A series of peptides that have similar abilities to activate thymocytes, but very different abilities to positively and negatively select thymocytes will be tested for their ability to induce TCR-CD8 interaction by FRET microscopy. Positive selecting ligands may have a slower rate of induction of this interaction than negative selecting ligands, but not necessarily a weaker response. Signaling will be compared between these ligands using a FRET Erk reporter construct, classical western blot techniques, and intracellular staining for phospho-proteins. Binding kinetics for MHCp to T cell derived membrane preparations will be measured to define the contribution of TCR-MHCp and CD8-MHC interactions in the different binding phases. A membrane-proximal region of the TCR ?-chain (?-CPM) is required for positive but not negative selection. The limits of positive and negative selection for thymocytes bearing ?-CPM mutant TCRs will be tested to see if they recognize negative selecting ligands of the same potency as wild type TCR, and if they can positively select with higher affinity ligands. The ability of ?-CPM TCRs to bind MHCp of varying avidities will be compared, and FRET microscopy will be used to quantify the likely defect in CD8-TCR interaction. Recruitment of signaling molecules to the immune synapse of ?-CPM mutant T cells will be imaged. T cells expressing different components of the TCR-CD3 complex as fluorescent chimeras will be made to investigate inter-subunit distances in the TCR, and how or if these are altered during antigen recognition. This strategy will allow investigation of potential clustering of TCRs on the cell surface in the resting state and during MHCp recognition. These strategies will allow investigation of potential conformation changes either within or between TCRs during antigen recognition. The TCR is crucial for recognizing virus-infected cells and cancer cells. Understanding its function, and how T cells develop in the thymus is of primary importance to controlling infectious disease, cancer and autoimmunity.
