This competing renewal investigates the involvement of the protein tyrosine phosphatase family of diabetic autoantigens, phogrin (IA-2 beta) and ICA-512 (IA-2) and their invertebrate homologs as targets of T-cells and antibodies in type 1diabetes in the NOD mouse and human subjects. We recently observed that the phogrin molecule incorporates at least two """"""""super-epitopes"""""""" that are presented to autoreactive T-cells in NOD mice and man by multiple class II molecules that confer susceptibility to T1D. To gain insight into the full range of naturally processed epitopes, studies are proposed to map the epitopes recognized by CD4+, and CD8+ T-cells and diabetic autoantibodies to phogrin and IA-2. The project builds upon our previous studies of structural and cell biology of these molecules and investigations of cell-mediated immunity in the NOD mouse. The emphasis here will be to relate these findings to the disease in humans. Thus we will exploit transgenic mouse models in which human MHC class I and II genes have been introduced to define relevant peptide epitopes and altered peptide ligands and proceed from there to studies with human subjects using resources available at the Barbara Davis Center for Childhood Diabetes.
Four specific aims are proposed:
Aim 1. Analysis of the naturally processed peptide epitopes of phogrin and IA-2-that are presented to T-cells in the NOD mouse in the context of MHC class I (H2-Kd, H2-Db) and class II (I-Ag7), and in vivo analysis of unmodified or modified versions of these peptides as tolerogens.
Aim 2. Analysis of the naturally processed peptide epitopes of phogrin and IA-2-that are presented to T-cells in transgenic mice that carry HLA transgenes that are associated with type 1 diabetes in man namely HLA-DQ8 (DQA 1 *0301, DQB 1*0302), -DR3 (DRB 1*0301), -DR4 (DRB 1*0401 ), and -A2 (A*0201).
Aim 3. Evaluation of spontaneous T-cell responses to such epitopes in prediabetic and new onset type 1 diabetic human subjects.
Aim 4. Structural mapping of autoantibody binding sites in the IA-2 and phogrin molecules, and analysis of the influence of these autoantibodies on processing and presentation of the intact antigens. The definition of potential """"""""super-epitopes"""""""" in IA-2 and phogrin has important implications for antigen-based therapies. The ability to present an autoantigenic peptide simultaneously on multiple HLA molecules overcomes one of theoretical limitation to the use of altered peptide ligands as tolerogens as it reduces the numbers and combinations that may be required to restore tolerance. Further understanding of B-cell epitopes at the structural level will likely improve the predictive power of this key diagnostic tool, and might identify potentially suppressive epitopes that could be targeted therapeutically.
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