The long term objective of our research program 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, leading to the development of immunocompetent T cells. The studies proposed here focus on the cells (pre-T cells) in murine bone marrow which are the immediate progenitors of thymocytes and are the cells which are ultimately responsible for replenishing the T cell pool. The data available to date suggest that pre-T cells are phenotypically heterogeneous, and that this heterogeneity may reflect unique developmental stages of pre-T cells within the bone marrow. Here we propose to characterize the various populations of pre-T cells in terms of their surface phenotype, their ability to respond to IL-3 and MGF in vitro and their pattern(s) of thymic colonization. The developmental relationship between the identifiable pre-T cell types will be examined to determine potential precursor-progeny relationships and to determine whether the various pre-T cell types are biased in their ability to give rise to CD4+ or CD8+ T cells, or T cells which express alpha, beta vs gamma, delta T cell receptor types. The major goal of this proposal is to conduct a detailed study of the sequential maturation of pre-T cells in vitro, and we will exploit the ability of pre-T cells to grow in IL-3 and/or MGF for this purpose. PCR will be used to detect the transcription of T cell-specific mRNA species in the various pre-T cell populations, either before or after culture in IL-3 and/or MGF under conditions which maintain the progenitor status of the pre-T cells. Once this has been accomplished, specific cytokines and stromal cells of bone marrow and thymic origin will be tested for their ability to induce the transcription of T cell-specific mRNA by pre-T cells, which will indicate the initial stages of T cell development. The extent of that development will be examined by subsequent phenotypic and functional analyses. The production of long term pre-T cell lines and clones, which may be used in parallel with the pre-T cells from primary cultures in the above studies, will also be undertaken during the course of the proposed work. These studies represent what we believe to be the next logical steps in defining the very earliest events in T cell development and, as such, may have a direct impact on the development of therapeutic approaches to T cell immunodeficiencies such as acquired immunodeficiency syndrome (AIDS).
