Role of Regulatory T Cells in the Prevention of Diabetes by Insulin in NOD Mice

Abstract

The use of insulin therapy to prevent development of type 1 diabetes in nondiabetic relatives of individuals with diabetes has been difficult. We demonstrate that in animal models, it was necessary to use high dose insulin to prevent diabetes. To mimic the conditions of the Diabetes Prevention Trial, DPT-1, we administered insulin without adjuvants by daily subcutaneous injections to young adult prediabetic mice. To confirm that diabetes was prevented (rather than just delayed) treatment was continued for 70 to 80 weeks and glucoses were monitored weekly. We evaluated T cell function and phenotype using adoptive transfer, and regulatory T cell staining by flow cytometry. High dose insulin therapy provided significant protection from diabetes (diabetes free survival at age 72 wks of 56% versus 17 % survival in the untreated control group (p =0.00034,). The number of pathogenic T cells were reduced but not completed eliminated by long-term insulin preventative therapy. In contrast, there was a significant increase in CD4 + FoxP3 + T cells with insulin therapy (7.6% + 0.4 SEM) compared to prediabetic mice (4.4% + 0.2) or overtly diabetic mice (3.6% + 0.2, P<0.0001). In untreated control mice, 15-20% of the mice did not develop disease. Analysis of these naturally-protected mice showed that they also had an increase in CD4 + FoxP3 + Tregs. Comparison of Tregs in insulin-protected compared to naturally-protected mice demonstrated presence of insulin specific CD4+FoxP3+ Tregs in insulin treated mice but not in naturally protected animals. At the initiation of insulin-preventative therapy, if mice were given anti-GITR antibody DTA-1, the insulin preventative therapy was less effective and diabetesonset was accelerated.Our results demonstrate that an animal model of spontaneous diabetes, longterm therapy with high dose insulin prevents diabetes, and that induction of a regulatory subset of lymphocytes may be an important factor to prevent the development of type 1 diabetes.