We recently established, using the NOD mouse model, 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.


R.L. Baker: None. T. Delong: None. P. Gottlieb: Advisory Panel; Self; Bristol-Myers Squibb Company. Research Support; Self; Caladrius Biosciences, Inc.. Advisory Panel; Self; Eli Lilly and Company. Board Member; Self; ImmunoMolecular Therapeutics. Consultant; Self; Kamada. Research Support; Self; JDRF, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc., MacroGenics, Inc., Pfizer Inc.. Advisory Panel; Self; Viacyte, Inc.. Research Support; Self; GlaxoSmithKline plc., Janssen Pharmaceuticals, Inc.. K.M. Haskins: None.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals.org/content/license.