Methods for Producing T lymphocytes in vitro from Stem Cells

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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, rheumatoid 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 and differentiate. In vitro models of T cell development rely on accessory factors derived from supporting stromal cells of which Notch-1 ligands are the best understood. However Notch-1 ligand is insufficient to allow T cell development in the absence of accessory cells indicating additional factors produced by the stromal cells are required. We have identified the chemokine CXCL12 as a co-factor in the process of beta-selection and this has enabled us to create an accessory cell free culture system which is permissive for beta-selection. We now propose to examine the effect of additional stromal derived components such as laminin and Wnt for their effect in a cell-free system. We also propose to test whether our insight into the development of thymocytes will permit the growth of T cells from stem cells without the need for stromal accessory cells. This will provide a simpler system to evaluate the molecular processes of differentiation. Furthermore, it may permit the expansion of T cells for cell-based therapy in the area of regenerative medicine.