T cell recognition of peptide/MHC complexes is a critical part of virtually all specific immune responses whether appropriate or inappropriate (autoimmune). This type of recognition is mediated by the T cell receptor for antigen, a heterodimeric surface structure resembling immunoglobulin. The focus of this application continues to be the biochemical nature of this interaction and in what ways it is similar and in what ways it is distinct from antibodies binding to their respective antigens. Consequently, one of our first priorities is to follow up our initial characterization of TCR affinity for a single peptide/MHC complex and extend this to include both affinity and kinetic information for a variety of other TCRs and ligands as well. In this way, we will be able to assess the useful range of parameters employed by TCR's and more efficiently compare them with other ligand receptor-pairs. We will also study the kinetic parameters of antagonist/differential signaling peptides/MHC complexes, using both the cytochrome c system and the hemoglobin system. This information may help to choose between the various models proposed for this phenomenon. We have also utilized Circular Dichroism to study TCR binding to peptide/MHC complexes and find evidence suggesting that a conformational change may occur in the TCR. We are interested in defining this phenomenon further and particularly in identifying the residues in the TCR which are responsible for the CD shift as this. would provide clues to the nature of this effect. An altered ability to induce conformational changes in TCRs has also been suggested as a possible mechanism of antagonism and we have begun to survey such complexes.
A third aim i nvolves following up our observation that a chemically cross-linked peptide/MHC dimer of heterodimers can specifically stimulate T cells. If general, this suggests that TCR dimers (induced by MHC dimers) may be sufficient for T cell activation. We will also continue to provide soluble TCRs and MHCs to Dr. Bjorkman's group to aid in crystallization efforts, particularly focused on obtaining a TCR/Ag/MHC co-crystal structure. In addition, we will explore 2D protein crystallization approaches as well. With respect to thymic differentiation we will continue our efforts to identify the natural peptide(s) ligand for a TCR in the thymus, utilizing an antibody raised against MCC/Ek complexes which blocks positive selection of an MCC/Ek specific TCR. Additionally, we have developed an 'MHC tetramer' reagent which can label T cells in an antigen specific manner. This will allow us to track specific T cell populations in the thymus and to address some of the major unresolved issues in thymic differentiation such as the stage at which negative selection is first evident and whether the decision to down regulate CD4 or CD8 is highly regulated or stochastic.
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