PI3K signalling and T lymphocyte development

T lymphocytes, so-called because they develop in the Thymus, are a type of white blood cell crucial for the function of the immune system. Aberrant function of these cells is associated with immunodeficiency (e.g. AIDS) or autoimmunity (e.g. type I diabetes, rheumatioid arthritis). T lymphocytes are derived from blood stem cells and complete their maturation in the thymus, an organ located near the heart that has evolved specifically to provide an environment that promotes T cell development. The developmental stages haematopoietic stem cells pass through as they mature into T lymphocytes have been relatively well-characterised. This has allowed the identification of key regulatory checkpoints that cells must pass through in order to develop further. One of these checkpoints is called beta-selection. In order to pass through this checkpoint cells must generate specific signals which bring about changes in gene expression and allows the cells to divide. The beta-selection signal is also necessary for cells to survive. We have identified some of the genes which are responsible for generating the beta-selection signal. These genes which are called phosphatidylinositol-3- kinases are potential drug targets which are being actively investigated by many biotechnology and pharmaceutical companies. Our project proposal is to pursue experiments which will increase our understanding of how signalling by phosphatidylinositol-3- kinases is regulated by cell surface receptors and how this is integrated with upstream and downstream processes. It is reasonable to assert that general principles learned from study of early T lympchocyte differentiation may be reiterated at subsequent developmental stages. Thus, we believe our work on early T cell development may be relevant to processes important for the activation of mature T lymphocytes in health and disease.

We have provided evidence that the gamma and delta isoforms of PI3K play an important role in T cell development at the beta-selection step where they regulate growth and proliferation. We show data that demonstrates the P101 protein is required in T cell development. As P101 is the adapter that links P110gamma to G-protein coupled receptors (GPCR) this suggests that a GPCR may be involved in beta-selection. We propose to test for the physical association of CXCR4 with the pre-T cell receptor (pre-TCR) by applying a combination of biochemistry, cell biology and imaging technologies to pre T cell tumour lines and to untransformed cells expanded from haematopoietic progenitor cells by culture on OP9-DL1 cells. We will use a specific inhibitor of CXCR4 called AMD3100 to test the requirement for signalling by this receptor in the context of preTCR signalling. We have also shown data in this proposal that demonstrates the input of activated Ras is required for the proper function of P110gamma in the context of beta-selection. We will extend this finding to determine if PI3K required for Ras mediated signalling in beta-selection. Furthermore, we will also the ability of PI3K effectors to rescue the phenotype of p110gamma/delta double mutant thymocytes. These studies should provide mechanistic insights to complement our genetic studies. Further mechanistic insights should be gained by measuring signalling and gene expression in mutant thymocytes sorted as pure populations by flow cytometry.