Dr. Hutton plans to identify antigens recognized by 20 different islet antigen-reactive T-cell clones that have been obtained from NOD mice. Some of these clones can adoptively transfer disease into prediabetic NOD mice or NOD.scid animals. The method he plans to use, termed TEASEL (T-cell epitope analysis using subtracted expression libraries) has previously been successfully used for identification of the epitope recognized by an islet antigen reactive T-cell from a patient with IDDM (imogen 38) and a similar approach has been used to identify another molecule recognized by autoantibodies in some patients with IDDM termed phogrin (phosphatase homologue of granules). In brief, the procedure involves preparation of a subtracted library of cDNA (e.g., bTC3 and aTC2), construction of cDNA libraries for screening in T-cell proliferation assays, and expression and purification of recombinant proteins. There are 5 specific aims. In the first, he will refine the TEASEL method to enhance its sensitivity and specificity. This will include: 1) use of normal tissue rather than cell lines as sources of cDNA, 2) use of tissue from the same species, strain, and MHC haplotype as the T-cell clones (NOD), 3) use of alternative restriction sites in the initial fragmentation of the cDNA, 4) Development of libraries based on longer cDNA fragments (this will reduce the complexity of the libraries), 5) perform additional rounds of subtraction with abundantly expressed cDNAs, and 6) perform additional rounds of subtraction with cDNAs from non-pancreatic tissues. Briefly, subtractive hybridization will be carried out of libraries prepared from the beta cell tumor lines as well as normal tissues. CDNA libraries will be used for screening in T-cell proliferation assays. The subtracted cDNA libraries will be sampled in triplicate batches of 100,000 clones. Limiting dilution analyses will be carried out to isolate reactive clones. The inclusion bodies from bacteria will be prepared as described previously, and the recombinant antigens will be added to the proliferation assays at concentrations of 10-100 ug/ml. This assay may be modified by use of latex beads to immobilize antigen and enhance the sensitivity of the proliferative response. This overall method will then be applied to mapping epitopes of CD4 T-cell clones from the NOD mouse. These clones will be obtained from Dr. Kathryn Haskins and Dr. Dale Wegmann. The clones have been isolated from the spleens and lymph nodes of spontaneously diabetic NOD mice. The phenotypes of the cells and their requirements for transfer of diabetes vary. The molecular cloning and expression of the cDNAs from which these T-cell epitopes are derived from an islet cDNA library will then be carried out, and the tissue specificity and subcellular localization of the antigen will be done. In instances where the cDNA is relatively short, overlapping 20 aa peptides will be synthesized and used to map the epitope recognized by the T-cell clone. Full length cloning and expression of the cDNA which incorporate the NOD T-cell epitope sequence will be done using cDNA's from other investigators or generated by PCR using reverse transcribed mRNA or islet cDNA library from other investigators. A number of vector systems which are currently in use will be used to express the full-length protein. Finally, the pathologic significance of the antigen will be tested by determining the responses of CD4 cells from NOD mice to these antigens, determining the serum titer and isotype of antibodies to the cloned antigens, establishment of CD4 cell lines from spontaneously diabetic NOD mice, and testing these lines for their ability to transfer diabetes.
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