becoming Tregs. The observations in this study suggest that whilst the role of SHIP-1 in primary human T cells has some similarities to its role in both murine T cells and leukaemic cell lines, there are also significant differences which it will be crucial to take into account when designing drugs to target SHIP-1. The final results section comprises work completed for Novartis, the sponsor of this PhD. In this section it was found that knockout of PI3Kγ in mice resulted in elevated production of IL17A and IL-17F along with abberant expression of IL-17 receptors and loss of PI3K signalling in response to IL-17A. These findings are of particular relevance for PI3Kγ inhibitors that are in clinical trials for inflammation and autoimmune diseases.EThOS - Electronic Theses Online ServiceGBUnited Kingdo