Type 1 diabetes (T1D) develops as control of autoreactive CD4 cells is lost, which occurs in part because of escalating responses of cells that escape regulation and are highly pathogenic. While many cell types contribute to this disease, ameliorating the autoimmune response of CD4 cells continues to be an important strategy for treatment of T1D. The goal of the proposed studies is to assess changes in development and function of CD4 cell subsets during progression to T1D. It has become increasingly clear that a changing dynamic of CD4 cell responses occurs in this autoimmune disease. The objective is to determine whether checkpoints can be identified where autoreactive CD4 cells can be modulated to prevent or reverse diabetes onset. Using the NOD mouse model of T1D, our studies show that Th1 and Th17 subsets of CD4 cells, which are distinguished by the production of IFN-3 vs IL-17, respectively, are present at T1D onset and that both subsets can induce T1D. Moreover, we find CD4 cells that differentially secrete these cytokines throughout the prediabetic phase. Recent studies suggest that FoxP3-expressing regulatory CD4 cells (TR cells) can lose function and may even have the potential to convert to Th17 cells in a proinflammatory milieu, which could also contribute to the loss of control that precipitates T1D onset. These findings underscore the need for better understanding of the responses of CD4 cells and how they are altered as the autoimmune response progresses. We find that persisting CD4 cells become indefinitely established in NOD mice with adoptive transfer studies when allelically marked CD4 populations from NOD or TCR-transgenic BDC 2.5 mice are injected into 1 wk old recipients. These cells are thus subject to in situ regulation by the environment which promotes the development of T1D. This powerful in vivo model will enable us to follow changes that arise in CD4 cell subsets with progression to disease. We will also make use of IFN-3 and FoxP3 reporter mice to track subsets of CD4 cells. We propose to: 1) test the hypothesis that effector CD4 cells that are generated in T1D prone individuals in the prediabetic phase over time increase in number and differentiate into highly pathogenic Th1 and Th17 cells with capacity to mount a potent cytokine-driven effector response culminating in disease onset;and 2) determine if TR cells lose function or convert to pathogenic Th17 cells with the advancing autoimmune response that leads to T1D. These basic studies are essential to provide new insights into the development of the autoreactive CD4 cell response in T1D, and may distinguish a late stage of effector differentiation where intervention could inhibit the responses of pathogenic CD4 cells and control or reverse the disease process so that mechanisms of tolerance can be restored. PUBLIC HEALTH RELEVANCE: Type 1 diabetes is a lifelong, incurable autoimmune disease which can be treated with insulin. However, treatment does not prevent the eventual and devastating effects of this disease, which may include kidney failure, blindness, nerve damage, amputations, heart attack, and stroke. The best hope for achieving better treatments and even a cure is to better understand the autoimmune response. The goals of the proposed studies are to investigate whether checkpoints can be identified where responses of pathogenic CD4 cells can targeted to prevent complete destruction of insulin-producing islet cells so that tolerance can be established.