The goal of this project is to characterize those elements within the thymus that are involved in inducing the maturation and selecting the repertoire of thymocytes. We have focused on the role of T cell receptor (TCR) positive cells and their factors in thymic development. We will employ mice in which the recombinase activating gene (RAG) has been inactivated (RAG-/- mice). Lymphocytes from these animals are unable to rearrange and express TCRs and fail to enter the CD4/CD8 developmental pathway. To investigate the role of TCR+ cells in thymocyte development, we will assess the ability of an exogenous source of intrathymic AKR Thy1.1+ TCR+ cells to promote the differentiation of RAG-/- Thy1.2+ thymocytes. We have created AKR->RAG-/- bone marrow chimeras, and are using flow cytometric analysis to determine if the RAG-/- cells have been induced to express CD4 and/or CD8. Preliminary studies indicate that RAG-/- thymocytes can be induced to enter the CD4/CD8 pathway in the presence of TCR+ cells. To further evaluate the nature of cells able to induce T cell development, we will examine the ability of different T cell subset (CD4+, CD8+ TCRgd+ cells) to induce maturation of RAG-/- thymocytes. Further, because the ability of TCR+ cell to induce thymocyte development may be via an indirect effect on the thymic epithelium, we will use immunohistologic methods to evaluate maturation of thymic epithelium in reconstituted RAG-/- mice.. Finally, we plan to investigate the cytokines that may be involved in the inductive process. Since it difficult to maintain high levels of cytokines in vivo by injection, we plan to use an in vitro culture system in which thymi from fetal mice can be maintained in media containing specific cytokines and subsequently analyzed by immunofluorescence and flow cytometry after culture . Results from these studies will be important to understating the signals involved in the early maturational stages of T cell development.
