Methods for Producing T lymphocytes in vitro from Stem Cells

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.

We will identify factors that may be important for the generation of T lymphocytes from HSC. We will test the effects of recombinant Wnt proteins following addition to the culture on the proliferation and differentiation of DN3 cells. We will examine the role of laminin-5, which signals via the integrin alpha6beta4. The combination of rDL4 and CXCL12 defined minimal requirements for a stromal cell-free in vitro system that promoted thymocyte differentiation but this was less efficient than the OP9-DL1 stromal cell line. This inefficiency may be due to the suboptimal presentation of the Notch ligand or the CXCL12 which is normally associated with extracellular matirix. To improve the efficiency of Notch-ligand presentation we will examine a protein in which the extracellular domain of DL4 is fused to the Fc-portion of human IgG1. By using an anti-human IgG to capture the recombinant protein, this system effectively immobilises the DL4 so that it can effectively activate Notch-1. We will immobilise CXCL12 on extracellular matrices such as the heparan sulphate, fibronectin or laminin to increase its local concentration. We propose to expand this in vitro method and develop a culture system for the development of DP T cells from the HSC stage. To achieve this we plan to incorporate our own findings with methods published by Irwin Bernstein's group who has shown differentiation of HSCs to the DN2/DN3 stage by combining immobilised Notch-ligand with cytokines. Our experiments would utllize murine HSCs. It will be important to show functionality of the T cells generated in our accessory cell-free system thus cultured T cells will be transferred into recipient mice using the marker CD45.1 to distinguish donor from host. Initially these will be autologous (MHC identical) transplants, subsequently we will perform allogeneic transplants which will allow the ability of the transferred T cells to become tolerant in a MHC-mismatched host.

The outputs of this project will be high quality basic research that will develop a novel method for growing T cells in vitro. This will provide a novel system for understanding the signals that regulate T cell development and activation and is necessary for the development of new strategies for manipulating T cell growth therapeutically. Age-related degeneration of the immune system may be countered by T cell replacement facilitated by the development of novel methods to expand T cells. Thus this work is relevant to the area of regenerative medicine and the personalization of medicine. The work will impact our understanding of mechanisms of cellular expansion and differentiation which are key aspects of the regenerative medicine agenda. Moreover, the work will address issues of cell production in vitro which is a fast-moving area of significant commercial interest. Manipulating stem cells is currently a priority area for the biotechnology and pharmaceutical industry. This sector, working together with the community of academic and clinical scientists represents the major beneficiaries for the results generated by this research. This project will provide training for a post-doctoral scientist in state of the art methods for the culture of mammalian stem cells and lymphocytes which are not in routine use in the community. At the conclusion of this research project we will have established a robust method for the in vitro generation of DP thymocytes from a HSC source. We anticipate that this project will yield sufficient new data for a publication in an academic journal and contribute to the strengthening of our patent position. Certainly, we will present the results at national and international conferences. We would like to extend our research into developing a method for the development of fully mature T lymphocytes in an accessory cell-free in vitro culture system. The Babraham Institute through BBT Ltd. (the Institute's wholly owned trading company) has secured IP related to the project (UK patent application 0911184.0): 'In vitro methods for the preparation of differentiated haematopoietic cells and uses thereof'. The commercial exploitation of the findings relating to this project will be undertaken with a view of generating commercial return and as a mean to increase the uptake and distribution of the technology. It is anticipated that the commercialisation route would be through licensing or a co-development program with an industrial partner best placed in the stem cell / regenerative medicine market. The work proposed has relevance to the 3R's agenda in that by harnessing the proliferative potential of stem cells fewer animals may be required for a given project. Moreover, in vitro methods that can reproduce in vivo processes present rational alternatives to live animal research.