Detection and Characterization of T cells Reactive to Hybrid Insulin Peptides in Patients with Type 1 Diabetes

Abstract

Abstract Our lab has recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Furthermore, these peptides are detected in the islets of NOD mice, indicating that islet-infiltrating T cells encounter their antigen at the site of immune injury. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors. In this study, using an IFN-g ELISPOT assay, we investigated whether T cells reactive to a panel of 16 HIPs could be observed in the PBMCs of T1D patients or control subjects. We observed that about a third of the T1D patients responded to at least one HIP; 70% of the responding patients (but only one control subject) showed reactivity to multiple HIPs. Responses to 4 HIPs were significantly elevated in T1D patients but not in control subjects. Using a multiplexed cytokine assay, we determined that T cells from responder patients had a bias towards a Th1 phenotype upon stimulation with HIPs. We isolated 8 non-redundant, antigen-specific T cell clones reactive to 6 different HIPs. These T cell clones react to their target HIPs in the low nanomolar range and secrete IFN-g, TNF-a and GM-CSF. This is the first report that HIP-reactive T cells with an inflammatory phenotype can be detected in the peripheral blood of patients with T1D, indicating that the presence of HIP-reactive T cells might serve as a biomarker of disease activity in the human setting.