The overall goal of this proposal is to understand how diabetogenic T cell receptors (TCRs) recognize the MHCII and hybrid peptide complexes. Peptide ligands of CD4 and CD8 T cells are conventionally derived from contiguous fragments of the parental proteins by the endocytic pathway and proteasome. We first suggested insulin (Ins) B:9-23 and WE14, a natural cleavage product of chromogranin A (ChgA), peptides may be spliced with other beta cell granule proteins and form neo-antigens. We also demonstrated that fusion peptides are the true ligands for many diabetogenic T cells. Both C-terminal modification of the Ins B:9-23 peptide and N- terminal additions to the WE14 peptide of ChgA can create superagonists for T cells. We proposed peptide fusion by transpeptidation as a means of creating the required modifications to the peptides and explain how the T cells driving autoimmunity escape negative selection in the thymus but find their antigen in the target tissue. Our structural and biophysical data showed that the EALYLV portion of Ins B:9-23 and WXRM(D/E) portion of WE14 may be common acceptors of these hybrid peptides. We determined the structures of two different types of mouse TCRs and a human TCR, bound to their optimal versions of the MHCII-Ins peptide ligands. All the Ins reactive TCRs engage the N-terminal portion (EALYL) of Ins B:9-23, indicating similarities in how these ligands are formed in both human and mouse. The structures showed that the specificity differences among mouse Type A and Type B T cells lies in how they interact with the amino acid at p8 (B:21) of the Ins peptide. The structures also show how the peptide modifications were essential to the formation of the complexes, bolstering a role for modification of the peptide in vivo to initiate the CD4 T cell response in T1D. We hypothesize that EALYLV and WXRM(D/E) are the acceptors of the fusion peptides and form the common diabetogenic TCR docking spots, in addition to being novel targets for T1D immunotherapy. Previously, we have shown aromatic amino acids play important role in TCR cross-reactivity. The aromatic amino acids (Y and W) may be the common epitopes for diabetogenic TCRs and critical to shape the broadness of hybrid peptide reactive T cell repertories. Whereas, the donor peptides of hybrid peptides contribute to diversity the T cell repertoire to both Ins and ChgA autoantigens. We will study the following specific aims: Determine how diabetogenic CD4 TCRs recognize IAg7 and ChgA WE14 hybrid peptides; Define the nature of the human Ins B:9-23 hybrid peptides presented to the diabetogenic CD4 TCRs; Test monoclonal antibodies (mAbs) specific for the acceptor portions of the hybrid peptides bound to MHC in the development of T1D. These data may guide therapeutic strategies towards specific modulation of TCR activation, which could be the basis for a novel therapeutic approach in treatment of T1D. Research Strategy Page 2
Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. Increasing evidence suggests the hybrid peptides derived from pancreatic proteins, such as insulin and chromogranin A, are potential epitopes for diabetogenic T cells. This project investigates how hybrid peptides are presented to diabetogenic T cells, and become autoantigens, triggering autoimmune diabetes. The results would provide a molecular basis to for development of novel therapeutic strategies to prevent or treat this disease. Research Strategy Page 3
