Proinsulin C-peptide is a major source of HLA-DQ8 restricted HIPs recognized by human Islet-Infiltrating CD4+T cells

ABSTRACT Type 1 diabetes (T1D) is an autoimmune disease that develops when T cells destroy the pancreatic insulin-producing beta cells that reside in the pancreatic islets. Immune cells, including T cells infiltrate the islets and gradually destroy the beta cells. Human islet-infiltrating CD4 + T cells recognize peptide epitopes derived from proinsulin, particularly C-peptide. Hybrid Insulin peptides (HIPs) are neoepitopes formed by the fusion of two peptides derived from beta-cell granule proteins and are known to be the targets of pathogenic CD4 + T cells in the NOD mouse and human islet-infiltrating CD4 + T cells. Proinsulin is widely recognized as a central antigen in T1D, but its role in forming HIPs is unclear. We developed a method to functionally screen TCRs derived from human islet-infiltrating CD4 + T cells and applied this to the identification of new proinsulin-derived HIPs. We generated a library of 4,488 candidate HIPs formed by fusion of proinsulin fragments and predicted to bind to HLA-DQ8. This library was screened against 109 islet-infiltrating CD4 + T-cell TCRs isolated from four organ donors who had T1D. We identified 13 unique HIPs recognized by 9 different TCRs from two organ donors. HIP specific T-cell avatars responded specifically to a peptide extract from human islets. These new HIPs predominantly stimulated CD4 + T-cell proliferation in PBMCs from people with T1D in contrast to HLA-matched controls. This is the first unbiased functional, islet-infiltrating T-cell based, screen to identify proinsulin derived HIPs. It has revealed many new HIPs and a central role of proinsulin C-peptide in their formation. SUMMARY Type 1 diabetes is an autoimmune disease caused by T cells destroying the pancreatic insulin-producing beta cells. The antigens/epitopes seen by disease promoting CD4 + T cells are poorly understood. Hybrid insulin peptides (HIPs) are a new class of CD4 + antigen recognized by pathogenic NOD mouse CD4 + T cells. In humans very few HIPs recognized by human islet-infiltrating CD4 + T cells are known. We show that proinsulin HIPs are recognized by human islet-infiltrating CD4 + T cells from T1D donors and describe 13 new HIPs formed by fusion of proinsulin peptides. This work shows that proinsulin, particularly C-peptide, is a major contributor to the pool HIPs recognized by human islet-infiltrating CD4 + T cells and are therefore central to autoimmunity in T1D.