Type I Diabetes (T1D) or juvenile diabetes, is an early onset autoimmune disease that culminates in the targeting and destruction of ? cells found within the pancreatic islets of Langerhans. Little is known about how autoreactive lymphocytes escape negative selection in the thymus, resulting in the breakdown in central tolerance. Understanding the mechanisms of autoimmune T cell selection and development is crucial for identifying the underlying issues in the breakdown in central tolerance and may be applicable in establishing peripheral tolerance to islet antigen specific autoimmune T cells. Due to the low clonal frequency of autoreactive T cells in the thymus and periphery, studies into underlying mechanisms in self-reactive T cell selection and survival have been challenging. To bypass this issue, T cell receptor (TCR) transgenic models have been utilized, however only a limited number of islet antigen specific TCR transgenic models have been described [1-4]. Therefore, there has been limited study into the mechanisms of autoreactive TCR selection in the thymus, and most of the knowledge we have about the role of negative selection in limiting self-reactivity is based on studies done with model antigens, not with TCRs obtained during an autoimmune response. To address this issue, we have composed a panel of ten insulin specific TCRs with variable reactivity to insulin, the majority of which have been isolated from the islets of spontaneously diabetic non-obese diabetic (NOD) mice. Insulin has been shown to be a major antigen in development of autoimmune diabetes on the NOD background, and antibodies to insulin are used as predictive biomarkers in human disease. In this proposal we plan to study the development of these T cell clones in the thymus through the use of a new approach for the rapid generation of TCR transgenic mice, called retrogenic mice, using retroviral-mediated stem cells gene transfer and novel 'self-cleaving'2A peptide-linked multicistronic retroviral vectors that express both TCR chains in a single vector [5-14]. This will allow us to quickly generate a large number of TCR retrogenic mice with ten different TCR's to study the fate of a single TCR during positive and negative selection. We will be able to elucidate the role of thymically expressed insulin in central tolerance by utilizing mice with reduced thymic expression of insulin and mice with mutated insulin TCR contact residue. In addition, we want to establish whether insulin reactive TCR's can be deleted in the thymus using DEC-205 insulin and insulin mimetope fusion peptide antibodies. Finally, we propose to express insulin in an inducible manner, under the Aire promoter, in an effort to establish central tolerance. We hypothesize that our unique panel of insulin reactive TCRs can be used to define the threshold for positive and negative selection of autoreactive insulin specific thymocytes.
To date, there has been limited study into the mechanisms of autoreactive TCR selection and escape in the thymus, eventually leading to a breakdown in central tolerance. Studies proposed herein will address the role of thymically expressed insulin on downstream TCR signaling pathways and its effects on thymocyte development which may lead to new insights about the peripheral state of self-reactive T cells and lead to novel and optimal approaches in establishing central and peripheral tolerance to insulin and diabetes prevention.