In neonatal rodents, the beta-cell mass undergoes a phase of remodeling that includes a wave of apoptosis. Using both mathematical modeling and histochemical detection methods, we have demonstrated that beta-cell apoptosis is significantly increased in neonates as compared with adult rats, peaking at approximately 2 weeks of age. Other tissues, including the kidney and nervous system, also exhibit neonatal waves of apoptosis, suggesting that this is a normal developmental phenomenon. We have demonstrated that increased neonatal beta-cell apoptosis is also present in animal models of autoimmune diabetes, including both the BB rat and NOD mouse. Traditionally, apoptosis has been considered a process that does not induce an immune response. However, recent studies indicate that apoptotic cells can do the following: 1) display autoreactive antigen in their surface blebs; 2) preferentially activate dendritic cells capable of priming tissue-specific cytotoxic T-cells; and 3) induce the formation of autoantibodies. These findings suggest that in some circumstances physiological apoptosis may, in fact, initiate autoimmunity. Initiation of beta-cell-directed autoimmunity in murine models appears to be fixed at approximately 15 days of age, even when diabetes onset is dramatically accelerated. Taken together, these observations have led us to hypothesize that the neonatal wave of beta-cell apoptosis is a trigger for beta-cell-directed autoimmunity.

Type 1 diabetes is an autoimmune disorder characterized by the selective destruction of pancreatic β cells, resulting in insulin deficiency and hyperglycemia. Studies of both humans (1) and the NOD mouse model (2, 3) have shown that β cell destruction is mediated largely by T lymphocytes. Despite the direct β cell cytotoxic role of T cells during diabetes progression, prediction of disease in both humans and the NOD mouse has been based primarily on the presence of circulating autoantibodies to putative T cell antigens (4, 5). Detection and characterization of antigen-specific T cells from peripheral blood during the progression of type 1 diabetes, or other autoimmune diseases, has thus far not been possible without in vitro manipulation. Consequently, little is known regarding the evolution of autoreactive T cell populations during the natural history of autoimmune diseases.Although the detection of antigen-specific T cells using MHC tetramers has permitted careful characterization of T cell responses to many viral and bacterial infections (6, 7), attempts to elucidate the in vivo dynamics of autoreactive T cells during the progression of autoimmune diseases have been less successful. With a single exception (8), attempts to visualize autoreactive T cells in peripheral blood or lymphoid organs ex vivo with MHC tetramers bearing naturally-occurring ligands have failed (9-14), possibly because of the lower avidity that T cells have for self versus foreign peptide/MHC ligands (15, 16). To overcome this limitation, we have engineered high-avidity peptide/MHC tetramers capable of stably interacting with low-avidity autoreactive T cells, thus facilitating their detection. The visualization of lowavidity T cell populations in peripheral blood would provide a simple, minimally invasive method for assessing the presence of autoreactive T cells within infiltrated tissues and might therefore be useful for predicting the development of autoimmune disease.The feasibility of employing a high-avidity MHC tetramer to detect relatively low-avidity autoreactive T cells was investigated using NOD mice, an extensively studied model of human type 1 diabetes (3,17). In these mice, diabetes develops by 16 to 20 weeks of age in approximately 80% of females and is preceded by a prolonged period of mononuclear cell inflammation of the Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic β cells by T lymphocytes. Prediction of cell-mediated autoimmune diseases by direct detection of autoreactive T cells in peripheral blood has proved elusive, in part because of their low frequency and reduced avidity for peptide MHC ligands. We demonstrate here that MHC class I tetramers complexed to a high-avidity analogue of an immunodominant β cell epitope detect diabetogenic CD8 + T cells in the peripheral blood of NOD mice ex vivo and that the quantification of this autoreactive T cell population in peripheral blood is a powerful predictor of autoimmune diabetes.This article was published online in advan...

Autoimmune (type 1) diabetes results from a loss of β cells that is mediated by self-reactive T cells. Previous studies have shown that a single injection of CFA prevents diabetes in nonobese diabetic (NOD) mice, but the mechanism(s) of protection remain unknown. We show here that NOD mice immunized with CFA have a markedly reduced incidence of diabetes and that this reduced incidence is associated with a decrease in the number of β cell-specific, autoreactive CTL. In addition, the adoptive transfer of diabetes into syngeneic NOD/SCID recipients was prevented by CFA immunization, and the protective effects of CFA were lost when cells expressing the NK cell marker, asialo GM1, were removed from both donor cells and recipient mice. Returning a population of CD3−DX5+ cells to the adoptive transfer restored the protective effects of CFA. Therefore, NK cells mediate the protective effects of CFA possibly through the down-regulation of autoreactive CTL and stimulation of NK cells represents a novel approach to the prevention of autoimmune diabetes.

CTLs are important mediators of pancreatic β cell destruction in the nonobese diabetic mouse model of type 1 diabetes. Cross-presentation of Ag is one means of priming CTLs. The death of Ag-bearing cells has been implicated in facilitating this mode of priming. The role of β cell death in facilitating the onset of spontaneous autoimmune diabetes is unknown. Here, we used an adoptive transfer system to determine the time course of islet-derived Ag presentation to naive β cell-specific CD8 T cells in nonobese diabetic mice and to test the hypothesis that β cell death enhances the presentation of β cell autoantigen. We have determined that β cell death enhances autoantigen presentation. Priming of diabetogenic CD8 T cells in the pancreatic lymph nodes was negligible before 4 wk, progressively increased until 8 wk of age, and was not influenced by gender. Administration of multiple low doses of the β cell toxin streptozotocin augmented in situ β cell apoptosis and accelerated the onset and magnitude of autoantigen presentation to naive CD8 T cells. Increasing doses of streptozotocin resulted in both increased pancreatic β cell death and significantly enhanced T cell priming. These results indicate that in situ β cell death facilitates autoantigen-specific CD8 T cell priming and can contribute to both the initiation and the ongoing amplification of an autoimmune response.