T-cell responses to hybrid insulin peptides prior to type 1 diabetes development

T-cell responses to posttranslationally modified self-antigens are associated with many autoimmune disorders. In type 1 diabetes, hybrid insulin peptides (HIPs) are implicated in the T-cell–mediated destruction of insulin-producing β-cells within pancreatic islets. The natural history of the disease is such that it allows for the study of T-cell reactivity prior to the onset of clinical symptoms. We hypothesized that CD4 T-cell responses to posttranslationally modified islet peptides precedes diabetes onset. In a cohort of genetically at-risk individuals, we measured longitudinal T-cell responses to native insulin and hybrid insulin peptides. Both proinflammatory (interferon-γ) and antiinflammatory (interluekin-10) cytokine responses to HIPs were more robust than those to native peptides, and the ratio of such responses oscillated between pro- and antiinflammatory over time. However, individuals who developed islet autoantibodies or progressed to clinical type 1 diabetes had predominantly inflammatory T-cell responses to HIPs. Additionally, several HIP T-cell responses correlated to worsening measurements of blood glucose, highlighting the relevance of T-cell responses to posttranslationally modified peptides prior to autoimmune disease development.

Type 1 diabetes (T1D) is a prototypical organ-specific autoimmune disease that develops in stages (1, 2). The stages are marked by the presence of islet autoantibodies directed against insulin and other β-cell proteins, followed by impaired glucose tolerance, and finally clinical diabetes marked by hyperglycemia and the need for insulin treatment (3). The T1D disease course provides a defined preclinical period and the ability to measure immune responses prior to clinical symptoms.Self-reactive T cells target pancreatic β-cells in both murine models of spontaneous autoimmune diabetes and human T1D (4), with a number of antigens implicated as T-cell epitopes (5, 6). Recently, posttranslationally modified (PTM) epitopes have been characterized as novel autoantigens that may lead to a break in tolerance, thus resulting in T-cell–mediated immunity to pancreatic islets. PTM of antigens is well-described in autoimmune diseases, such as celiac disease (gluten sensitivity), in which tissue transglutaminase mediates deamidation of glutamine to glutamic acid within gliadin to create immunogenic CD4 T-cell epitopes (7–10). In rheumatoid arthritis, citrullinated peptides form epitopes from cartilage proteins that both elicit antibody responses and activate CD4 T cells (11). Similarly, a novel class of epitopes within T1D are hybrid insulin peptides (HIPs) that are formed within lysozymes of β-cells through a covalent bond between an insulin peptide fragment and another β-cell peptide, thereby generating a neo-epitope (12, 13).Recent studies provide strong evidence for the role of HIPs in the development of diabetes in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes (12, 14–16). Notably, the antigen for the well-studied “diabetogenic” BDC2.5 T-cell clone and transgenic mouse model is a HIP formed between a peptide fusion of C-peptide and a cleavage product of chromogranin A, termed WE14 (12, 17, 18). C-peptide is cleaved from the A and B chains of insulin prior to secretion from the β-cell. In the NOD mouse, HIP-reactive CD4 T cells have a proinflammatory phenotype, can be detected prior to the onset of diabetes, and their frequency increases as the disease progresses (15). Another CD4 T-cell epitope critical for NOD diabetes development is a fragment of the insulin B chain, consisting of amino acids 9 to 23 (B:9–23) (19–21). A strongly stimulating T-cell epitope is very likely a HIP consisting of a fragment of this insulin B-chain peptide with a portion of C-peptide fused to the C-terminal end (22). HIP-reactive CD4 T cells have also been studied in the context of human T1D, with multiple CD4 T-cell clones and lines grown from the residual pancreatic islets of T1D organ donors subsequently responding to these neo-epitopes (12, 23, 24). A number of HIP-reactive T cells have also been measured from the peripheral blood in newly diagnosed T1D patients (25–30); however, the timing of when these T cells appear in the disease course and whether these responses directed at PTM peptides precede those toward native antigens remains to be addressed. We hypothesized that HIP T-cell responses precede clinical diabetes development and are more robust than responses to native insulin peptides.In this study, we longitudinally collected peripheral blood mononuclear cells (PBMCs) from genetically at-risk individuals and measured reactivity to a panel of HIPs and native antigens using sensitive enzyme-linked immunospot (ELISPOT) assays, which have previously been used to identify CD4 T-cell responses in T1D (31). We show that PBMCs respond to native insulin peptides, but the cells respond more robustly to specific HIPs, including the insulin B chain B:9–23 HIP (B22E) and two C-peptide–derived HIPs (C-peptide/islet amyloid polypeptide-2 C:IAPP-2] and C:A chain). We demonstrate that T-cell responses fluctuate between pro- and antiinflammatory during the preclinical phase prior to T1D development. Interestingly, individuals who progressed to clinical disease or who seroconverted to islet autoantibody positivity during the course of the study had a distinct polarization toward proinflammatory responses to specific HIPs. Remarkably, the T-cell response to the C:IAPP-2 HIP correlated with worsening measures of blood glucose. Overall, the data support a pathogenic role for PTM epitopes in the preclinical stage of T1D, and the fluctuating nature of the T-cell responses has implications for timing therapies to prevent T1D and potentially other autoimmune disorders.