The overall aim of this application is to study the developmental potential of T cell progenitor populations in the bone marrow and thymus and to characterize the thymic microenvironment. A key tool in these analyses will be a novel long-term thymocyte culture system in which a thymic stromal cell line, theta-35, supports the growth of immature CD4-CD8-CD3/TCR- triple negative (TN) thymocytes with T and B cell developmental potential. In view of preliminary data showing that theta-35 stromal cells can support the growth of bone marrow derived lymphoid cells, Aim 1 proposes to use clonal analysis to determine whether bone marrow contains T cell restricted progenitors or whether such progenitor activity derives from multipotential progenitors that can also generate other lineages. Single bone marrow cells highly enriched in thymus repopulating activity will be isolated by flow cytometry and seeded over theta-35 stroma. Progeny derived from the single precursors will then be tested for their capacity to differentiate into T, B, natural killer, dendritic cells and myeloid cells.
In Aim 2, a similar clonal strategy will be used to define whether the most immature intrathymic population, which has been shown to differentiate into B, natural killer and dendritic cells in addition to T lymphocytes, is multipotential or contains lineage restricted progenitors. Although thymopoiesis is sustained by bone marrow emigrants during adult life, upon arrival in that organ they differentiate into TN and must proliferate extensively to generate 100 million or more thymocytes. In order to define the replicative potential of subpopulations of TN cells, in Aim 3 the different CD44 and/or CD25 expressing TN subpopulations will be isolated and their self renewal/proliferative potential measured following seeding onto theta-35 stroma. Finally, the goal of Aim 4 is to identify thymic stromal cells that can support the maturation of TN thymocytes. TN cells, harvested from long term thymocyte cultures, will be seeded onto each of the 76 additional stromal cell lines available in the laboratory and their maturation will be evaluated at various times thereafter. Taken together, these studies will establish lineage relationships during early T cell development and hematopoiesis and further characterize the thymic microenvironment.
