The long-term objective of the research described in this application is to gain a better understanding of the process of T cell development from the early, uncommitted stem cells in the bone marrow, through the maturation and selection processes that take place within the thymus. The studies proposed in this application focus on the cells in murine bone marrow which are the immediate progenitors of thymocytes and are the cells that are responsible for replenishing of the T cell pool. Initially, the precise developmental status of the cells found in each of several marrow populations that contain pre-T cell activity will be determined. The primary focus of these studies will be on two cell populations that the investigator has recently shown to represent discrete stages of pre-T cell development. Each of these fractions will be characterized as to their pre-T cell activity and their transcription of developmentally critical genes. The extracellular signals that are responsible for driving the subsequent development of the pre-T cell subsets will next be examined, with specific emphasis on the control of TCR-associated gene expression and rearrangement. Here the ability of these cells to be propagated in vitro in IL-3 and MGF without any apparent differentiation or alteration in their developmental potential will be exploited. Specific cytokines and stromal cells will be used to supplement bone marrow cultures and the effects of these agents on pre-T cell development will be assessed by monitoring the transcription of T cell-specific mRNA. Previous work with this system has demonstrated the feasibility of this approach. The extent of any such development will be examined by subsequent phenotypic and functional analyses. Studies to establish experimental systems in which pre-T cells can be genetically manipulated will be initiated.
