T cells reactive to Hybrid Insulin Peptides in the Peripheral Blood of T1D Patients are Proinflammatory

We recently established that hybrid insulin peptides (HIPs), formed spontaneously in islet beta-cells by fusion of insulin C-peptide fragments to peptides of Chromogranin A (ChgA) or Islet Amyloid Polypeptide (IAPP), are ligands for diabetogenic CD4 T cell clones. The goal of this study was to investigate whether HIP-reactive T cells were indicative of ongoing autoimmunity in human T1D patients. To investigate their relevance to human disease, we used IFN-gamma ELISPOT to determine whether HIP-reactive T cells with an inflammatory phenotype were present in PBMCs of new onset T1D patients. We observed that PBMCs from new onset T1D patients responded to 8 of 16 HIPs tested and that nearly half of the patients tested responded to one or more HIPs. We used a CFSE based assay to further characterize T cells reactive to HIP peptides. CD4+CFSEdimCD25+ T cells were sorted single into single wells using FACS and cloned. T cell clonality was confirmed using TCR deep sequencing and T cell reactivity to specific HIPs was confirmed by ELISA. A total of 6 T cell clones were isolated and show a variety of TCR usage. One T cell clone was isolated from the same patient on two different blood draws, indicating a high frequency of this T cell clone in the peripheral blood. While all the T cell clones isolated secreted IFN-gamma in response to HIPs, they also produced other inflammatory cytokines such as TNF-alpha and GM-CSF and reacted to HIPs in the low nanomolar range. Our findings provide new evidence that HIP-reactive T cells are critical players in the pathogenesis of T1D and indicate that HIP-reactive T cells present in the peripheral blood can potentially serve as a biomarker of disease in a subset of patients.