Pathogenesis and Prevention of Iddm

McDevitt, Hugh

Abstract

The purpose of this research program is to bring together five investigators in the Departments of Microbiology and Immunology, and Pathology to develop a series of collaborative, research projects. These research projects are designed to apply the techniques of molecular immunology to understanding the development and specificity of the CD4+ T responses to islet cell antigens in type I diabetes in the NOD mouse, in NOD transgenic models expressing human IDDM susceptibility genes, and to develop novel methods of altering the immune responses to prevent IDDM. Drs. Yueh-Hsiu Chien will develop specific T cell staining reagents by producing soluble biotinylated I-A/g7 molecules with a covalently bound peptide coupled to fluorescent streptavidin. The peptide epitopes of glutamic acid decarboxylase and pre pro-insulin will be identified in Dr. McDevitt's laboratory (under the support of DK-51667). Dr. McDevitt will utilize NOD mice transgenic for HLA-DQ8, 7, and 6 to identify the immunodeficient peptide epitopes of human GAD 65 and human PPI presented by IDDM susceptible (DQ8), 7, and 6 to identify the immunodominant peptide epitopes of human GAD 65 and human PPI presented by IDDM susceptible (DQ8) and resistant (DQ7,6) alleles in man. The same T cell receptor staining reagents will be developed for detection of GAD and PPI specific, DQ8 restricted T cells in pre-diabetic and recent onset diabetic patients. In a collaboration between Dr. Chien and Dr. Mark Davis, the polymerase chain reaction (PCR) will be used to characterize and sequence T cell receptor V alpha and V beta sequences from single T cells isolated from the islets of 14-18 day old NOD mice. These T cell receptor V beta and V alpha sequences have detectable shared CDR 3 sequence motifs, suggesting an antigen specific T cell response. T cell receptors from single cells will be expressed in transfected T cell lines and transgenic mice to permit a determination of their function. Transgenic T cells will be tested for their antigenic specificity using a panel of recombinant murine islet cell antigens produced in Dr. McDevitt's laboratory. Dr. Crabtree will produce transgenic mice expressing a mutant cyclophlin able to bind and inhibit calcineurin only when complexed to a modified cyclosporin A. Modified cyclosporin A will be used to inhibit signaling in T cells, to inhibit T cell activation and T cell development, and to induce tolerance to transplanted tissues and islet cell antigens. In collaboration with Dr. McDevitt, Dr. Crabtree will introduce the modified cyclophilin gene into the NOD mouse strain. Modified cyclosporin A will be used to block T cell signaling at various time points following birth to induce tolerance to islet cell antigens. Dr. Weissman and Dr. Judith Shizuru will isolate of hematopoietic stem cells to analyze the effects of whole bone marrow (WBM) versus hematopoietic stem cell (HSC) transplantation in replacing the endogenous lymphoid compartment. H2 matched or syngeneic bone marrow will be used. These experiments will explore the potential for utilizing bone marrow transplantation to prevent the development of type I diabetes in susceptible individuals. Dr. Weissman will also use targeted recombination to analyze the development of the T cell receptor repertoire in NOD mice with endogenous and transplanted bone marrow. These studies will rely on the same techniques used by Drs. Chien and Davis to trace the development of islet cell specific T cells in the NOD mouse.