CD8 T CELLS IN INITIATION OF IDDM

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DESCRIPTION (Adapted from Investigator's abstract): The overall goal of this project is to explore the role of CD8 T-cells in the pathogenesis of IDDM in NOD mice. Based on the diabetogenic qualities of several CD8 T-cell clones recently produced in the investigator's laboratory, the hypothesis being presented is that CD8 T-cells play an important role in early events leading to diabetes. These clones will be investigated to determine their T-cell receptor (TCR) gene usage, the autoantigens and their peptides recognized, their homing in vivo, and mechanisms of activation. Another goal is to generate a TCR transgenic mouse with the TCR from one of the clones. Other clones will be isolated to investigate whether this TCR represents a marker for T-cells that initiate beta cell damage. Mechanisms of activation of CD8 T-cells will be explored using transgenic mice bearing the costimulatory molecules B7-1 and B7-2 expressed in the pancreatic islets. The interaction of pathogenic T-cells with regulatory cells will be studies by crossing the CD8 TCR transgenic mice to RAG knock out mice to look for acceleration of disease in the absence of other modifying lymphocytes, and in collaborative efforts looking at the interaction of these cells with regulatory T-cell clones. The specific aims of the application are (1) to examine the process of activation and homing of islet-specific CD8 T-cells in young NOD mice; (2) to identify the antigen recognized by pathogenic CD8 T-cells; and (3) to study the interaction of CD8 T-cell with CD4 T-cells and antigen-presenting cells (APC) involved in the disease process. Under aim 1, the major goal is to produce a TCR transgenic mouse using TCR a and b chain constructs derived from the CD8 clone, G9. To characterize this TCR transgenic (tg) mouse, experiments will be carried out to determine the proportion of T-cells bearing the tg TCR, whether these T-cells respond to islet cells as antigen, whether the mice develop accelerated diabetes, if anti-TCR antibodies can block disease, and the fate of tg T-cells after transfer to non-transgenic mice. Also to be investigated under this aim are other CD8 T-cell lines and clones to determine whether the TCR on these cells can be used as a marker for disease and whether anti-TCR antibodies can block diabetes; what factors affect the pathogenicity of the CD8 T-cells; the role of costimulatory molecules (B7-1, B7-2) in the activation of CD8 T-cells; and the importance of MHC class I on beta cells in initiation of insulitis. Specific Aim 2 is to identify the antigen(s) recognized by pathogenic CD8 T-cell lines through screening of a beta cell cDNA library. Subsequent experiments would include identification of peptide determinants, investigating the extent of CD8 T-cell specificity for the antigen and whether other pathogenic clones could be propagated with it, and determining how islet-specific the antigen is. Under the third specific aim to study the interactions of CD8 with CD4 T-cells and antigen-presenting cells in the pathogenic process, the chief goal will be to determine the importance of B-cells in IDDM. Experiments will include collaborative studies with Dr. Flavell's laboratory to backcross CD40L knock out (ko) mice on to NOD mice, determination of IDDM incidence in mice without B-cells, and generation of mice with B-cells that can present antigen but cannot secrete antibodies. Another goal of this aim is to examine the effects of regulatory T-cells (specifically an insulin-specific protective clone isolated by the Sherwin laboratory) on the pathogenic CD8 T-cell clones.