Regulatory vs. inflammatory cytokine T-cell responses to mutated insulin peptides in healthy and type 1 diabetic subjects

Abstract

Certain class II MHC (MHCII) alleles in mice and humans confer risk for or protection from type 1 diabetes (T1D). Insulin is a major autoantigen in T1D, but how its peptides are presented to CD4 T cells by MHCII risk alleles has been controversial. In the mouse model of T1D, CD4 T cells respond to insulin B-chain peptide (B:9-23) mimotopes engineered to bind the mouse MHCII molecule, IA g7 , in an unfavorable position or register. Because of the similarities between IA g7 and human HLA-DQ T1D risk alleles, we examined control and T1D subjects with these risk alleles for CD4 T-cell responses to the same natural B:9-23 peptide and mimotopes. A high proportion of new-onset T1D subjects mounted an inflammatory IFN-γ response much more frequently to one of the mimotope peptides than to the natural peptide. Surprisingly, the control subjects bearing an HLA-DQ risk allele also did. However, these control subjects, especially those with only one HLA-DQ risk allele, very frequently made an IL-10 response, a cytokine associated with regulatory T cells. T1D subjects with established disease also responded to the mimotope rather than the natural B:9-23 peptide in proliferation assays and the proliferating cells were highly enriched in certain T-cell receptor sequences. Our results suggest that the risk of T1D may be related to how an HLA-DQ genotype determines the balance of T-cell inflammatory vs. regulatory responses to insulin, having important implications for the use and monitoring of insulinspecific therapies to prevent diabetes onset.T ype 1 diabetes (T1D), the autoimmune form of diabetes, results from T cell-mediated destruction of insulin-producing β-cells within pancreatic islets (1). The disease is dramatically increasing in incidence, doubling in the last two decades (2, 3), and now predictable with the measurement of antibodies directed against insulin and other proteins found in β-cells (4). Major efforts at disease prevention have been undertaken using preparations of insulin (s.c., oral, and intranasal) to induce tolerance and delay the onset of clinical symptoms (5-7). Measuring insulin-specific T-cell responses from the peripheral blood has been challenging but would allow for assessment of therapeutic response, which has been a major obstacle in these trials. In our study, we sought to detect peripheral T-cell responses to insulin in T1D patients and nondiabetic controls using a modified insulin B-chain peptide.Insulin is a major self-antigen for both T and B cells in murine and human T1D, with insulin B-chain amino acids 9-23 (B:9-23) being a key epitope presented by major histocompatibility complex class II (MHCII) molecules to CD4 T cells targeting pancreatic β-cells (8-10). There is strong evidence from the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes that the NOD MHCII molecule, IA g7 , is required for development of T1D and that pathogenic CD4 T cells recognize insulin B:9-23 presented in an unfavorable position or register (Reg3) in the IA g7 peptide binding groove (9, 11,...

Tissue distribution and clonal diversity of the T and B cell repertoire in type 1 diabetes

“…The acquisition of additional intraislet and antigen-specific T cells, as well as single-cell analyses, are currently ongoing in our laboratory along with several groups within the nPOD network and the Integrated Islet Distribution Program (12,13,36,54). We expect these consortium efforts will improve resolution of the adaptive signature in T1D, particularly given the heterogeneous distribution of insulitis within donor pancreata (50,53,55).…”

Autoreactive T cells in type 1 diabetes

“…Despite unresolved differences related to registers used in these studies, these observations support the concept that differences in MHC register use are key to disease development and lead to the presentation of native antigens as neoepitopes. Similar mechanisms may be operative in patients: HLA-DQ8-restricted CD4 + T lymphocytes may target insulin peptides in a low-affinity binding register similar to the NOD register 3 (170,171). Ultimately, differences in HLA-peptide complex interactions can impact responding T cell activation and phenotypes and synergize with the reduced insulin expression in the thymus that is associated with predisposing insulin gene variants.…”

Methyldopa blocks MHC class II binding to disease-specific antigens in autoimmune diabetes

“…To measure primary T cell responses, as opposed to the antigen presentation described above, we performed indirect enzyme-linked immunospot (ELISPOT) assays on cryopreserved samples using an insulin B:9-23 (B22E) mimotope, which is presented by DQ8 (37,38). Seven study subjects showed a response to the mimotope at baseline, and their responses decreased upon completion of the study ( Figure 6A).…”