This proposal continues investigations on antigen presentation events in NOD diabetogenesis. It is based on recent progress that led to the identification of insulin (INS) as a major autoantigen. We identified different sets of anti-INS T cells, and in particular, one set that bypassed thymic control and which was a component of diabetogenesis. Furthermore, we identified the segment of the INS molecule recognized by such T cells and provided a cellular and biochemical explanation for its presentation in islets. The set of anti-INS T cells that escape thymus clonal deletion recognize a peptide segment of the B chain and are reactive only with peptides from the B chain found in secretory granules as byproducts of INS catabolism. Our goal is to study these T cells, which all evidence points to having a role in initiating the diabetogenic process. We propose examining their early interaction with APC in islets; and to determine the consequence of this early interaction, in part using new T cell receptor (TCR) transgenic mice directed to INS peptides. One issue is the role of the pancreatic lymph node (pLN) and the effects it has on the subsequent response of islets. Importantly, we plan an evaluation of the response of the islets to the entrance of INS reactive T cells, a process which may have consequences for the subsequent stage of diabetogenesis. The examination of the anti-INS T cells will be carried out in two complementary aims: aim 1 examines the transgenic mice for early diabetogenic events, the entrance of the T cells into islets, the requirement for pLN, and the response of the islets. The early events are examined in regular TCR transgenic mice as well as in the same mice lacking lymph nodes (nodeless). Aim 2 examines these events by transferring the T cells, a procedure that will give better control of the processes involved. Here we probe whether the anti-INS T cells need prior activation in the pLN. In parallel to the use of the INS transgenic mice, we will be examining regular NOD mice in aim 3 in which the two previous aims are placed in the context of normal diabetogenesis. The ultimate goal is to unravel the initiating events in NOD autoimmunity. PUBLIC HEALTH RELEVANCE: We study experimental autoimmune diabetes in a mouse model very much akin to type 1 diabetes, an important human disease. Examining some of the fundamental steps in the process should allow us to better understand the human counterpart and start developing rational ways to control the disease.