A Unique Insulin Derived Epitope Contributes to Enhanced Pathogenic Activity of the Autoreactive Diabetogenic CD8 T-Cell Clonotype AI4 in NOD Mice

Type 1 diabetes (T1D) develops due to T cell mediated autoimmune destruction of pancreatic beta cells. Several CD8+ T cell clones important for the initiation of disease have been isolated from prediabetic islets of NOD mice. One of these is the beta cell autoreactive AI4 CD8 T cell clone. In addition to its role in driving the very earliest stages of development, this T cell clone is capable of adoptively transferring disease independently of CD4+ T cell help. An important question is the relative importance of AI4 T cells compared to other pathogenic CD8 T cell clones that contribute to diabetes. Thus, we set out to assess the hierarchy of AI4 T cell pathogenesis in relation to T cell clones recognizing two documented beta cell autoantigens, proinsulin 2 (PI) and IGRP. Chimeric mice were generated by combining BM from NOD mice transgenically expressing, the AI4 TCR with BM from mice transgenically overexpressing in APCs PI (NOD.PI) or IGRP (NOD.EA.IGRP) to determine if systemic tolerance to either insulin or IGRP can prevent AI4 driven T1D development. Compared to NOD syngenic BM control or NOD.EA.IGRP BM recipients, NOD.PI BM recipients revealed a reduction in AI4 mediated T1D and fewer AI4 T cells in the spleen and thymus of the mixed chimeras. This would suggest the ability of AI4 T cells to induce T1D in NOD mice is upstream of the autoreactive T cell response to IGRP. Additional in vitro proliferation studies determined that the reduction in AI4 T cell numbers in mixed BM chimeras may in fact be the result of negative selection driven by a novel transgenically expressed insulin epitope presented by intrathymic APCs. Subsequent analyses by our collaborators determined the AI4 TCR has the unique ability to recognize a 6mer insulin 2 epitope in the context of H2-Db. Furthermore, the TCR appears to focus recognition on the epitope with minimal interaction with the class I molecule. Of the multiple beta cell autoantigenic epitopes the AI4 T cell clone has been shown to recognize, this epitope is likely the most important to induce beta cell cytotoxicity. In addition to the discovery of a novel insulin epitope, these results further support the hypothesis that the promiscuity of TCR antigen recognition may be an important factor in determining the pathogenicity of autoreactive CD8 T cells contributing to T1D development