On the pathway from hematopoietic stem cell to functional T-lymphocyte traffic of T-lymphocyte progenitors through the thymic microenvironment appear to be an obligatory step. This study proposed to investigate the nature and consequences of the interactions between the lymphocyte progenitor population and the non-lymphoid cells of the thymus. The capacity of the non-lymphoid cells derived in culture from the mouse thymus, for sustaining and directing the proliferation and differentiation of T-lymphocyte progenitors will be confirmed in vivo by evaluating the functional reconstitution of immunodeficient mice following grafting with such cultured non-lymphoid cells. Primary cultures of heterogenous thymic non-lymphoid cells (TNLC) will be characterized using morphological and functional techniques directed at the detection and enumeration of epithelial cells and macrophages. The individual cell types comprising the heterogenous TNLC will be isolated by cloning from enriched monolayers. Pure populations thus established will be maintained in vitro and their functional capacity assessed in vivo. Thus, adult thymectomized, """"""""lethally"""""""" irradiated, hematopoietically reconstituted mice will be grafted with either heterogenous or purified populations of TNLC. The reconstitution of cell mediated function in grafted mice will be assessed by the survival of allogeneic skin grafts, and by evaluating their peripheral lymphoid populations for T-cell function using the in vitro assays of mitogen and MLR responsiveness as well as an in vivo assay for GVH reactivity. Using in vitro techniques of organ culture and co-culture, as well as in vivo diffusion chambers, the nature of the progenitor-microenvironmental cell interaction (i.e., contact versus humoral) will be determined. Preliminary studies of homing, kinetics and differentiation of function in the progenitor population will be pursued. The role of the thymic microenvironment in tolerance induction will be assessed by using allogeneic combinations of TNLC and reconstituting hematopoietic cells in the in vivo model. The demonstration by Zinkernagel et al (85) that the non-lymphoid cells of the thymus play an important role in the differentiation of anti-self-H2 specification of T-cells adds impetus to the project.
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