One of the goals of investigations of the immunobiology of type 1 diabetes is the design of therapeutic agents that target pathogenic T cells reactive to beta cell antigens. In order to facilitate this approach, more information is needed regarding dominant epitopes of candidate antigens recognized by autoreactive T cells in this disease. Of equal importance is the development of assay systems to monitor antigen-specific T cells responses in subjects undergoing treatment with autoantigen-based immunotherapeutic agents. Unfortunately, analyses utilizing the only practical source of human T cells, i.e. peripheral blood, have proved to be unreliable, primary because of the lack of correlation between T cells responses to candidate antigens and disease progression. Furthermore, the difficulty of obtaining stable antigen-reactive clones or lines from patients has greatly limited attempts to obtain information on the fine specificity of autoreactive T cells. Recently an innovative strategy for the investigation of antigen-specific T cells has been reported which involves construction of tetramers of recombinant MHC-peptide complexes. In this proposal we describe a series of experiments designed to evaluate the potential for utilization of MHC-peptide complexes to identify populations of T cells specific to epitopes of defined beta cell antigens and restricted to high risk class II MHC alleles. The preliminary results presented here indicate that covalent complexes of IA/g7-insulin B:9-23 can be recognized by T cells derived from NOD mice, a murine model of type 1 diabetes. In other investigations we have derived T cells specific to insulin B:9-23 and islet IA-2 from DQ8 transgenic mice. These reagents provide a means to develop and use DQ8-peptide complexes to detect autoreactive T cells in patients with type 1 diabetes.
The Specific Aims of this proposal are: 1) To define epitopes for the T cell response to insulin B:9-23 and islet IA-2 in immunized DQ8 transgenic mice and compare these epitopes to those recognized by disease-associated T cells in MOD mice; 2) To construct covalent DQ8-peptide complexes and tetrameters to strain antigen-specific T cell hybridomas T cell hybridomas derived from immunized DQ8-peptide tetramers to detect and enrich antigen-specific T cells hybridomas derived from immunized DQ8 transgenic mice; 3) To construct tetramers of IA/g7-B:9-23 and IA/g7-TT:830-843 to characterize the distribution of diabetes-relevant autoreactive and exogenous antigen-stimulated T cells in NOD mice; 4) To use DQ8- peptide tetramers to detect and enrich antigen-specific T cells from immunized DQ8 transgenic mice and to analyze these cells for specificity in bulk and limiting-dilution cultures; and (5) To use the information gained from the preceding Aims to design and use MHC-peptide complexes to detect, isolate, and characterize autoreactive T cells in the peripheral blood of patients with type 1 diabetes.
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