The goal of the proposed research is to use a liposome reconstitution system to investigate the interaction of the antigen receptor on Major Histocompatibility Complex (MHC)-restricted T cells with antigen, MHC products, and other molecules involved in activation of these cells.
The specific aims of the proposal are: 1. To incorporate purified, functionally active receptor into liposomes. 2. To probe receptor interactions with other cell surface molecules which may influence T cell activation. 3. To carry out structure and function correlation studies by investigating binding activity of discrete peptide regions of the receptor. 4. To demonstrate and investigate the binding of isolated receptor to its ligand, i.e., antigen/MHC. These objectives will be carried out be incorporating the purified receptor protein into lipid vesicles for fusion to receptor-negative cells. Receptor will be isolated from T cell hybrids which respond to antigen/MHC by producing IL-2. Receptor protein will be isolated by affinity chromatography on Sepharose beads to which an anti-receptor antibody has been coupled. Purified receptor will be combined with lipids and fusogenic agents and the liposomes formed by removal of detergent during dialysis. The receptor-containing liposomes will then be fused to receptor-negative cells and tested for (a) binding to anti-receptor antibodies and (b) functional activity determined by the fused cells' ability to produce IL-2 in response to the antigen/MHC specificity of the transferred receptor. Using the liposome reconstitution system described above, it will be possible to address such questions as (a) Does the T cell receptor interact separately with the antigen and MHC molecule or does it recognize antigen/MHC as one entity? (b) Is the T cell receptor one protein species or is it part of a complex? (c) How is the interaction of the T cell receptor with antigen/MHC affected by other cell surface molecules thought to be involved in activation? The information thus gained on how the T cell receptor interacts on a molecular level with antigen, MHC, and other membrane proteins will be of great importance to our understanding of how the immune system works.
