In order to avoid autoimmune disease, T cells that recognize self- components must be destroyed. Autoreactive T cells can be eliminated in the thymus by the process of negative selection. I have been studying the regulation of negative selection in a transgenic mouse model in which all of the T cells and thymocytes express the same T cell receptor for antigen (TCR). By using an in vitro culture system, I have found that, like mature T cell activation, negative selection requires delivery of two signals to a thymocyte. One signal is delivered through the TCR and the other is delivered through a ligand that is expressed on antigen presenting cells (APC). Preliminary results indicate that Thy1 may be involved in delivery of the TCR-dependent signal, but the costimulatory signal remains unidentified. Thy1 is a glycoprotein that is linked to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor. It is abundantly expressed on thymocytes, T cells, and brain tissue. Both Thy1 and other GPI-linked proteins have been shown to be involved in mature T cell activation, but their function in the thymus and brain is unknown. Herein, I have proposed to follow two lines of investigation into the signaling requirements for negative selection of autoreactive thymocytes. First, I will identify the costimulatory signal involved in thymocyte deletion by examining whether antibodies against the costimuli known to play a role in mature T cell activation also affect negative selection. Failing to identify a known costimulus, I will use well-defined APC populations and the in vitro culture system in concert to make and screen for an antibody against the ligand on APC that costimulates negative selection. Second, I will investigate the role of Thy1 and other GPI-linked proteins in negative selection and identify the ligand for Thy1. These experiments will be performed by first assessing the effect of antibodies against these proteins on negative selection in both the in vitro culture system and in fetal thymic organ cultures. Next, the ability of thymocytes from mice which lack Thyl to undergo negative selection will be addressed. Finally, ligands for Thy1 will be identified by the use of a soluble Thy1 -Ig reagent. The success of the proposed experiments will be aided by the following: l) culture systems which are already set up, 2) antibodies to known costimuli Thy1, and GPI-linked proteins which have already been obtained, 3) the gift of the mice lacking Thy1, and 4) collaboration with local experts for isolating Thy1 ligands. Results from these experiments are important for two reasons. First, they will further our understanding of how the immune system regulates the growth of autoreactive T cells. Second, by identifying ligands for Thy1, these experiments will probe a cell-cell interaction which may be involved in both the nervous and immune systems.
