T lymphocytes reactive against pancreatic antigens are considered the primary source of autoimmune dysfunction in Type 1 Diabetes (T1D) patients. Insulitic lesions provoked by the infiltration of these T cells into the pancreatic islets leads to destruction of the insulin-producing a-cells. Such T cells constitute only a minor fraction of the total repertoire, in particular in the blood, the only sample source that is realistically accessible in T1D patients. In spite of intense efforts, reliable quantitation of these cells has proven difficult, although highly desirable: 1. To evaluate the true risk and the temporal evolution of disease in first-degree relatives of T1D patients, particularly when considering immunointervention. 2. To monitor disease progression in recent onset patients. 3. To measure the effectiveness of therapeutic protocols, in particular in the context of clinical trials. These studies are hampered by their long-term endpoints of beta-cell destruction several years after treatment (disease onset, deteriorating C-peptide production). In the first phase of this Phased Innovation Award, the Immunology and Eye sections of the Joslin Diabetes Center's Research Division will design and implement an instrument for time-lapse measurement of T cell activation and sensing of the earliest signs of antigen recognition (Ca flux, mobility changes). For these proof of principle studies, T cells of known specificity will be challenged with cognate peptide on presenting cells or in MHC-peptide recombinant molecules (tetramers). A high-speed computer-controlled microscope will scan through large numbers of T cells (500,000), recording intracellular Calcium over time. Computational pattern recognition analysis of these images will identify individual cell objects, track their movements, and compute their Calcium and mobility traces. These traces will be converted into vectors of mathematical descriptors of the cellular responses, and cluster analysis of these data will identify and quantitate the various response profiles in naive, memory or tolerant T cells. In the second phase of this project, this instrument will be applied to autoreactive T cells of diabetic patients, in the three contexts listed above: breadth, prognostic value in recent-onset patients; risk evaluation and natural history in """"""""at risk"""""""" pre-diabetic subjects; efficacy in patients undergoing therapy. We will quantitate and characterize the response profiles of cells reactive against islet antigens (insulin, GAD, others being characterized). These investigations will be performed in relation with tetramer analysis of diabetic autoreactivity (Seattle). They will apply to the patient population of the Joslin Diabetes Center/Children's Hospital study group and will be directly connected to screening and therapy efforts within the TrialNet consortium.
